Статті в журналах: "Sequential Link Activation"

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Martinuzzi, Emanuela, Georgia Afonso, Marie-Claude Gagnerault, Gaetano Naselli, Diana Mittag, Béhazine Combadière, Christian Boitard, et al. "acDCs enhance human antigen–specific T-cell responses." Blood 118, no. 8 (August 25, 2011): 2128–37. http://dx.doi.org/10.1182/blood-2010-12-326231.

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Abstract Detection of human Ag-specific T cells is limited by sensitivity and blood requirements. As dendritic cells (DCs) can potently stimulate T cells, we hypothesized that their induction in PBMCs in situ could link Ag processing and presentation to Ag-specific T-cell activation. To this end, unfractionated PBMCs (fresh or frozen) or whole blood were incubated for 48 hours with protein or peptide Ag together with different DC-activating agents to rapidly and sequentially induce, pulse, and mature DCs. DC activation was therefore lined up with Ag recognition by neighboring T cells, thus telescoping the sequential steps of T-cell activation. Efficient processing of protein Ags made prior knowledge of epitopes and HLA restrictions dispensable. While reducing stimulation time, manipulation and blood requirements, in situ DC induction specifically amplified Ag-specific T-cell responses (cytokine secretion, proliferation, CD137/CD154 up-regulation, and binding of peptide-HLA multimers). IL-1β, although released by DCs, was also secreted in an Ag-specific fashion, thus providing an indirect biomarker of T-cell responses. These accelerated cocultured DC (acDC) assays offered a sensitive means with which to evaluate T-cell responses to viral and melanoma Ag vaccination, and may therefore find application for immune monitoring in viral, tumor, autoimmune, and transplantation settings.

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Vachharajani, Vidula T., Tiefu Liu, Xianfeng Wang, Jason J. Hoth, Barbara K. Yoza, and Charles E. McCall. "Sirtuins Link Inflammation and Metabolism." Journal of Immunology Research 2016 (2016): 1–10. http://dx.doi.org/10.1155/2016/8167273.

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Sirtuins (SIRT), first discovered in yeast as NAD+ dependent epigenetic and metabolic regulators, have comparable activities in human physiology and disease. Mounting evidence supports that the seven-member mammalian sirtuin family (SIRT1–7) guard homeostasis by sensing bioenergy needs and responding by making alterations in the cell nutrients. Sirtuins play a critical role in restoring homeostasis during stress responses. Inflammation is designed to “defend and mend” against the invading organisms. Emerging evidence supports that metabolism and bioenergy reprogramming direct the sequential course of inflammation; failure of homeostasis retrieval results in many chronic and acute inflammatory diseases. Anabolic glycolysis quickly induced (compared to oxidative phosphorylation) for ROS and ATP generation is needed for immune activation to “defend” against invading microorganisms. Lipolysis/fatty acid oxidation, essential for cellular protection/hibernation and cell survival in order to “mend,” leads to immune repression. Acute/chronic inflammations are linked to altered glycolysis and fatty acid oxidation, at least in part, by NAD+ dependent function of sirtuins. Therapeutically targeting sirtuins may provide a new class of inflammation and immune regulators. This review discusses how sirtuins integrate metabolism, bioenergetics, and immunity during inflammation and how sirtuin-directed treatment improves outcome in chronic inflammatory diseases and in the extreme stress response of sepsis.

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Bogatcheva, Natalia V., Peiyi Wang, Anna A. Birukova, Alexander D. Verin, and Joe G. N. Garcia. "Mechanism of fluoride-induced MAP kinase activation in pulmonary artery endothelial cells." American Journal of Physiology-Lung Cellular and Molecular Physiology 290, no. 6 (June 2006): L1139—L1145. http://dx.doi.org/10.1152/ajplung.00161.2005.

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In this study, we demonstrate that challenge of endothelial cells (EC) with NaF, a recognized G protein activator and protein phosphatase inhibitor, leads to a significant Erk activation, with increased phosphorylation of the well-known Erk substrate caldesmon. Inhibition of the Erk MAPK, MEK, by U0126 produces a marked decrease in NaF-induced caldesmon phosphorylation. NaF transiently increases the activity of the MEK kinase known as Raf-1 (∼3- to 4-fold increase over basal level), followed by a sustained Raf-1 inhibition (∼3- to 4-fold decrease). Selective Raf-1 inhibitors (ZM-336372 and Raf-1 inhibitor 1) significantly attenuate NaF-induced Erk and caldesmon phosphorylation. Because we have previously shown that Ca2+/calmodulin-dependent protein kinase II (CaMKII) participates in Erk activation in thrombin-challenged cells, we next explored if CaMKII is involved in NaF-induced EC responses. We found that in NaF-treated EC, CaMKII activity increases in a time-dependent manner with maximal activity at 10 min (∼4-fold increase over a basal level). Pretreatment with KN93, a specific CaMKII inhibitor, attenuates NaF-induced barrier dysfunction and Erk phosphorylation. The Rho inhibitor C3 exotoxin completely abolishes NaF-induced CaMKII activation. Collectively, these data suggest that sequential activation of Raf-1, MEK, and Erk is modulated by Rho-dependent CaMKII activation and represents important NaF-induced signaling response. Caldesmon phosphorylation occurring by an Erk-dependent mechanism in NaF-treated pulmonary EC may represent a link between NaF stimulation and contractile responses of endothelium.

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Jeschke, Andreas, and Albert Haas. "Sequential actions of phosphatidylinositol phosphates regulate phagosome-lysosome fusion." Molecular Biology of the Cell 29, no. 4 (February 15, 2018): 452–65. http://dx.doi.org/10.1091/mbc.e17-07-0464.

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Phagosomes mature into phagolysosomes by sequential fusion with early endosomes, late endosomes, and lysosomes. Phagosome-with-lysosome fusion (PLF) results in the delivery of lysosomal hydrolases into phagosomes and in digestion of the cargo. The machinery that drives PLF has been little investigated. Using a cell-free system, we recently identified the phosphoinositide lipids (PIPs) phosphatidylinositol 3-phosphate (PI(3)P) and phosphatidylinositol 4-phosphate (PI(4)P) as regulators of PLF. We now report the identification and the PIP requirements of four distinct subreactions of PLF. Our data show that (i) PI(3)P and PI(4)P are dispensable for the disassembly and activation of (phago)lysosomal soluble N-ethylmaleimide-sensitive factor attachment protein receptors, that (ii) PI(3)P is required only after the tethering step, and that (iii) PI(4)P is required during and after tethering. Moreover, our data indicate that PI(4)P is needed to anchor Arl8 (Arf-like GTPase 8) and its effector homotypic fusion/vacuole protein sorting complex (HOPS) to (phago)lysosome membranes, whereas PI(3)P is required for membrane association of HOPS only. Our study provides a first link between PIPs and established regulators of membrane fusion in late endocytic trafficking.

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Okamoto, Isamu, Yoshiaki Kawano, Daizo Murakami, Takashi Sasayama, Norie Araki, Toru Miki, Albert J. Wong, and Hideyuki Saya. "Proteolytic release of CD44 intracellular domain and its role in the CD44 signaling pathway." Journal of Cell Biology 155, no. 5 (November 19, 2001): 755–62. http://dx.doi.org/10.1083/jcb.200108159.

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CD44 is a widely distributed cell surface adhesion molecule and is implicated in diverse biological processes. However, the nature of intracellular signaling triggered by CD44 remains to be elucidated. Here, we show that CD44 undergoes sequential proteolytic cleavage in the ectodomain and intracellular domain, resulting in the release of a CD44 intracellular domain (ICD) fragment. Consequently, CD44ICD acts as a signal transduction molecule, where it translocates to the nucleus and activates transcription mediated through the 12-O-tetradecanoylphorbol 13-acetate–responsive element, which is found in numerous genes involved in diverse cellular processes. Expression of an uncleavable CD44 mutant as well as metalloprotease inhibitor treatment blocks CD44-mediated transcriptional activation. In search of the underlying mechanism, we have found that CD44ICD potentiates transactivation mediated by the transcriptional coactivator CBP/p300. Furthermore, we show that cells expressing CD44ICD produce high levels of CD44 messenger RNA, suggesting that the CD44 gene is one of the potential targets for transcriptional activation by CD44ICD. These observations establish a novel CD44 signaling pathway and shed new light on the functional link between proteolytic processing of an adhesion molecule at the cell surface and transcriptional activation in the nucleus.

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Romanelli, Angela, Kathleen A. Martin, Alex Toker та John Blenis. "p70 S6 Kinase Is Regulated by Protein Kinase Cζ and Participates in a Phosphoinositide 3-Kinase-Regulated Signalling Complex". Molecular and Cellular Biology 19, № 4 (1 квітня 1999): 2921–28. http://dx.doi.org/10.1128/mcb.19.4.2921.

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ABSTRACT p70 S6 kinase (p70S6K) is an important regulator of cell proliferation. Its activation by growth factor requires phosphorylation by various inputs on multiple sites. Data accumulated thus far support a model whereby p70S6K activation requires sequential phosphorylations at proline-directed residues in the putative autoinhibitory pseudosubstrate domain, as well as threonine 389. Threonine 229, a site in the catalytic loop is phosphorylated by phosphoinositide-dependent kinase 1 (PDK-1). Experimental evidence suggests that p70S6K activation requires a phosphoinositide 3-kinase (PI3-K)-dependent signal(s). However, the intermediates between PI3-K and p70S6K remain unclear. Here, we have identified PI3-K-regulated atypical protein kinase C (PKC) isoform PKCζ as an upstream regulator of p70S6K. In coexpression experiments, we found that a kinase-inactive PKCζ mutant antagonized activation of p70S6K by epidermal growth factor, PDK-1, and activated Cdc42 and PI3-K. While overexpression of a constitutively active PKCζ mutant (myristoylated PKCζ [myr-PKCζ]) only modestly activated p70S6K, this mutant cooperated with PDK-1 activation of p70S6K. PDK-1-induced activation of a C-terminal truncation mutant of p70S6K was also enhanced by myr-PKCζ. Moreover, we have found that p70S6K can associate with both PDK-1 and PKCζ in vivo in a growth factor-independent manner, while PDK-1 and PKCζ can also associate with each other, suggesting the existence of a multimeric PI3-K signalling complex. This work provides evidence for a link between a phorbol ester-insensitive PKC isoform and p70S6K. The existence of a PI3-K-dependent signalling complex may enable efficient activation of p70S6K in cells.

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Lacanà, E., J. K. Ganjei, P. Vito, and L. D'Adamio. "Dissociation of apoptosis and activation of IL-1beta-converting enzyme/Ced-3 proteases by ALG-2 and the truncated Alzheimer's gene ALG-3." Journal of Immunology 158, no. 11 (June 1, 1997): 5129–35. http://dx.doi.org/10.4049/jimmunol.158.11.5129.

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Abstract Recent attention has been focused on the members of the IL-1beta-converting enzyme (ICE)/Ced-3 family of cysteine protease as the key components of programmed cell death. However, the molecular events that lead to protease activation and link it to the final apoptotic processes remain poorly characterized. We have shown recently that ALG-2 is a Ca2+-binding protein required for apoptosis. ALG-2 depletion protects the mouse T cell hybridoma 3DO from programmed cell death induced by several stimuli, such as synthetic glucocorticoids, TCR, and Fas triggering. In this work, we show that in the ALG-2-depleted clones the ICE/Ced-3 proteases are normally activated upon TCR, Fas, and dexamethasone stimulation, as determined by cleavage of the endogenous substrate poly(ADP-ribose) polymerase and of a fluorogenic substrate. ALG-3, a truncated form of the familial Alzheimer's disease gene PS2, confers resistance to TCR- and Fas-induced apoptosis. Of interest, it also reduces protease activity and inhibits poly(ADP-ribose) polymerase cleavage upon Fas triggering. Our results suggest that, during apoptosis, ALG-2 functions downstream of, and that ALG-3 interferes with the sequential activation of members of the ICE family proteases.

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Würtz, R. P., and C. von der Malsburg. "A Hierarchical Dynamic Link Network to Solve the Visual Correspondence Problem." Perception 25, no. 1_suppl (August 1996): 183. http://dx.doi.org/10.1068/v96l0702.

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Conventional neural networks try to solve the problem of object recognition in a single step by building a stimulus — response system that codes its result as cell activities. We take a different approach assuming that recognition is an active process with temporal dynamics and results in an ordered state. We present a structure of neuronal layers, interconnected by dynamic links (von der Malsburg, 1985 Berichte der Bunsengesellschaft für Physikalische Chemie89 703 – 710) that solves the correspondence problem between two images and thus constitutes an important building block for a model of recognition. Images as well as stored models are represented as Gabor pyramids. This allows the dynamics to proceed from coarse to fine scale and reduces the sequential processing time inherent in the concept. Invariance under background changes is also made possible. On the lowest frequency level, a single blob of activity moves across the image and model layer, respectively. Dynamic links between these layers are initialised to the (highly ambiguous) feature similarities. Links grow or decline according to a combination of feature similarity and correlated activation. This enforces correct neighbourhood relationships in addition to feature similarity. On the higher levels the established correspondences are refined by several blobs in parallel. We present an improved version of the dynamical system proposed by Würtz [1995 Multilayer Dynamic Link Networks for Establishing Image Point Correspondences and Visual Object Recognition (Thun, Frankfurt a.M.: Harri Deutsch)] and show, with examples of human faces, that it evolves from an unordered link distribution to any ordered state where only corresponding point pairs are connected by strong links. Correspondences between sample points are population-coded by a set of neighbouring links.

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Rescan, Claude, Alexandre Coutant, Hélène Talarmin, Nathalie Theret, Denise Glaise, Christiane Guguen-Guillouzo, and Georges Baffet. "Mechanism in the Sequential Control of Cell Morphology and S Phase Entry by Epidermal Growth Factor Involves Distinct MEK/ERK Activations." Molecular Biology of the Cell 12, no. 3 (March 2001): 725–38. http://dx.doi.org/10.1091/mbc.12.3.725.

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Cell shape plays a role in cell growth, differentiation, and death. Herein, we used the hepatocyte, a normal, highly differentiated cell characterized by a long G1 phase, to understand the mechanisms that link cell shape to growth. First, evidence was provided that the mitogen-activated protein kinase kinase (MEK)/extracellular signal-regulated kinase (ERK) cascade is a key transduction pathway controlling the hepatocyte morphology. MEK2/ERK2 activation in early G1 phase did not lead to cell proliferation but induced cell shape spreading and demonstration was provided that this MAPK-dependent spreading was required for reaching G1/S transition and DNA replication. Moreover, epidermal growth factor (EGF) was found to control this morphogenic signal in addition to its mitogenic effect. Thus, blockade of cell spreading by cytochalasin D or PD98059 treatment resulted in inhibition of EGF-dependent DNA replication. Our data led us to assess the first third of G1, is exclusively devoted to the growth factor-dependent morphogenic events, whereas the mitogenic signal occured at only approximately mid-G1 phase. Moreover, these two growth factor-related sequential signaling events involved successively activation of MEK2-ERK2 and then MEK1/2-ERK1/2 isoforms. In addition, we demonstrated that inhibition of extracellular matrix receptor, such as integrin β1 subunit, leads to cell arrest in G1, whereas EGF was found to up-regulated integrin β1 and fibronectin in a MEK-ERK–dependent manner. This process in relation to cytoskeletal reorganization could induce hepatocyte spreading, making them permissive for DNA replication. Our results provide new insight into the mechanisms by which a growth factor can temporally control dual morphogenic and mitogenic signals during the G1 phase.

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Wei, Xiumei, Yu Zhang, Cheng Li, Kete Ai, Kang Li, Huiying Li, and Jialong Yang. "The evolutionarily conserved MAPK/Erk signaling promotes ancestral T-cell immunity in fish via c-Myc–mediated glycolysis." Journal of Biological Chemistry 295, no. 10 (January 29, 2020): 3000–3016. http://dx.doi.org/10.1074/jbc.ra119.012231.

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The mitogen-activated protein kinase (MAPK) cascade is an ancient and evolutionarily conserved signaling pathway involved in numerous physiological processes. Despite great advances in understanding MAPK-mediated regulation of adaptive immune responses in mammals, its contribution to T-cell immunity in early vertebrates remains unclear. Herein, we used Nile tilapia (Oreochromis niloticus) to investigate the regulatory roles of MAPK/extracellular signal–regulated kinase (Erk) signaling in ancestral T-cell immunity of jawed fish. We found that Nile tilapia possesses an evolutionarily conserved MAPK/Erk axis that is activated through a classical three-tier kinase cascade, involving sequential phosphorylation of RAF proto-oncogene serine/threonine-protein kinase (Raf), MAPK/Erk kinase 1/2 (Mek1/2), and Erk1/2. In Nile tilapia, MAPK/Erk signaling participates in adaptive immune responses during bacterial infection. Upon T-cell activation, the MAPK/Erk axis is robustly activated, and MAPK/Erk blockade by specific inhibitors severely impairs T-cell activation. Furthermore, signals from MAPK/Erk were indispensable for primordial T cells to proliferate and exert their effector functions. Mechanistically, activation of the MAPK/Erk axis promoted glycolysis via induction of the transcriptional regulator proto-oncogene c-Myc (c-Myc), to ensure the proper activation and proliferation of fish T cells. Our results reveal the regulatory mechanisms of MAPK/Erk signaling in T-cell immunity in fish and highlight a close link between immune signals and metabolic programs. We propose that regulation of T-cell immunity by MAPK/Erk is a basic and sophisticated strategy that evolved before the emergence of the tetrapod lineage. These findings shed light on the evolution of the adaptive immune system.

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Soriano, Marco, Andrea Cavallo, Alessandro D’Ausilio, Cristina Becchio, and Luciano Fadiga. "Movement kinematics drive chain selection toward intention detection." Proceedings of the National Academy of Sciences 115, no. 41 (September 21, 2018): 10452–57. http://dx.doi.org/10.1073/pnas.1809825115.

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The ability to understand intentions based on another’s movements is crucial for human interaction. This ability has been ascribed to the so-called motor chaining mechanism: anytime a motor chain is activated (e.g., grasp-to-drink), the observer attributes to the agent the corresponding intention (i.e., to drink) from the first motor act (i.e., the grasp). However, the mechanisms by which a specific chain is selected in the observer remain poorly understood. In the current study, we investigate the possibility that in the absence of discriminative contextual cues, slight kinematic variations in the observed grasp inform mapping to the most probable chain. Chaining of motor acts predicts that, in a sequential grasping task (e.g., grasp-to-drink), electromyographic (EMG) components that are required for the final act [e.g., the mouth-opening mylohyoid (MH) muscle] show anticipatory activation. To test this prediction, we used MH EMG, transcranial magnetic stimulation (TMS; MH motor-evoked potentials), and predictive models of movement kinematics to measure the level and timing of MH activation during the execution (Experiment 1) and the observation (Experiment 2) of reach-to-grasp actions. We found that MH-related corticobulbar excitability during grasping observation varied as a function of the goal (to drink or to pour) and the kinematics of the observed grasp. These results show that subtle changes in movement kinematics drive the selection of the most probable motor chain, allowing the observer to link an observed act to the agent’s intention.

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Kim, Yeun Ju, Maria Luisa Guzman-Hernandez, Eva Wisniewski, Nicolas Echeverria, and Tamas Balla. "Phosphatidylinositol and phosphatidic acid transport between the ER and plasma membrane during PLC activation requires the Nir2 protein." Biochemical Society Transactions 44, no. 1 (February 9, 2016): 197–201. http://dx.doi.org/10.1042/bst20150187.

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Phospholipase C (PLC)-mediated hydrolysis of the limited pool of plasma membrane (PM) phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P2] requires replenishment from a larger pool of phosphatidylinositol (PtdIns) via sequential phosphorylation by PtdIns 4-kinases and phosphatidylinositol 4-phosphate (PtdIns4P) 5-kinases. Since PtdIns is synthesized in the endoplasmic reticulum (ER) and PtdIns(4,5)P2 is generated in the PM, it has been postulated that PtdIns transfer proteins (PITPs) provide the means for this lipid transfer function. Recent studies identified the large PITP protein, Nir2 as important for PtdIns transfer from the ER to the PM. It was also found that Nir2 was required for the transfer of phosphatidic acid (PtdOH) from the PM to the ER. In Nir2-depleted cells, activation of PLC leads to PtdOH accumulation in the PM and PtdIns synthesis becomes severely impaired. In quiescent cells, Nir2 is localized to the ER via interaction of its FFAT domain with ER-bound VAMP-associated proteins VAP-A and–B. After PLC activation, Nir2 also binds to the PM via interaction of its C-terminal domains with diacylglycerol (DAG) and PtdOH. Through these interactions, Nir2 functions in ER–PM contact zones. Mutations in VAP-B that have been identified in familial forms of amyotrophic lateral sclerosis (ALS or Lou-Gehrig's disease) cause aggregation of the VAP-B protein, which then impairs its binding to several proteins, including Nir2. These findings have shed new lights on the importance of non-vesicular lipid transfer of PtdIns and PtdOH in ER–PM contact zones with a possible link to a devastating human disease.

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Ohba, Reiko, David J. Steger, James E. Brownell, Craig A. Mizzen, Richard G. Cook, Jacques Côté, Jerry L. Workman, and C. David Allis. "A Novel H2A/H4 Nucleosomal Histone Acetyltransferase in Tetrahymena thermophila." Molecular and Cellular Biology 19, no. 3 (March 1, 1999): 2061–68. http://dx.doi.org/10.1128/mcb.19.3.2061.

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ABSTRACT Recently, we reported the identification of a 55-kDa polypeptide (p55) from Tetrahymena macronuclei as a catalytic subunit of a transcription-associated histone acetyltransferase (HAT A). Extensive homology between p55 and Gcn5p, a component of the SAGA and ADA transcriptional coactivator complexes in budding yeast, suggests an immediate link between the regulation of chromatin structure and transcriptional output. Here we report the characterization of a second transcription-associated HAT activity from Tetrahymenamacronuclei. This novel activity is distinct from complexes containing p55 and putative ciliate SAGA and ADA components and shares several characteristics with NuA4 (for nucleosomal H2A/H4), a 1.8-MDa, Gcn5p-independent HAT complex recently described in yeast. A key feature of both the NuA4 and Tetrahymena activities is their acetylation site specificity for lysines 5, 8, 12, and 16 of H4 and lysines 5 and 9 of H2A in nucleosomal substrates, patterns that are distinct from those of known Gcn5p family members. Moreover, like NuA4, the Tetrahymena activity is capable of activating transcription from nucleosomal templates in vitro in an acetyl coenzyme A-dependent fashion. Unlike NuA4, however, sucrose gradient analyses of the ciliate enzyme, following sequential denaturation and renaturation, estimate the molecular size of the catalytically active subunit to be ∼80 kDa, consistent with the notion that a single polypeptide or a stable subcomplex is sufficient for this H2A/H4 nucleosomal HAT activity. Together, these data document the importance of this novel HAT activity for transcriptional activation from chromatin templates and suggest that a second catalytic HAT subunit, in addition to p55/Gcn5p, is conserved between yeast and Tetrahymena.

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Haubold, Katja, Michael Rink, Brigitte Spath, Ali Amirkhosravi, John L. Francis, Barbara Eifrig, Carsten Bokemeyer, and Florian Langer. "Microparticle-Associated Tissue Factor: A Molecular Link Between Coagulation Activation, Inflammation and Disease Progression in Early-Stage Prostate Cancer?" Blood 112, no. 11 (November 16, 2008): 3813. http://dx.doi.org/10.1182/blood.v112.11.3813.3813.

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Abstract Activation of coagulation and inflammation is a characteristic finding in patients with advanced malignancies, including prostate cancer. Tissue factor (TF), a molecule involved in hemostasis, thrombosis and pro-inflammatory signaling pathways, is over-expressed on tumor cells and cells of the tumor microenvironment (i.e. endothelial cells, fibroblasts and tissue macrophages). Moreover, the enhanced release of TF into plasma in association with sub-cellular membrane vesicles, so-called plasma microparticles (MPs), has recently been associated with key events in molecular oncogenesis and cancer progression. In this study, we measured TF-specific procoagulant activity (PCA) of plasma MPs in 58 consecutive patients with clinically localized prostate cancer (mean age, 64±5 years) to explore its potential as a prognostic marker in this tumor entity. MPs were isolated from pre-operative plasma samples by sequential high-speed centrifugation for 1 h at 16,100 × g. TF-specific PCA of plasma MPs was quantified using a highly sensitive one-stage clotting assay in the presence and absence of inhibitory TF monoclonal antibody and calibration of clotting times against serial dilutions (1:10–1:105) of lipidated recombinant human full-length TF (rhTF1–263), showing a linear correlation in a log-log plot with R2>0.99. The lower detection limit of this assay for rhTF1–263 (33 kDa) was <5 pg/ml (<150 fM), and the intra- and inter-assay coefficients of variation were 7.3% and 5.4%, respectively. Total numbers of TF-positive MPs were measured by single-color flow cytometry using PE-conjugated TF monoclonal antibody (HTF-1) and microspheres for size calibration (1 μm) and sample flow standardization. TF antigen was quantified in plasma by ELISA. Calibrated automated thrombography (CAT) was used to monitor thrombin generation in platelet-free plasma samples over a 2-h period without the addition of exogenous TF or phospholipids. Intravascular coagulation activation was assessed by measuring plasma D-dimer. All assay systems were validated using MPs spontaneously shed from prostate cancer cell lines (PC-3, LNCaP and DU145) or from whole blood monocytes after challenge with endotoxin. Based on plasma fibrinogen and C-reactive protein levels, patients were stratified into those with (n=26) and those without (n=32) laboratory evidence of an acute-phase reaction (APR). Compared to healthy male controls (n=20), patients had significantly increased levels of both D-dimer (0.46±0.19 vs. 0.21±0.05 mg/l) and TF-specific PCA of plasma MPs (563±301 vs. 292±74 U/ml) (P<0.001). Among patients, laboratory evidence of an APR was associated with a significant increase in MP-associated TF PCA (699±351 vs. 452±196 U/ml) (P=0.001). Overall, we found a significant correlation between MP-associated TF PCA and plasma D-Dimer (P=0.015), suggesting that plasma MPs contributed to in-vivo coagulation activation in a TF-dependent manner. CAT also revealed significantly increased thrombin generation in patient compared to control plasmas, as indicated by a shortening in lag phase (25±4 vs. 29±5 min) and an increase in both peak thrombin generation (184±76 vs. 127±71 nM) and the endogenous thrombin potential, defined as the area under the thrombin generation curve (3576±509 vs. 2980±562 nM*min) (P<0.01). Importantly, TF-specific PCA of plasma MPs correlated neither with absolute numbers of TF-positive MPs nor with plasma TF antigen, suggesting that a substantial and variable fraction of the total plasma TF pool circulated as an inactive variant. Interestingly, systemic levels of IL-8, an inflammatory cytokine involved in TF/FVIIa-dependent, PAR-2-mediated pro-migratory signaling pathways in tumor cells and shown to be of biological relevance in advanced, hormone-refractory prostate cancer, were elevated in patients compared to controls (9±11 vs. 4±6 pg/ml) (P<0.01). In summary, our findings suggest that TF-specific PCA of plasma MPs contributes to intravascular coagulation activation in patients with early-stage prostate cancer and may represent an important molecular link between hypercoagulability, inflammation and disease progression. The above-described assay for the quantification of MP-associated TF PCA could thus be of prognostic value in the risk stratification of patients with localized prostate cancer with respect to thromboembolic complications and/or tumor recurrence.

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Nikoobin, A., and M. Moradi. "Indirect solution of optimal control problems with state variable inequality constraints: finite difference approximation." Robotica 35, no. 1 (July 2, 2015): 50–72. http://dx.doi.org/10.1017/s0263574715000521.

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SUMMARYIn this paper, a method for the indirect solution of the optimal control problem (OCP) in the presence of pure state variable inequality constraints (SVICs) and mixed state-control inequality constraints (SCIC), without a need for a close initial guess is presented. In the proposed method, using the finite difference approximation (FDA), the pure SVICs are converted to SCIC. Here, the distance of the constraint function to the feasibility bounds of the constraint is computed in every situation and the control signal is chosen appropriately to facilitate the constraint stays safe. In this method, prior knowledge of the numbers and sequences of activation times is not required. So, it can be simply implemented in continuous boundary value problem (BVP) solvers. The proposed method simply applies the SVICs and since the constraint is directly applied on the control signal, it improves the convergence. On the other hand, because of the convergence problem in the indirect solution of OCP, the simple homotopy continuation method (HCM) is used to overcome the initial guess problem by deploying a secondary OCP for which the initial guess can be zero. The proposed approach is applied on a few comprehensive problems in the presence of different constraints. Simulations are compared with the direct solution of the OCP to confirm the accuracy and with the penalty function method and the sequential constraint-free OCP to confirm the convergence. The results indicate that the FDA method for handling the constraints along with the HCM is easy to apply with acceptable accuracy and convergence, even for highly nonlinear problems in robotic systems such as the constrained time optimal control of a two-link manipulator (TLM) and a three-link common industrial robot.

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Lengerke, Claudia, Sabine Schmitt, Teresa Bowman, Leila Maouche-Chretien, Shannon L. McKinney-Freeman, Il Ho Jang, Alan J. Davidson, et al. "BMP and Wnt Induce Embryonic Blood Fate by Activation of the Cdx-Hox Pathway." Blood 110, no. 11 (November 16, 2007): 199. http://dx.doi.org/10.1182/blood.v110.11.199.199.

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Abstract The hematopoietic morphogen BMP4 has been implicated in early blood development, however the precise role and the underlying genetic pathways that link BMP action to blood fate specification remain poorly understood. In this study, we have employed the in vitro system of murine embryonic stem (mES) cell differentiating into embryoid bodies (EBs) as a model to explore mesoderm induction and hematopoietic specification during mammalian embryogenesis. We show that BMP plays a role in blood fate specification in two sequential phases: initially, BMP acts as a dorsoventral patterning agent, inducing ventroposterior mesoderm; at a later phase, BMP is required for hematopoietic fate specification from mesoderm. In dissecting this second specification phase, we discovered a previously unsuspected coordination between BMP4 and Wnt3a signaling, and linked these morphogens directly to Cdx and Hox genes. Using chromatin immunoprecipitation assays performed on EB-derived cells, we detected the association of the Wnt signaling effector LEF1 with the proximal Cdx1 promoter. This association was modulated by BMP signaling. In promoter reporter assays both BMP4 and Wnt3a activated the Cdx1 promoter while Wnt inhibitors or point mutations in the LEF1 responsive elements of the Cdx1 promoter abrogated this response. Together, these results suggest that BMP4 activates the Cdx1 promoter via LEF1 and the Wnt pathway. In functional assays, inhibition of Wnt signaling strongly suppressed the formation of hematopoietic colony initiating cells, suggesting that activation of the Wnt-Cdx-Hox pathway is a central downstream component of the BMP dependent blood fate specification program. Using ES cell lines engineered for conditonal Cdx1 or Cdx4 gene overexpression, we demonstrate that ectopic activation of Cdx genes is sufficient to establish hematopoietic fate decisions in the absence of BMP signaling during mesoderm specification to blood. Finally, to explore the relevance of our findings in vivo, we studied the effects of conditional BMP overexpression on cdx4 expression and embryonic blood formation during zebrafish embryogenesis. Matching our results with EBs, we find that overexpression of bmp2b at midgastrulation expands the cdx4 expression domain and strongly enhances blood formation. Taken together our results reveal an interaction between the BMP and Wnt signaling pathways, upstream of the Cdx genes, that is critical for the induction of the embryonic blood cell program. Given the evolutionary conservation of the BMP-Wnt-Cdx-Hox effect on blood formation from fish to mice, we anticipate that similar pathways can be exploited to direct the differentiation of human ES cells into blood.

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17

Yamamoto, Shinji, Hiroki Hikasa, Hirofumi Ono, and Masanori Taira. "Molecular link in the sequential induction of the Spemann organizer: direct activation of the cerberus gene by Xlim-1, Xotx2, Mix.1, and Siamois, immediately downstream from Nodal and Wnt signaling." Developmental Biology 257, no. 1 (May 2003): 190–204. http://dx.doi.org/10.1016/s0012-1606(03)00034-4.

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18

Duygu, Sari, Nikolaos Patsoukis, and Vassiliki A. Boussiotis. "Phosphorylation of Tyrosine 340 in the Pleckstrin Homology Domain of RIAM Is Required for Inside-Out Activation of LFA-1 and LFA-1: ICAM-1-Mediated Adhesion." Blood 120, no. 21 (November 16, 2012): 837. http://dx.doi.org/10.1182/blood.v120.21.837.837.

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Abstract Abstract 837 Adhesion of lymphocytes to antigen presenting cells (APCs) is a critical event linking innate and adaptive immunity and a mandatory step for the initiation of T cell immune responses. Control of lymphocyte adhesion to APC is accomplished through the regulation of the principle adhesion molecule on the lymphocyte surface, the β2 integrin designated lymphocyte functional antigen 1 (LFA-1), which binds to intercellular adhesion molecule 1 (ICAM-1) on the surface of APCs. In order to mediate its adhesive function LFA-1 must be activated via a process referred to as inside-out signaling, which results in conformation changes of the receptor that extends the ectodomains of the α and β chains leading to a high affinity state. Because LFA-1-mediated adhesion is a central link between innate and adaptive immunity, understanding the mechanisms implicated in inside-out-mediated LFA-1 activation is a subject of intense investigation. Currently, it is poorly understood how signals originating from the TCR are linked to specific mechanisms that regulate inside-out activation of LFA-1. Among the few signaling molecules that have been implicated in this process in hematopoietic cells are the small GTPase Rap1 and its downstream effector RIAM. RIAM is a multidomain protein that includes a talin binding region, two coiled-coiled regions, a small N-terminus proline-rich region, sequential Ras association (RA) and pleckstrin homology (PH) domains, and a large C-terminus proline-rich region, via which interacts with Ena/VASP family proteins and profilin and is recruited to the sites of actin turnover. Through its C-terminus RIAM also interacts with the SH3-domain of PLC-γ1. The RA and PH domains of RIAM function as an integral unit and as a proximity detector since both are required for translocation of RIAM to the plasma membrane. The RA domain of RIAM has specificity for Rap1-GTP whereas the PH domain binds to the PLC-γ1 substrate PI(4,5)P2. We determined that the integral RA-PH structural unit of RIAM has also an integral functional role because both the RA and PH regions are required for LFA-1-mediated adhesion. Here we sought to determine how TCR proximal signaling events regulate RIAM function leading to inside-out activation of LFA-1 and LFA-1:ICAM-1 mediated adhesion. Using primary human T lymphocytes and Jurkat T cells we determined that upon TCR/CD3-mediated stimulation, activated Src family kinases Fyn and Lck associate with and induce tyrosine phosphorylation of RIAM. To identify the precise domain(s) of RIAM, which undergo phosphorylation by these kinases, we co-expressed individual truncation constructs of RIAM N-terminus, RA-PH, and C-terminus regions along with the active or inactive form of Fyn or Lck in COS cells. Immunoprecipitations and immunoblot assays revealed that active Fyn and Lck mediated robust and selective tyrosine phosphorylation of the RA-PH structural unit of RIAM. Detailed analysis by mass spectrometry identified that tyrosine 340 (Y340) within the PH domain of RIAM was the specific target. The PH domain of RIAM has specificity for PI(4,5)P2 and is an integral component of the RA-PH proximity detector. We hypothesized that phosphorylation of tyrosine 340 in the PH domain might have a regulatory role in the function of the RA-PH structural unit in mediating LFA-1 activation. To address this issue we used site directed mutagenesis and we introduced a tyrosine to phenylalanine mutation in site 340 (Y34F) of RIAM, rendering this residue resistant to phosphorylation mediated by Fyn and Lck. Expression of RIAM Y340F in Jurkat T cells abrogated LFA-1 activation and LFA-1-mediated adhesion in response to TCR/CD3 stimulation. Under these conditions, activation of LFA-1 in response to PMA, which bypasses TCR-mediated signaling, remained intact. Thus, besides recruitment of RIAM to the plasma membrane, TCR-mediated activation of Src family kinases and phosphorylation of RIAM PH domain is a mandatory requirement for the function of the RA-PH proximity detector as a regulator of LFA-1 activation. Our results provide a biochemical, molecular and mechanistic link between TCR-mediated signaling and inside-out activation of LFA-1 thereby initiating LFA-1: ICAM-1-mediated adhesion and cross-talk between T cells and APC. Disclosures: No relevant conflicts of interest to declare.

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19

Barata, Joao T., Angelo A. Cardoso, Lee M. Nadler, and Vassiliki A. Boussiotis. "Interleukin-7 promotes survival and cell cycle progression of T-cell acute lymphoblastic leukemia cells by down-regulating the cyclin-dependent kinase inhibitor p27kip1." Blood 98, no. 5 (September 1, 2001): 1524–31. http://dx.doi.org/10.1182/blood.v98.5.1524.

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In normal T-cell development interleukin-7 (IL-7) functions as an antiapoptotic factor by regulating bcl-2 expression in immature thymocytes and mature T cells. Similar to what occurs in normal immature thymocytes, prevention of spontaneous apoptosis by IL-7 in precursor T-cell acute lymphoblastic leukemia (T-ALL) cells correlates with up-regulation of bcl-2. IL-7 is also implicated in leukemogenesis because IL-7 transgenic mice develop lymphoid malignancies, suggesting that IL-7 may regulate the generation and expansion of malignant cells. This study shows that in the presence of IL-7, T-ALL cells not only up-regulated bcl-2 expression and escaped apoptosis but also progressed in the cell cycle, resulting in sequential induction of cyclin D2 and cyclin A. Down-regulation of p27kip1 was mandatory for IL-7–mediated cell cycle progression and temporally coincided with activation of cyclin-dependent kinase (cdk)4 and cdk2 and hyperphosphorylation of Rb. Strikingly, forced expression of p27kip1 in T-ALL cells not only prevented cell cycle progression but also reversed IL-7–mediated up-regulation of bcl-2 and promotion of viability. These results show for the first time that a causative link between IL-7–mediated proliferation and p27kip1 down-regulation exists in malignant T cells. Moreover, these results suggest that p27kip1 may function as a tumor suppressor gene not only because it is a negative regulator of cell cycle progression but also because it is associated with induction of apoptosis of primary malignant cells.

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20

Sari, Duygu, Niko Tsopoulidis, John M. Asara, Nikolaos Patsoukis, and Vassiliki A. Boussiotis. "Phosphorylation of Tyrosine 340 in the Plekstrin Homology Domain of RIAM Is Required for Translocation of RIAM to the Plasma Membrane, Phosphorylation of RIAM-Associated PLC-g1 and LFA-1 Activation." Blood 124, no. 21 (December 6, 2014): 2743. http://dx.doi.org/10.1182/blood.v124.21.2743.2743.

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Abstract Adhesion of lymphocytes to antigen presenting cells (APCs) is a critical step linking innate and adaptive immunity. Lymphocyte-APC adhesion is accomplished through the principle adhesion molecule on the lymphocyte surface, the β2 integrin designated lymphocyte functional antigen 1 (LFA-1), which binds to intercellular adhesion molecule 1 (ICAM-1) on the surface of APCs. LFA-1 must be activated via a process referred to as inside-out signaling, which results in conformation changes leading to a high affinity state. Among the few signaling molecules implicated in inside-out signaling in hematopoietic cells are the small GTPase Rap1 and its downstream effector RIAM. RIAM is a multidomain protein that includes a talin binding region, two coiled-coiled regions, a small N-terminus proline-rich region, sequential Ras association (RA) and pleckstrin homology (PH) domains, and a large C-terminus proline-rich region, via which interacts with Ena/VASP family proteins and profilin. Through its C-terminus, RIAM constitutively interacts with PLC-γ1. The RA domain of RIAM has specificity for Rap1-GTP whereas the PH domain binds to the PLC-γ1 substrate PI(4,5)P2. The RA-PH domain region of RIAM functions as an integral unit and as a proximity detector, and both RA and PH are required for translocation of RIAM to the plasma membrane. Using primary human T lymphocytes and Jurkat T cells we determined previously that RIAM undergoes tyrosine phosphorylation by Src family kinases upon TCR stimulation. In the present study we sought to determine the role of tyrosine phosphorylation in RIAM function. To identify the precise region(s) of RIAM, which undergo phosphorylation by these kinases, we co-expressed individual truncation constructs of RIAM N-terminus, RA-PH or C-terminus regions along with the active or inactive form of Fyn or Lck in COS cells. Immunoprecipitations and immunoblot assays revealed that active Fyn and Lck mediated robust and selective tyrosine phosphorylation of the RA-PH structural unit of RIAM. Tandem mass spectrometry (LC-MS/MS) identified that tyrosine 340 (Y340) within the PH domain was the specific target. Because this tyrosine is localized within the RA-PH integral unit, we examined whether phosphorylation of Y340 in the PH domain might have an active role in the function of RIAM. Using site directed mutagenesis, we introduced a tyrosine-to-phenylalanine mutation (Y340F) rendering this residue resistant to phosphorylation, and FLAG-tagged RIAM-WT or FLAG-tagged RIAM-Y340F constructs were expressed in Jurkat T cells. Anti-FLAG immunoprecipitation followed by immunoblot showed that RIAM-WT and RIAM-Y340F displayed comparable interaction with PLC-γ1. However, phosphorylation of PLC-γ1 associated with RIAM-Y340F was impaired. Because Src family kinases and Itk, which are involved in PLC-γ1 phosphorylation and activation localize at the lipid rafts upon T cell stimulation, we examined whether RIAM-Y340F might display differential translocation to the plasma membrane thereby altering the ability of RIAM-associated PLC-γ1 to undergo activating phosphorylation. Isolation of membranous and cytosolic fractions by nitrogen cavitation revealed that in contrast to RIAM-WT, which rapidly translocated to the membrane fraction upon T cell stimulation, RIAM-Y340F remained exclusively in the cytosolic fraction. To investigate whether RIAM-Y340F displayed altered plasma membrane localization in vivo, we used mCherry-RIAM-WT or mCherry-RIAM-Y340F and live cell imaging. Although RIAM-WT readily translocated to the plasma membrane and colocalized with Rap1-GTP, RIAM-Y340F was unable to translocate to the plasma membrane and was detectable only in the cytoplasm. Because RIAM translocation to the plasma membrane and PLC-γ1 activation are required for inside-out activation of LFA-1, we examined the effects of RIAM-Y340F on LFA-1 activation. We determined that expression of RIAM Y340F abrogated LFA-1 activation and LFA-1-mediated adhesion in response to TCR/CD3 stimulation. Thus, TCR-mediated phosphorylation of Y340 in RIAM PH domain by Src family kinases is a mandatory requirement for activation of RIAM-associated PLC-γ1 and LFA-1 activation. Our results provide a mechanistic link between TCR-mediated signaling and inside-out activation of LFA-1, thereby initiating LFA-1: ICAM-1-mediated adhesion and cross-talk between T cells and APC. Disclosures No relevant conflicts of interest to declare.

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21

Yamaguchi, Yasuto, John Morser, and Lawrence L. Leung. "Chemerin Bioactivity Is Regulated by Factor XIa: A Novel Interface Linking Between Coagulation, Hemostasis and Immunity." Blood 118, no. 21 (November 18, 2011): 2258. http://dx.doi.org/10.1182/blood.v118.21.2258.2258.

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Abstract Abstract 2258 Proteolytic modulation of chemokines by extracellular proteases, either released upon leukocyte degranulation or activated by the coagulation and fibrinolytic cascades, plays an important role in inflammation and immunity. Chemerin is a novel chemoattractant involved in innate and adaptive immunity and adipogenic differentiation via its receptor, CMKLR1, expressed on dendritic cells, macrophages and adipocytes. Chemerin circulates as an inactive precursor (chem163S) in blood whose bioactivity is regulated through proteolytic processing at its C-terminus. The most potent form of chemerin (chem157S) can be generated from chem163S in vitro by elastase directly or by sequential cleavages by plasmin to chem158K and then plasma carboxypeptidase-2 (CPB2, also termed thrombin-activatable fibrinolysis inhibitor TAFIa) or CPN to chem157S. Thus proteolytic processing is essential for chemerin activation. We have recently developed specific ELISAs for chem163S, 158K and 157S and showed that chem158K is present in plasma at a low level compared to chem163S. In contrast chem158K is the dominant form in synovial fluids of patients with inflammatory arthritis, suggesting that cleavage of chem163S at position 158K is a major step in the activation of chemerin in vivo. Although chem158K is present in vivo, it is unknown if it is generated by plasmin with a second cleavage by CPB2 or CPN to generate the active isoform, chem157S, as CPB2 inhibits plasmin generation. In this study we screened a panel of 21 proteases involved in coagulation, fibrinolysis and inflammation to determine which one could cleave the chemerin precursor chem163S to modulate its bioactivity. We carried out in vitro enzyme assays using 100 nM enzyme and 10 μM chem163S as a substrate, determining the results by SDS-PAGE and mass spectroscopy. Among the 21 proteases tested, cathepsin G, elastase, tryptase, plasmin, chymase, protease 3 and factor XIa could cleave chem163S. Mass spectroscopy identified tryptase, plasmin and factor XIa as enzymes that can digest chem163S to generate chem158K. Based on our previous observation that platelets are a rich cellular source of chemerin and release partially cleaved chemerin upon thrombin activation, we focused on factor XIa and compared its potency to plasmin. Factor XIa converted chem163S into chem158K in a dose-dependent manner whereas incubation with high concentrations of plasmin (300 nM) resulted in production of a non-specific smaller fragment, showing that the specificity of factor XIa was better than that of plasmin. Time-course studies showed that factor XIa and plasmin cleaved chem163S at comparable rates. Finally, we found that the fraction of chem158K to levels of total chemerin as determined by ELISA in factor XI-deficient plasma was lower than that in factor II-, IX-, X-deficient plasmas as well as normal pooled plasma. These data demonstrated that factor XIa could be the enzyme responsible for the first step of chemerin activation in the plasma compartment, ultimately leading to downstream inflammatory responses and provide a novel molecular link between coagulation, hemostasis and immunity. Disclosures: No relevant conflicts of interest to declare.

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22

Bologna, Cinzia, Roberta Buonincontri, Sara Serra, Tiziana Vaisitti, Valentina Audrito, Davide Brusa, Davide Rossi, et al. "Slamf-1/CD150 Is a Signaling Receptor Expressed by a Subset of Chronic Lymphocytic Leukemia Patients Characterized by a Favorable Prognosis." Blood 120, no. 21 (November 16, 2012): 1770. http://dx.doi.org/10.1182/blood.v120.21.1770.1770.

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Abstract Abstract 1770 Chronic lymphocytic leukemia (CLL) is characterized by the expansion of a monoclonal population of mature CD5+/CD23+ B lymphocytes, with a highly variable clinical course. Gene expression profiling studies identified SLAMF-1 (signaling lymphocytic activation molecule aka CD150) as part of the genetic signature characterizing CLL patients with favorable prognosis. Human SLAMF-1 is the prototype member of a family of receptors that act as co-activators through self-interactions on hematopoietic cell surface. SLAMF-1 activation is essential for full T cell functions, including cytokine secretion and development. The role of SLAMF-1 in antigen presenting cells, including B cells, is less well characterized, though it is known that the molecule initiates a signaling pathway that leads to B cell proliferation or CD95-mediated apoptosis. More recently, SLAMF-1 has been attributed a novel function as a microbial sensor that regulates bacterial phagosome functions by recruiting a supra-molecular complex, part of the ubiquitous cellular autophagic machinery. The analysis on the CD19+ fraction of 292 clinically and molecularly characterized CLL patients revealed highly variable levels of SLAMF-1 expression (1–95%). Statistical analyses of the data indicated that patients characterized by a good prognosis (in terms of disease stage at diagnosis or treatment requirements) express higher levels of SLAMF-1 compared to the other subgroups. Moreover, patients with > 6% SLAMF-1+/CD19+ CLL cells had a significantly longer treatment free survival (median 6.4 in SLAMF-1+vs 1.2 years in SLAMF-1− patients, P=.002). Consistently, SLAMF-1 expression inversely correlates with CD38 and CD49d, two molecular markers of unfavorable prognosis, and positively associates with the presence of somatic mutations in the IgHV genes. Functional experiments showed that the engagement of SLAMF-1 by an agonistic mAb started a well-characterized signaling pathway. Co-immunoprecipitation experiments demonstrated a direct interaction between the receptor and the adaptor molecule EAT-2, with its consequent phosphorylation and the following downstream activation of Vav-1, p38 and JNK. Moreover, co-crosslinking of SLAMF-1 and sIgM prolonged the phosphorylation of p38 and JNK and resulted in an increased percentage of CLL cells undergoing apoptosis, as compared to either signals alone or the basal condition. Furthermore, the engagement of SLAMF-1 for a period of 6 hours led to an increased appearance of autophagic vesicles, as confirmed by confocal and transmission electron microscopy. The modulation of apoptosis and autophagy was mediated by the sequential phosphorylation of JNK and Bcl-2: the final result is the activation of Bcl-2 (phospho-Ser70) and the release of Beclin-1, an essential member of the autophagic complex. In conclusion, i) SLAMF-1 is expressed at higher levels by patients with a good prognosis, ii) and performs as an immunoreceptor on CLL cells, starting a signaling pathway that involves the adaptor molecule EAT-2, Vav-1 and MAP kinases, like p38 and JNK. iii) SLAMF-1+ CLL patients respond to receptor engagement modulating an autophagic pathway mediated by the phosphorylation of JNK and Bcl-2. Taken together, these results suggest that SLAMF-1 could represent a novel marker for the subset of CLL patients characterized by an indolent clinical course and highlight a hypothetical link between the activation of the autophagic process and a better clinical outcome in CLL. Disclosures: No relevant conflicts of interest to declare.

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23

May, Andreas, Franz-Josef Neumann, and Klaus Preissner. "The Relevance of Blood Cell-Vessel Wall Adhesive Interactions for Vascular Thrombotic Disease." Thrombosis and Haemostasis 82, no. 08 (1999): 962–70. http://dx.doi.org/10.1055/s-0037-1615939.

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IntroductionFollowing an inflammatory or infectious stimulus, the body’s defense mechanism initiates recruitment of circulating leukocytes toward the inflammatory stimulus. The emigration of leukocytes into extravascular tissues occurs in a highly coordinated fashion in multiple steps, including rolling and tethering of blood cells along the vascular endothelium and their firm attachment and subsequent transmigration and invasion toward the inflammatory site.1 During these sequential steps, transcellular recognition of different adhesion receptor/counterligand pairs, such as selectins/sialyl LewisX-carbohydrates,2 integrins/ immunoglobulin superfamily cell adhesion molecules (ICAMs),3 or binding to (provisional) extracellular matrix components, such as fibrinogen/fibrin, vitronectin, or fibronectin, control the strength and duration of interactions between leukocytes (neutrophils [polymorphonucleocytes (PMN)], eosinophils, monocytes and macrophages, mast cells, lymphocytes) and the vessel wall.4 The importance of these cellular interactions is evident from patients with the rare congenital disorders of “leukocyte-adhesion-deficiency,” which are either caused by a lack or dysfunction of ß2-integrins (LAD I) or a deficiency in the expression of sialyl-LewisX carbohydrates (LAD II).5 The interdependent adhesion processes are regulated by vascular cell-derived chemokines and chemoattractants that may directly influence the expression profile and activation state of adhesion molecules, such as ß2- and ß1-integrins, the shedding of selectins, and the nonthrombogenic properties of endothelial cells.6 Prior to transmigration, leukocyte adhesion may induce the disruption of vascular endothelial (VE)-cadherin mediated endothelial cell-to-cell junctions7 involving the proteasome machinery.8 The spatio-temporal cellular expression of juxtacrine adhesion and signaling receptors–particularly on PMN, endothelial cells, and platelets–contribute to the coordination of adhesion and inflammatory mechanisms required for vascular homeostasis9 and prothrombotic outcome under imbalanced conditions. Not only do monocytes express tissue factor (a receptor for the protease factor VII/VIIa) on their surface after stimulation with endotoxin or cytokines, but PMN contain cell surface receptors, such as the factor X/Xa-binding ß2-integrin Mac-1 or effector cell protease receptor (EPR)-1, that link cellular activation and inflammation with the induction of the blood clotting cascade and serve as an alternate pathway for thrombin formation.10,11 Moreover, defects in natural anticoagulant mechanisms, such as the thrombomodulin/protein C pathway, are potential risk factors for vascular thrombotic complications, as in myocardial infarction.12 Pathophysiological stimuli, such as dysregulated direct (i.e., adhesive contact) or indirect (i.e., release of soluble factors) activation of leukocytes, serious infectious agonists, or autoantibodies, may result in endothelial cell dysfunction or injury with the amplification of inflammatory and prothrombotic responses. In the following, some of the principal juxtacrine interactions between leukocytes, platelets, and endothelium, together with their direct or indirect influence on hemostasis and consequences for vascular thrombotic disease, will be discussed. Further understanding of the bidirectional cross-talk of adhesion receptors and the contribution of connecting points, such as protease receptors, may lead to promising therapeutic strategies that aim to protect or regain the endothelial defense mechanisms.

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Schleiss, Cédric, Raphael Carapito, Luc Mathieu Fornecker, Leslie Muller, Paul Nicodeme, Ouria Tahar, Angélique Pichot, et al. "A Core Proliferative Program Induced By B-Cell Receptor Stimulation in Chronic Lymphocytic Leukemia Cells." Blood 134, Supplement_1 (November 13, 2019): 3777. http://dx.doi.org/10.1182/blood-2019-126779.

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B-cell receptor (BCR) engagement is widely acknowledged to sustain aberrant cell behavior and uncontrolled monoclonal proliferation in chronic lymphocytic leukemia (CLL), as well as in most leukemias and lymphomas arising from mature B lymphocytes. The precise mechanisms by which BCR signaling controls neoplastic B-cell proliferation are ill characterized. In this work, primary leukemic cells of untreated patients at initial stage of CLL (Binet stage A / Rai 0) presenting biological characteristics of aggressive form of the disease (unmutated IGHV genes and ZAP70 protein expression) were studied. Proliferation of CLL cells was induced ex vivo in six CLL samples using anti-IgM, together with mandatory co-stimulating factors (CD40L, IL-4 and IL-21) (Schleiss, Sci Rep, 2019). Using this model, we generated a unique set of 108 transcriptional and proteomic profiles during four days after activation (9 points from T0 to 96 hours after stimulation). A total of 23,348 transcripts and 50,503 unique peptides, the latter corresponding to 4,664 unique proteins, were identified and quantified. Statistical analysis of genes and proteins expression patterns identified a structured proliferative signature. In unsupervised analysis, principal component analysis (PCA) representation of the whole transcriptional profile showed the temporality of the response. Moreover, unsupervised temporal gene expression analysis using iterative optimization revealed clusters of temporal patterns exhibiting structured expression modulations during the time course after cell stimulation. Although the overall proteomic response appeared less structured than the transcriptional one at early time points after stimulation, samples showed a tendency for segregation with respect of their proliferative response at the two later time points. Hierarchical clustering, aimed to search for correlations between the transcriptional profiles, confirmed the temporal organization of the samples, while proliferative and non-proliferative cells were still distinguishable. Also, we identified a CLL cell activation signature corresponding to 3,097 differentially expressed genes (DE) and 1,209 differentially-abundant (DA) proteins. This unique dataset provided a unique opportunity to model the proliferative program of CLL cells after BCR engagement. We used the reverse engineering approach based on regression and system of equations previous published (Vallat et al., PNAS 2013). Thus, a temporal label was assigned to each gene and protein, restricting its potential link to actors with later temporal labels. The temporal model inferred with the genes and proteins datasets of proliferating CLL cells resulted in a regulatory network composed of 2,167 genes and 1,074 proteins representing 2,848 unique symbols, among which 395 gene-protein pairs, connected by 53,131 oriented links. Different temporal layers of actors were identified. At the earliest time-point after cell activation a first group was identified, involving transcriptional repression, negative regulation of BCR signaling, apoptotic process actors and a second group involving G1/S transition, DNA-replication genes. Later, expression of G0/G1 switch genes and regulators of cell proliferation and differentiation were identified. In conclusion, using a large dataset of temporal transcriptional and proteomic measurements coupled with mathematical modelling, we were able to unravel the molecular program downstream the signaling cascade activated by the engagement of the BCR and triggering primary CLL cell proliferation. This program was proven to organize around a limited number of hub genes and proteins whose sequential commitment drives the cellular response leading to proliferation days after cell activation. These hubs represent potential candidates for the development of novel therapeutic strategies for the treatment of aggressive CLL. Disclosures No relevant conflicts of interest to declare.

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Schober, Romana, Daniel Bonhenry, Victoria Lunz, Jinhui Zhu, Adela Krizova, Irene Frischauf, Marc Fahrner, et al. "Sequential activation of STIM1 links Ca2+ with luminal domain unfolding." Science Signaling 12, no. 608 (November 19, 2019): eaax3194. http://dx.doi.org/10.1126/scisignal.aax3194.

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The stromal interaction molecule 1 (STIM1) has two important functions, Ca2+ sensing within the endoplasmic reticulum and activation of the store-operated Ca2+ channel Orai1, enabling plasma-membrane Ca2+ influx. We combined molecular dynamics (MD) simulations with live-cell recordings and determined the sequential Ca2+-dependent conformations of the luminal STIM1 domain upon activation. Furthermore, we identified the residues within the canonical and noncanonical EF-hand domains that can bind to multiple Ca2+ ions. In MD simulations, a single Ca2+ ion was sufficient to stabilize the luminal STIM1 complex. Ca2+ store depletion destabilized the two EF hands, triggering disassembly of the hydrophobic cleft that they form together with the stable SAM domain. Point mutations associated with tubular aggregate myopathy or cancer that targeted the canonical EF hand, and the hydrophobic cleft yielded constitutively clustered STIM1, which was associated with activation of Ca2+ entry through Orai1 channels. On the basis of our results, we present a model of STIM1 Ca2+ binding and refine the currently known initial steps of STIM1 activation on a molecular level.

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26

Ma, Jie, Karen M. Harnett, Jose Behar, Piero Biancani, and Weibiao Cao. "Signaling in TRPV1-induced platelet activating factor (PAF) in human esophageal epithelial cells." American Journal of Physiology-Gastrointestinal and Liver Physiology 298, no. 2 (February 2010): G233—G240. http://dx.doi.org/10.1152/ajpgi.00409.2009.

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Transient receptor potential channel, vanilloid subfamily member 1 (TRPV1) receptors were identified in human esophageal squamous epithelial cell line HET-1A by RT-PCR and by Western blot. In fura-2 AM-loaded cells, the TRPV1 agonist capsaicin caused a fourfold cytosolic calcium increase, supporting a role of TRPV1 as a capsaicin-activated cation channel. Capsaicin increased production of platelet activating factor (PAF), an important inflammatory mediator that acts as a chemoattractant and activator of immune cells. The increase was reduced by the p38 MAP kinase (p38) inhibitor SB203580, by the cytosolic phospholipase A2 (cPLA2) inhibitor AACOCF3, and by the lyso-PAF acetyltransferase inhibitor sanguinarin, indicating that capsaicin-induced PAF production may be mediated by activation of cPLA2, p38, and lyso-PAF acetyltransferase. To establish a sequential signaling pathway, we examined the phosphorylation of p38 and cPLA2 by Western blot. Capsaicin induced phosphorylation of p38 and cPLA2. Capsaicin-induced p38 phosphorylation was not affected by AACOCF3. Conversely, capsaicin-induced cPLA2 phosphorylation was blocked by SB203580, indicating that capsaicin-induced PAF production depends on sequential activation of p38 and cPLA2. To investigate how p38 phosphorylation may result from TRPV1-mediated calcium influx, we examined a possible role of calmodulin kinase (CaM-K). p38 phosphorylation was stimulated by the calcium ionophore A23187 and by capsaicin, and the response to both agonists was reduced by a CaM inhibitor and by CaM-KII inhibitors, indicating that calcium induced activation of CaM and CaM-KII results in P38 phosphorylation. Acetyl-CoA transferase activity increased in response to capsaicin and was inhibited by SB203580, indicating that p38 phosphorylation in turn causes activation of acetyl-CoA transferase to produce PAF. Thus epithelial cells produce PAF in response to TRPV1-mediated calcium elevation.

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Moreno-Félix, Carolina, Griselda Wilson-Sánchez, Susana-Gabriela Cruz-Ramírez, Carlos Velázquez-Contreras, Maribel Plascencia-Jatomea, Ana Acosta, Lorena Machi-Lara, et al. "Bioactive Lipidic Extracts from Octopus (Paraoctopus limaculatus): Antimutagenicity and Antiproliferative Studies." Evidence-Based Complementary and Alternative Medicine 2013 (2013): 1–12. http://dx.doi.org/10.1155/2013/273582.

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Fractions from an organic extract from fresh octopus (Paraoctopus limaculatus) were studied for biological activities such as antimutagenic and antiproliferative properties usingSalmonellatester strains TA98 and TA100 with metabolic activation (S9) and a cancer cell line (B-cell lymphoma), respectively. A chloroform extract obtained from octopus tentacles was sequentially fractionated using thin layer chromatography (TLC), and each fraction was tested for antimutagenic and antiproliferative activities. Organic extract reduced the number of revertants caused by aflatoxin B1showing a dose-response type of relationship. Sequential TLC fractionation of the active extracts produced several antimutagenic and/or antiproliferative fractions. Based on the results obtained, the isolated fractions obtained from octopus contain compounds with chemoprotective properties that reduce the mutagenicity of AFB1and proliferation of cancer cell lines.

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Paulnock, D. M., and L. E. Lambert. "Identification and characterization of monoclonal antibodies specific for macrophages at intermediate stages in the tumoricidal activation pathway." Journal of Immunology 144, no. 2 (January 15, 1990): 765–73. http://dx.doi.org/10.4049/jimmunol.144.2.765.

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Abstract Macrophage activation for tumor cell killing is a multistep pathway in which responsive macrophages interact sequentially with priming and triggering stimuli in the acquisition of full tumoricidal activity. A number of mediators have been identified which have activating capability, including in particular IFN-gamma and bacterial LPS. Although the synergistic functional response of normal macrophages to sequential incubation with these activation signals has been well-established, characterization of the intermediate stages in the activation pathway has been difficult. We have developed a model system for examination of various aspects of macrophage activation, through the use of the murine macrophage tumor cell line, RAW 264.7. These cells, like normal macrophages, exhibit a strict requirement for interaction with both IFN-gamma and LPS in the development of tumor cytolytic activity. In addition, these cells can be stably primed by the administration of gamma-radiation. In the studies reported here, we have used RAW 264.7 cells treated with IFN-gamma alone or with IFN-gamma plus LPS to stimulate the production of rat mAb probes recognizing cell surface changes occurring during the activation process. In this way we have identified three Ag associated with intermediate stages of the activation process. One Ag, TM-1, is expressed on RAW 264.7 cells primed by IFN-gamma or gamma-radiation. This surface Ag thus identifies cells at the primed cell intermediate stage of the tumoricidal activation pathway regardless of the mechanism of activation. A second Ag, TM-2, is expressed on IFN-treated RAW 264.7 cells but not on RAW 264.7 cells primed with gamma-radiation alone. Expression of this Ag can be induced by treatment of irradiated cells with IFN-gamma, but is not induced by IFN-gamma treatment of a noncytolytic cell line, WEHI-3. This Ag thus appears to be an IFN-inducible cell surface protein associated specifically with macrophage activation for tumoricidal activity. Finally, Ag TM-3 is detectable on RAW 264.7 cells primed by either IFN-gamma or gamma-radiation, after subsequent triggering of the primed cells with LPS. The addition of the mAb recognizing this antigen to the function assay of tumor cell killing can inhibit they lytic activity of both triggered cells. Thus, this Ag may play a role in the antitumor effector functions of activated macrophages. Overall, the results suggest that these mAb can serve as useful tools for identification of molecules associated with the process of macrophage activation for tumor cell killing.

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Kumar, G., S. Wang, S. Gupta, and A. Nel. "The membrane immunoglobulin receptor utilizes a Shc/Grb2/hSOS complex for activation of the mitogen-activated protein kinase cascade in a B-cell line." Biochemical Journal 307, no. 1 (April 1, 1995): 215–23. http://dx.doi.org/10.1042/bj3070215.

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Ligation of membrane immunoglobulin M (mIgM) receptor in the Ramos B-cell line induced tyrosine phosphorylation of several intracellular substrates, including the adaptor protein. Shc. Phosphorylated Shc could be seen to associate with Grb2 in a complex which included hSOS. Inasmuch as hSOS is involved in p21ras activation, we also demonstrated that mIgM ligation activated a Ras-dependent kinase cascade in which sequential activation of Raf-1 and MEK-1 culminates in the activation of p42 mitogen-activated protein (MAP) kinase (ERK-2). The tumour promoter and protein kinase C agonist, phorbol 12-myristate 13-acetate (PMA), also activated Raf-1, MEK-1, and MAP kinase in Ramos cells, but did not induce tyrosine phosphorylation of Shc or Shc/Grb2 association. Okadaic acid, another tumour promoter and serine/threonine phosphatase inhibitor, activated p42 MAP kinase without activating Raf-1 or MEK-1, suggesting the existence of a serine/threonine phosphatase which directly regulates MAP kinase activity.

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30

Jin, Fengyan, Shaji K. Kumar, and Yun Dai. "The Lysine-Specific Demethylase KDM4A/JMJD2A Acts As a Tumor Suppressor in Multiple Myeloma." Blood 132, Supplement 1 (November 29, 2018): 191. http://dx.doi.org/10.1182/blood-2018-191.

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Abstract Introduction: Histone lysine methylation, a reversible event dynamically and reciprocally regulated by lysine methyltransferases (KMTs) and demethylases (KDMs), represents one of the major epigenetic mechanisms for regulation of chromatin remodeling and gene expression re-programming. The KDM4 family, which belongs to the Jumonji C (JmjC)-domain-containing proteins (JMJDs), consists of five members, including KDM4A-E that demethylate H3K9me2/3 and/or H3K36me2/3 in a Fe2+- and α-ketoglutarate-dependent manner. KDM4 proteins are involved in various cellular processes such as gene transcription and translation, DNA replication, DNA repair, apoptosis, and stem cell renewal. Notably, increasing evidence implicates KDM4 dysregulation in promoting genomic instabilities and oncogenesis, thereby which is considered as a potential target for emerging cancer epigenetic therapy. Although KDM4A, a member of the KDM4 family, has been widely studied in many solid tumors including breast, prostate, bladder cancer, its role in hematopoietic malignancies, including multiple myeloma (MM), remains unknown. Materials and Methods: Human MM cell lines (U266, RPMI8226, H929, OPM-2) were employed. After exposed to hypoxia (or the chemical hypoxia mimetic lactic acid) and anti-MM agents (e.g., bortezomib/Btz), cells were analyzed by flow cytometry, qPCR, Western blot to monitor apoptosis, cell cycle, proliferation (Ki67), DNA double-strand break/DSB (γH2A.X), expression of 1q21 and anti-apoptotic genes, as well as activation of the NF-κB and HIF pathways. The shRNA approach was used to knock down KDM4A for functional evaluation. The findings from in vitro experiments involving cell lines were then validated in primary MM samples to link KDM4A expression to disease progression and therapeutic response. Results: Analysis of the MM genome-wide GEP databases revealed that KDM4A mRNA was significantly up-regulated in MGUS and MM, but not SMM, compared to normal control, as well as in relapsed MM, compared to newly-diagnosed MM. To our surprise, KDM4A expression rather favored overall survival of MM patients, including those carrying 1q21 gain in whom KDM4A expression was indeed lower than those who did not have this high risk cytogenetic abnormality. Moreover, KDM4A expression correlated adversely with expression of 1q21 genes (e.g., CKS1B, MCL1, PSMD4, ARNT). Whereas basal KDM4A protein level was moderately but clearly higher in MM cell lines carrying 1q21 gain or acquired drug resistance than their counterparts, exposure to hypoxia or lactic acid (but not cobalt chloride) resulted in marked KDM4A up-regulation, accompanied by NF-κB and HIF pathway activation. However, while NF-κB inhibition and to a lesser extent ARNT/HIF-1β knockdown led to a robust increase in hypoxia-induced KDM4A expression, shRNA knockdown or pharmacological inhibition of KDM4A triggered NF-κB activation and HIF expression, as well as up-regulated anti-apoptotic proteins (e.g., Mcl-1, TNFAIP3/A20, CKS1B), in association with increased H3K36me3 rather than H3K9me3. Furthermore, KDM4A knockdown or inhibition sharply diminished Btz lethality and overrode hypoxia-mediated cytoprotection. Interestingly, KDM4A knockdown also increased MM cell proliferation, promoted S phase entry, and attenuated Btz-induced DSB. Last, IHC of sequential bone marrow biopsies revealed that while KDM4A protein was relatively low at diagnosis, its level was markedly increased when patients achieved CR and then fell to the baseline low level at relapse. Conclusion: KDM4A/JMJD2A, a lysine demethylase that has been recognized as an pro-oncogenic protein via its epigenetic and/or non-epigenetic properties, is identified for the first time as a potential tumor suppressor in MM, particularly in a high risk subtype carrying 1q21 gain. Whereas KDM4A is expressed in MM and can be further induced by hypoxia that naturally exists in bone marrow niche, it seems to play multiple inhibitory roles in cell growth, cell cycle, DNA repair, and drug resistance by suppressing expression of oncogenic and anti-apoptotic genes (especially 1q21 genes), likely via H3K36me3 demethylation, and antagonizing NF-κB and HIF activation. These findings suggest that in contrast to its pro-oncogenic role in certain solid tumors, KDM4A might instead act as a tumor suppressor in MM. This work was supported by NNSFC (81471165, 81670189, and 81670190). Disclosures Kumar: AbbVie: Membership on an entity's Board of Directors or advisory committees, Research Funding; KITE: Membership on an entity's Board of Directors or advisory committees, Research Funding; Janssen: Membership on an entity's Board of Directors or advisory committees, Research Funding; Celgene: Membership on an entity's Board of Directors or advisory committees, Research Funding.

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Morris, Gerald P., Geoffrey L. Uy, David L. Donermeyer, Paul M. Allen, and John F. DiPersio. "Donor Dual TCR T Cells Preferentially Expand and Mediate Pathologic Alloreactivity in Acute Graft Versus Host Disease." Blood 118, no. 21 (November 18, 2011): 1972. http://dx.doi.org/10.1182/blood.v118.21.1972.1972.

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Abstract Abstract 1972 The nature of the T cell repertoire mediating pathologic in vivo alloreactivity is an important question for understanding the development of acute graft-versus-host disease (aGvHD) following clinical allogeneic transplantation. We have previously demonstrated that the small proportion of T cells that naturally express 2 T cell receptors (TCR) as a consequence of incomplete TCRa allelic exclusion during thymic development contribute disproportionately to the alloreactive T cell repertoire, both in vitro and in vivo in a mouse model of graft versus host disease (GvHD) (J. Immunol., 182:6639, 2009). Here, we extend these findings to human biology, examining dual TCR T cells from healthy volunteer donors (n = 12) and patients who have undergone allogeneic hematopoietic stem cell transplantation (HSCT) (n = 19). Peripheral blood was collected at day 30 post-HSCT or at the time of presentation with symptomatic acute GvHD. Dual TCR T cells were measured in peripheral blood by pair-wise staining with 3 commercially-available and 2 novel TCRa mAbs. Dual TCR T cells were consistently and significantly expanded in patients with symptomatic aGvHD, representing 5.3±3.8 % of peripheral T cells, compared to 1.7±0.8 % of T cells in healthy controls (p < 0.005) (Figure 1). There was no correlation between dual TCR T cell frequency and GvHD severity. Furthermore, sequential analysis of peripheral blood in 2 patients demonstrated expansion of dual TCR T cells concurrent with the development of aGvHD (Figure 2). Dual TCR T cells from patients with symptomatic aGvHD demonstrated increased expression of CD69 as compared to T cells expressing a single TCR, indicative of preferential activation of dual TCR T cells during aGvHD. Similarly, dual TCR T cells isolated from patients with symptomatic aGvHD demonstrate increased production of IFN-g ex vivo, indicative of the ability to mediate pathogenic alloreactive responses. Dual TCR T cell clones isolated from healthy donors and patients post-HSCT by single cell FACS sorting demonstrate alloreactive responses against a range of allogeneic cell lines in vitro. We propose that the increased alloreactivity of dual TCR T cells results from the less stringent thymic selection for secondary TCR, and thus provides a link between thymic selection, the TCR repertoire, and alloreactivity. These findings may lead to simple ways of phenotypically identifying specific T cells predisposed to inducing aGvHD for subsequent examination of T cell repertoires and functional studies. Furthermore, these data suggest that dual TCR T cells represent a potential predictive biomarker for aGvHD and a potential target for selective T cell depletion in HSCT. Disclosures: No relevant conflicts of interest to declare.

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Hamzeh, Mahsa, and Bernard Robaire. "Androgens activate mitogen-activated protein kinase via epidermal growth factor receptor/insulin-like growth factor 1 receptor in the mouse PC-1 cell line." Journal of Endocrinology 209, no. 1 (January 10, 2011): 55–64. http://dx.doi.org/10.1530/joe-10-0223.

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Androgens are the primary regulators of epididymal structure and functions. In the classical view of androgen action, binding of androgen to the intracellular androgen receptor (AR) produces the receptor-steroid complex that has high affinity for DNA response elements and regulates the transcription of target genes. In this study, we demonstrate that in epididymal cells, 5α-dihydrotestosterone (DHT) can cause an alternative and rapid response that is independent of AR–DNA interactions and is mediated by activation of signaling pathways through the AR. We examined changes in AKT and extracellular signal-regulated protein kinases (ERK1/2) activation at early time points after DHT supplementation in the mouse proximal caput epididymis-1 cell line. DHT had no significant effect on AKT activation at any time point. However, DHT activated the ERK pathway as early as at 1 min, the pathway remained activated at 10 min, but activation was not sustained at later time points. Interestingly, ERK activation was blocked by hydroxyflutamide (HF), indicating that early ERK activation was an AR-mediated response. DHT phosphorylates steroid receptor co-activator (SRC) kinase, and this activation was required for the ERK response. EGFR and IGF1R were downstream of SRC, and these two receptors together contributed to enhance ERK and cAMP response element-binding protein (CREB) phosphorylation. We postulate that this rapid action of androgen may ultimately act to modulate the transcription of genes regulated by AR in the nucleus. These results support the hypothesis that DHT can activate a pathway involving the sequential activation of MEK, ERK1/2, and CREB through the EGFR/IGF1R in an epididymal cell line.

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Ohgushi, Masatoshi, Shunsuke Kuroki, Hiroshi Fukamachi, Lorraine A. O'Reilly, Keisuke Kuida, Andreas Strasser та Shin Yonehara. "Transforming Growth Factor β-Dependent Sequential Activation of Smad, Bim, and Caspase-9 Mediates Physiological Apoptosis in Gastric Epithelial Cells". Molecular and Cellular Biology 25, № 22 (15 листопада 2005): 10017–28. http://dx.doi.org/10.1128/mcb.25.22.10017-10028.2005.

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ABSTRACT Transforming growth factor β (TGF-β) has been implicated in the maintenance of homeostasis in various organs, including the gastric epithelium. In particular, TGF-β-induced signaling was shown to be required for the differentiation-associated physiological apoptosis of gastric epithelial cells, but its mechanism has not been well understood. In this study, the molecular mechanism of TGF-β-induced apoptosis was analyzed in a human gastric epithelial cell line, SNU16, as an in vitro model. Expression of Smad7 and Bcl-XL, but not viral FLIP, was shown to prevent TGF-β-induced apoptosis, indicating an exclusive requirement of the activation of Smad signaling pathway and mitochondrial dysfunction followed by activation of caspase-9. In addition, treatment with TGF-β induced binding of Bim, a proapoptotic Bcl-2 homology domain 3 (BH3)-only protein, to Bcl-XL, which is dependent on the activation of Smad, and reduction in the expression of Bim by RNA interference decreased the sensitivity to TGF-β-induced apoptosis. Moreover, we found abnormalities in the gastric epithelium of both Bim and caspase-9 knockout mice; these abnormalities were associated with a defect of physiological apoptosis in gastric epithelial cells. These results indicate for the first time that TGF-β is involved in the physiological loss of gastric epithelial cells by activating apoptosis mediated by Smad, Bim, and caspase-9.

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Tarafdar, Anuradha, Ashfia Fatima Khan, Emilio Cosimo, Hothri A. Moka, Karen Dunn, and Alison M. Michie. "Subversion of Pkca and Pkcbii Upregulation Play an Essential Role in Chronic Lymphocytic Leukaemia Development, Inducing Constitutive B Cell Receptor-Mediated Signals." Blood 126, no. 23 (December 3, 2015): 488. http://dx.doi.org/10.1182/blood.v126.23.488.488.

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Abstract B cell receptor (BCR)-mediated signals orchestrate key events during the life cycle of B lymphocytes enabling normal B cell development and maturation. Chronic lymphocytic leukaemia (CLL), an incurable malignancy of mature B cells, displays deregulated BCR-mediated signalling, the intensity of which correlates disease pathogenesis. As such, signals generated upon BCR engagement represent promising targets for novel therapies. Of note, the expression profile of selected protein kinase C (PKC) isoforms, which link proximal BCR mediated signals with downstream pathways, exhibit altered expression patterns in CLL cells: upregulation of PKCβII, PKCε, PKCζ and downregulation of PKCα and PKCβI compared with normal B cells. We previously demonstrated that introduction of a kinase inactive PKCα (PKCα-KR) construct in mouse lymphoid progenitor cells resulted in development of a CLL-like disease both in vitro and in vivo. This model resembles the more aggressive subset of CLL, exhibiting an upregulation of ZAP-70 and elevated ERK-MAPK-mTOR signalling resulting in enhanced proliferation and increased tumor load in the lymphoid organs. Interestingly, reduced PKCα function resulted in PKCβII upregulation, a key pathogenic feature of CLL. Inhibition of PKCβII in these cells with enzastaurin resulted in cell cycle arrest in vitro, reduced tumour load and elevated apoptosis in vivo indicating that PKCβII plays a vital role in maintaining cell survival in our model. To further elucidate the role of PKCβ in leukaemogenesis, we have performed sequential prkcb knockdown (KD) and PKCα-KR expression in the lymphoid progenitor cells. prkcb KD resulted in reduced proliferation and survival concurrent with reduced expression of leukaemic markers (CD23 and CD5) compared to control cells indicating that prkcb plays an essential role in initiation of leukaemogenesis in our model. To identify targets responsible for the regulation for prkcb transcription we performed global gene analysis (Affymetrix GeneChip mouse gene 1.0 ST) comparing MIEV (empty vector control) and PKCα-KR transduced cells at successive time-points mapping critical stages of B-cell transformation, pre- and post PKCβII upregulation. MetaCoreTM analysis revealed that upon upregulation of PKCβII, the BCR-mediated signalling pathway was significantly upregulated in PKCα-KR expressing cells. At the protein level, key hubs proximal to the BCR - Lyn, Btk and Akt - were upregulated, indicating constitutive activation of BCR signalling in the CLL-like PKCα-KR expressing cells. Additionally, proliferative signals downstream of the BCR (mTOR, NF-kB and c-myc) were also upregulated. Treatment of PKCα-KR expressing cells with the Btk inhibitor Ibrutinib (PCI-32765), which has recently been approved for the treatment of CLL, reduced cellular proliferation and inhibited phosphorylation of BtkY223, AktS473 and S6S235/236. Overall, we demonstrate that PKCβII expression is essential for leukaemogenesis and identify key signalling pathways that drive the initiation/development of CLL in the PKCα-KR model. Disclosures No relevant conflicts of interest to declare.

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35

Gleitz, Hélène F. E., Aurélien J. F. Dugourd, Nils B. Leimkühler, Inge A. M. Snoeren, Stijn N. R. Fuchs, Sylvia Menzel, Susanne Ziegler, et al. "Increased CXCL4 expression in hematopoietic cells links inflammation and progression of bone marrow fibrosis in MPN." Blood 136, no. 18 (October 29, 2020): 2051–64. http://dx.doi.org/10.1182/blood.2019004095.

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Abstract Primary myelofibrosis (PMF) is a myeloproliferative neoplasm (MPN) that leads to progressive bone marrow (BM) fibrosis. Although the cellular mutations involved in the pathogenesis of PMF have been extensively investigated, the sequential events that drive stromal activation and fibrosis by hematopoietic–stromal cross-talk remain elusive. Using an unbiased approach and validation in patients with MPN, we determined that the differential spatial expression of the chemokine CXCL4/platelet factor-4 marks the progression of fibrosis. We show that the absence of hematopoietic CXCL4 ameliorates the MPN phenotype, reduces stromal cell activation and BM fibrosis, and decreases the activation of profibrotic pathways in megakaryocytes, inflammation in fibrosis-driving cells, and JAK/STAT activation in both megakaryocytes and stromal cells in 3 murine PMF models. Our data indicate that higher CXCL4 expression in MPN has profibrotic effects and is a mediator of the characteristic inflammation. Therefore, targeting CXCL4 might be a promising strategy to reduce inflammation in PMF.

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Lee, Jinho, Marilyne Labrie, Gen Yong, Todd Camp, Hongli Ma, Megan Grout, Wei Xu, et al. "Multiomics profiling of longitudinal melanoma specimens unravels molecular mechanisms of resistance to sequential targeted and cancer immunotherapies." Journal of Clinical Oncology 38, no. 15_suppl (May 20, 2020): e22015-e22015. http://dx.doi.org/10.1200/jco.2020.38.15_suppl.e22015.

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e22015 Background: We evaluated spatially-resolved protein profiling of longitudinal tumor specimens derived from two patients with metastatic melanoma who progressed on sequential therapies including targeted therapy (TT) and immune checkpoint blockade (ICB) therapy targeting T cell-surface antigens (CTLA-4 and PD-1). The purpose of this study was to identify molecular determinants of resistance to sequential TT and ICB therapies. Methods: We performed multiplexed and multidimensional spatial protein profiling using NanoString’s GeoMx Digital Spatial Profiling (DSP) platform and single-cell level imaging analysis with Cyclic Immunofluorescence (CycIF) to simultaneously determine dynamic changes in tumor intrinsic signaling pathways and immune response in the tumor microenvironment (TME). Results: The first patient presented with a BRAFV600E-positive brain metastasis. This patient was sequentially treated with ipilimumab (Ipi), the combination of dabrafenib and trametinib, and pembrolizumab (Pembro) and progressed despite of high expression of CD56 NK cell after all treatments. In addition, CycIF analysis revealed drug resistance in a subpopulation of cells that had continued activation of mTOR (pS6) and EGFR pathways. The second patient presented with dermal metastases on the flank with NRASQ61K mutation. This patient was sequentially treated with Pembo, Talimogene Laherparepvec, the combination of Ipi plus nivolumab, and two different investigational agents combined with Pembro. This patient displayed stable disease (SD) on Pembro but eventually progressed on the subsequent therapies. DSP analysis demonstrated CD68/CD40 myeloid cell infiltrates as well as HLA-DR and CD44 in the TME after the last treatment. CycIF analysis revealed dynamic changes in tumoral characteristics including DNA damage and proliferation during treatment. Furthermore, the analysis suggested that there might be a resistant subpopulation in the last tumor biopsy, which is in line with progression of the disease. Conclusions: In this study, we conducted detailed analyses on serial specimens from two patients to precisely define the spatial distribution of immune responses and cancer signaling pathways. The findings propose that concurrent proteomics analysis and immune monitoring of longitudinal tumor biopsies can be informative in clinical evaluation in order to identify salvage therapies to overcome drug resistance.

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Lambert, L. E., and D. M. Paulnock. "Modulation of macrophage function by gamma-irradiation. Acquisition of the primed cell intermediate stage of the macrophage tumoricidal activation pathway." Journal of Immunology 139, no. 8 (October 15, 1987): 2834–41. http://dx.doi.org/10.4049/jimmunol.139.8.2834.

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Abstract Macrophage activation for tumor cell killing is a multistep pathway in which responsive macrophages interact sequentially with priming and triggering stimuli in the acquisition of full tumoricidal activity. Although this synergistic response of normal macrophages to sequential incubation with activation signals has been well established, characterization of the intermediate stages in this pathway has been difficult, due in large measure to the instability of the intermediate cell phenotypes. We have developed a model system for examination of macrophage-mediated tumor cell lysis, with the use of the murine macrophage tumor cell line RAW 264.7. These cells, like normal macrophages, exhibit a strict requirement for interaction with both interferon-gamma (IFN-gamma, the priming signal) and bacterial lipopolysaccharide (LPS, the triggering signal) in the development of tumor cytolytic activity. In this system, the priming effects of IFN-gamma decay rapidly after withdrawal of this mediator and the cells become unresponsive to LPS triggering. We have recently observed that gamma-irradiation of the RAW 264.7 cells also results in development of a primed activation state for tumor cell killing. The effects of gamma-radiation on the RAW 264.7 cell line are strikingly similar to those resulting from incubation with IFN-gamma, with the exception that the irradiation-induced primed cell intermediate is stable and responsive to LPS triggering for at least 24 hr. Treatment with gamma-radiation also results in increased cell surface expression of major histocompatibility complex-encoded class I antigens; however, class II antigen expression is not induced. Irradiation-induced development of the primed phenotype is not solely the result of cytostatic effects as treatment of the cells with a radiomimetic drug, mitomycin C, results in decreases in [3H]thymidine incorporation that are similar to those observed after irradiation, without concomitant development of cytolytic potential. In addition, priming by gamma-radiation does not appear to be mediated by the release of soluble autoregulatory factors. This alternate pathway for induction of the primed macrophage activation state should serve as a useful tool for identification of molecules important to the functional potential of primed cells, and for elucidation of the biochemical mechanisms of the priming event in tumoricidal activation.

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Hase, Hidenori, Yumiko Kanno, Masaru Kojima, Kaoru Hasegawa, Daisuke Sakurai, Hidefumi Kojima, Naoyuki Tsuchiya, et al. "BAFF/BLyS can potentiate B-cell selection with the B-cell coreceptor complex." Blood 103, no. 6 (March 15, 2004): 2257–65. http://dx.doi.org/10.1182/blood-2003-08-2694.

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Abstract The tumor necrosis factor (TNF)–like ligand BAFF/BLyS (B-cell activating factor of the TNF family/B-lymphocyte stimulator) is a potent B-cell survival factor, yet its functional relationship with other B-cell surface molecules such as CD19 and CD40 is poorly understood. We found that follicular dendritic cells (FDCs) in human lymph nodes expressed BAFF abundantly. BAFF up-regulated a B cell–specific transcription factor Pax5/BSAP (Pax5/B cell–specific activator protein) activity and its target CD19, a major component of the B-cell coreceptor complex, and synergistically enhanced CD19 phosphorylation by B-cell antigen receptor (BCR). BAFF further enhanced B-cell proliferation, immunoglobulin G (IgG) production, and reactivity to CD154 by BCR/CD19 coligation and interleukin-15 (IL-15). Our results suggest that BAFF may play an important role in FDC–B-cell interactions through the B-cell coreceptor complex and a possibly sequential link between the T cell–independent and –dependent B-cell responses in the germinal centers.

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39

Salkuti, Surender Reddy, P. R. Bijwe, and A. R. Abhyankar. "Coordinated Action of Fast and Slow Reserves for Optimal Sequential and Dynamic Emergency Reserve Activation." International Journal of Emerging Electric Power Systems 17, no. 2 (April 1, 2016): 117–29. http://dx.doi.org/10.1515/ijeeps-2015-0150.

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Abstract This paper proposes an optimal dynamic reserve activation plan after the occurrence of an emergency situation (generator/transmission line outage, load increase or both). An optimal plan is developed to handle the emergency situation, using coordinated action of fast and slow reserves, for secure operation with minimum overall cost. This paper considers the reserves supplied by generators (spinning reserves) and loads (demand-side reserves). The optimal backing down of costly/fast reserves and bringing up of slow reserves in each sub-interval in an integrated manner is proposed. The simulation studies are performed on IEEE 30, 57 and 300 bus test systems to demonstrate the advantage of proposed integrated/dynamic reserve activation plan over the conventional/sequential approach.

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40

Kitten, A. M., T. K. Hymer, and M. S. Katz. "Bidirectional modulation of parathyroid hormone-responsive adenylyl cyclase by protein kinase C." American Journal of Physiology-Endocrinology and Metabolism 266, no. 6 (June 1, 1994): E897—E904. http://dx.doi.org/10.1152/ajpendo.1994.266.6.e897.

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The temporal pattern with which phorbol 12-myristate 13-acetate (PMA), an activator of protein kinase C (PKC), modulates parathyroid hormone (PTH)-responsive adenylyl cyclase (AC) was evaluated in a clonal osteoblast-like cell line (UMR-106). Brief (< or = 1 h) exposure of UMR-106 cells to PMA enhanced PTH stimulation of AC, whereas more prolonged PMA treatment decreased the PTH response, with maximum inhibition occurring at < or = 6 h. PMA treatment also resulted in initial activation followed by downregulation of PKC. Exposure of cells to 1,2-dioctanoyl-sn-glycerol, which activated but did not downregulate PKC, resulted in bidirectional modulation of PTH-responsive AC identical to that produced by PMA. Prolonged PMA exposure decreased PTH receptor number, as determined by radioligand binding studies, and reduced PTH receptor mRNA levels, assessed by Northern blot analysis. Forskolin activation of the catalytic subunit of AC was also decreased after prolonged PMA treatment. The results suggest that activation of PKC sequentially stimulates and then inhibits PTH responsiveness. Inhibition of the PTH response occurs by PKC actions exerted on the PTH receptor and the AC catalytic subunit.

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41

Banerjee, Pinaki P., Rahul Pandey, Rena Zheng, Megan M. Suhoski, Linda Monaco-Shawver, and Jordan S. Orange. "Cdc42-interacting protein–4 functionally links actin and microtubule networks at the cytolytic NK cell immunological synapse." Journal of Experimental Medicine 204, no. 10 (September 4, 2007): 2305–20. http://dx.doi.org/10.1084/jem.20061893.

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An essential function of the immunological synapse (IS) is directed secretion. NK cells are especially adept at this activity, as they direct lytic granules to the synapse for secretion, which enables cytotoxicity and facilitates host defense. This initially requires rearrangement of the actin cytoskeleton and, subsequently, microtubule-dependent trafficking of the lytic granules. As these two steps are sequential, specific linkages between them are likely to serve as critical regulators of cytotoxicity. We studied Cdc42-interacting protein–4 (CIP4), which constitutively interacts with tubulin and microtubules but focuses to the microtubule organizing center (MTOC) after NK cell activation, when it is able to associate with Wiskott-Aldrich syndrome protein (WASp) and the actin filament–rich IS. WASp deficiency, overexpression of CIP4, or parts of CIP4 interfere with this union and block normal CIP4 localization, MTOC polarization to the IS, and cytotoxicity. Reduction of endogenous CIP4 expression using small interfering RNA similarly inhibits MTOC polarization and cytotoxic activity but does not impair actin filament accumulation at the IS, or Cdc42 activation. Thus, CIP4 is an important cytoskeletal adaptor that functions after filamentous actin accumulation and Cdc42 activation to enable MTOC polarization and NK cell cytotoxicity.

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42

Laxminarayana, D., and G. M. Kammer. "Activation of type I protein kinase A during receptor-mediated human T lymphocyte activation." Journal of Immunology 156, no. 2 (January 15, 1996): 497–506. http://dx.doi.org/10.4049/jimmunol.156.2.497.

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Abstract The experiments reported herein have characterized the signaling pathway leading to stimulation of type I protein kinase A isozyme (PKA-I) activity during the early events of Ag receptor-mediated T cell activation. Inhibitor studies demonstrated that receptor-initiated activation of nonreceptor protein tyrosine kinases, phosphorylation and activation of phospholipase C-gamma 1, and activation of protein kinase C occur temporally and precede PKA-I activation. Bypass of both the TCR/CD3 complex and IL-1R and direct activation of protein kinase C by a phorbol ester can also activate PKA-I. To confirm that PKA-I activation via the TCR/CD3 complex and IL-1R requires antecedent protein tyrosine kinase-catalyzed phosphorylation of phospholipase C-gamma 1, we used wild-type and CD45-deficient (mutant J45.01) Jurkat T cell lines. Unlike wild-type Jurkat T cells, the absence of CD45 tyrosine phosphatase resulted in the failure of receptor-mediated activation of PKA-I activity and of IL-2 mRNA transcription in the mutant J45.01 Jurkat cell line. In conclusion, our data support the concept that a signal derived from ligand binding to both the TCR/CD3 complex and IL-1R receptor mediates rapid activation of the PKA-I isozyme in primary T lymphocytes by sequential activation of an intracellular pathway comprised of CD45 phosphatase/protein tyrosine kinase/polyphosphoinositide/Ca2+/protein kinase C pathway rather than via the conventional surface receptor/stimulatory G protein system.

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43

Yang, Zhantao, Sioban B. Keel, Brent L. Wood, Bart L. Scott, and Janis L. Abkowitz. "Pathophysiology of Macrocytic Anemia in Diamond Blackfan Anemia and Del(5q) Myelodysplastic Syndrome." Blood 126, no. 23 (December 3, 2015): 670. http://dx.doi.org/10.1182/blood.v126.23.670.670.

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Abstract Twenty-five percent of Diamond Blackfan anemia (DBA) cases result from haploinsufficiency of RPS19 and ~30% from haploinsufficiencies of other ribosomal proteins. The macrocytic anemia of del(5q) MDS results from RPS14 haploinsufficiency. When tested, these mutations disrupt ribosome assembly, yet how this causes macrocytic anemia remains uncertain. The dominant hypothesis is that P53 pathway activation leads to cell apoptosis, however, why erythropoiesis is severely impacted, while other hematopoietic as well as non-hematopoietic cells function appropriately, is difficult to explain. These observations led us to predict that DBA and del(5q) MDS share a pathogenesis that reflects the rapid kinetics of red cell and hemoglobin production, and specifically that poor ribosome assembly leads to an inability to quickly translate mRNA to protein, delayed globin synthesis in early erythroid precursors, and excess heme. Since free heme is toxic, cell death ensues. To explore this, we studied marrow from DBA (n=3) and del(5q) MDS (n=6) patients in cultures that optimize erythroid differentiation and showed that (a) cell numbers significantly decrease between stages I (lin-CD71+CD36-GlyA-)(BFU-E/early CFU-E) and II, (lin-CD71+CD36+GlyA-)(CFU-E/early proerythroblasts; Li et al. Blood 2014;124:3636); and (b) the cells that survive this insult differentiate normally to stages III (lin-CD36+GlyA+) and IV (lin-CD36-GlyA+). Using marrow from the index DBA patient, a 28-year old transfusion-dependent female with inherited RPS19 haploinsufficiency, and (as available) marrow from other DBA and del(5q) MDS patients (n=2-7 patients per study), we demonstrated that heme synthesis (assessed by ALAS2mRNA and protein) initiates normally while globin production (α- and β-globin mRNA and protein) is slowed, and that stage I and II cells have excess heme. Next, to discern the cell-specific characteristics that allow a DBA or del(5q) MDS cell to survive and fully mature, we compared those cells able to advance to stages III and IV in patient and concurrent control cultures. Stage III-IV GlyA+ DBA cells expressed 2.4-fold less ALAS2 mRNAand 2-fold more FLVCR mRNA than GlyA+ control cells (p values<0.05). Since the levels of ALAS2 and FLVCR mRNA were similar in stage I DBA and control cells, the time when heme synthesis initiates, these data imply that DBA cells capable of down-regulating their heme synthesis (have less ALAS2) or content (have moreFLVCR and increased heme export) preferentially survive. Data from del(5q) MDS patients are similar - cells progressing to stages III and IV have 2.9-fold less ALAS2 mRNA (p<0.05) and 9.4-fold higher FLVCR mRNA (p<0.01) than control cells. That heme synthesis proceeds normally, while globin production initiates slowly, was confirmed by sequential sampling of marrow cultures at days 3, 7, 10, 13, and 17; and as expected, DBA and del(5q) MDS cells at day 7 had increased reactive oxygen species (ROS). Consistent with these observations, treatment with 10 μM succinylacetone, a specific inhibitor of heme synthesis, improved the erythroid cell output of DBA and del(5q) MDS cultures by 68-95% (p values<0.05), while the erythroid cell output of concurrent control cultures decreased by 4-13%. Cell death appears to result both from apoptosis (not shown) and ferroptosis, a ROS-induced process previously characterized in kidney epithelial cells and cancer cell lines (Dixon et al. Cell 2012;149:1060). Recent data showing P53 sensitizes cells to ferroptosis by impeding the transcription of SCL7A11, a cysteine-glutamate antiporter, leading to less cysteine uptake, less GSH synthesis, less GPX4, and less antioxidants (Jiang et al. Nature 2015;520:57) link our observations to work implicating P53 activation in DBA and del(5q) MDS anemia. In support of this connection, DBA marrow cells at culture day 10 have 57% less SLC7A11 mRNA than control cells in preliminary experiments. Together, our studies demonstrate that erythropoiesis fails when heme exceeds globin. The data also suggest that decreasing heme synthesis, either directly or by chelating iron, may improve these anemias. They further argue that lenalidomide (destabilizes P53) and therapies decreasing heme should be synergistic. Since diverse mutations could impede brisk protein transcription or translation, and hence brisk globin production, heme toxicity might also contribute to anemia in MDS patients without del(5q). Disclosures No relevant conflicts of interest to declare.

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44

Kefas, BA, Y. Cai, Z. Ling, H. Heimberg, L. Hue, D. Pipeleers, and M. Van de Casteele. "AMP-activated protein kinase can induce apoptosis of insulin-producing MIN6 cells through stimulation of c-Jun-N-terminal kinase." Journal of Molecular Endocrinology 30, no. 2 (April 1, 2003): 151–61. http://dx.doi.org/10.1677/jme.0.0300151.

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We have recently shown that conditions known to activate AMP-activated protein kinase (AMPK) in primary beta-cells can trigger their apoptosis. The present study demonstrates that this is also the case in the MIN6 beta-cell line, which was used to investigate the underlying mechanism. Sustained activation of AMPK was induced by culture with the adenosine analogue AICA-riboside or at low glucose concentrations. Both conditions induced a sequential activation of AMPK, c-Jun-N-terminal kinase (JNK) and caspase-3. The effects of AMPK on JNK activation and apoptosis were demonstrated by adenoviral expression of constitutively active AMPK, a condition which reproduced the earlier-described AMPK-dependent effects on pyruvate kinase and acetyl-coA-carboxylase. The effects of JNK activation on apoptosis were demonstrated by the observations that (i). its inhibition by dicumarol prevented caspase-3 activation and apoptosis, (ii). adenoviral expression of the JNK-interacting scaffold protein JIP-1/IB-1 increased AICA-riboside-induced JNK activation and apoptosis. In primary beta-cells, AMPK activation was also found to activate JNK, involving primarily the JNK 2 (p54) isoform. It is concluded that prolonged stimulation of AMPK can induce apoptosis of insulin-producing cells through an activation pathway that involves JNK, and subsequently, caspase-3.

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45

Pandey, Dharmendra, Pankaj Goyal, James R. Bamburg, and Wolfgang Siess. "Regulation of LIM-kinase 1 and cofilin in thrombin-stimulated platelets." Blood 107, no. 2 (January 15, 2006): 575–83. http://dx.doi.org/10.1182/blood-2004-11-4377.

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Abstract Cofilin is a regulator of actin filament dynamics. We studied whether during platelet activation Rho kinase stimulates LIM kinase (LIMK) leading to subsequent phosphorylation and inactivation of cofilin. Platelet shape change and aggregation/secretion were induced by low and high concentrations of thrombin, respectively. We found that during these platelet responses Rho kinase activation was responsible for mediating rapid Thr508 phosphorylation and activation of LIMK-1 and for the F-actin increase during shape change and, in part, during secretion. Surprisingly, during shape change cofilin phosphorylation was unaltered, and during aggregation/secretion cofilin was first rapidly dephosphorylated by an okadaic acid–insensitive phosphatase and then slowly rephosphorylated by LIMK-1. LIMK-1 phosphorylation and cofilin dephosphorylation and rephosphorylation during aggregation were independent of integrin αIIbβ3 engagement. Cofilin phosphorylation did not regulate cofilin association with F-actin and was unrelated to the F-actin increase in thrombin-activated platelets. Our study identifies LIMK-1 as being activated by Rho kinase in thrombin-stimulated platelets. Two counteracting pathways, a cofilin phosphatase and LIMK-1, are activated during platelet aggregation/secretion regulating cofilin phosphorylation sequentially and independently of integrin αIIbβ3 engagement. Rho kinase–mediated F-actin increase during platelet shape change and secretion involves a mechanism other than LIMK-1–mediated cofilin phosphorylation, raising the possibility of another LIMK substrate regulating platelet actin assembly.

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46

Fitz, J. G., and A. H. Sostman. "Nucleotide receptors activate cation, potassium, and chloride currents in a liver cell line." American Journal of Physiology-Gastrointestinal and Liver Physiology 266, no. 4 (April 1, 1994): G544—G553. http://dx.doi.org/10.1152/ajpgi.1994.266.4.g544.

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By use of whole cell patch-clamp techniques, the effects of extracellular ATP on membrane ion currents of HTC cells from a rat liver tumor line were evaluated. ATP (500 microM) or the nonhydrolyzable analogue adenosine 5'-O-(3-thiotriphosphate) caused sequential activation of three currents: Icat (-1,325 +/- 255 pA at -80 mV) occurred early, was due to increased Na+ and K+ permeability, was present in 56% of 64 consecutive cells, and rapidly inactivated; IK (274 +/- 45 pA at 0 mV) was present in 59% of cells and also inactivated; and ICl (1,172 +/- 237 pA at +60 mV) was present in 94% of studies, was sustained, and exhibited outward rectification of the current-voltage relation. All three currents were present in 39% of cells. Increasing intracellular Ca2+ concentration ([Ca2+]i) by exposure to the 5'-nucleotide receptor agonist UTP (500 microM) or to thapsigargin activated Icat and IK but not ICl, whereas increasing ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid in the pipette (> or = 5 mM) inhibited ATP-dependent activation of Icat and IK but not ICl. A P2x-preferring agonist alpha, beta-methylene ATP (500 microM) did not activate currents; a P2y-preferring agonist 2-methylthioadenosine triphosphate activated Icat and IK at concentrations of 500 microM but not 50 microM. In perforated patch recordings, ATP produced triphasic changes in membrane potential with initial depolarization due to Icat, subsequent hyperpolarization due to IK, and a later sustained depolarization due to ICl. These findings indicate that ATP modulates HTC cell ion permeability through initial activation of Icat and IK mediated by 5'-nucleotide receptors which mobilize [Ca2+], and sustained activation of ICl through a separate Ca(2+)-independent mechanism.

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47

GÓMEZ DEL PULGAR, Teresa, Guillermo VELASCO, and Manuel GUZMÁN. "The CB1 cannabinoid receptor is coupled to the activation of protein kinase B/Akt." Biochemical Journal 347, no. 2 (April 10, 2000): 369–73. http://dx.doi.org/10.1042/bj3470369.

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Cannabinoids exert most of their effects in the central nervous system through the CB1 cannabinoid receptor. This G-protein-coupled receptor has been shown to be functionally coupled to inhibition of adenylate cyclase, modulation of ion channels and activation of extracellular-signal-regulated kinase. Using Chinese hamster ovary cells stably transfected with the CB1 receptor cDNA we show here that ∆9-tetrahydrocannabinol (THC), the major active component of marijuana, induces the activation of protein kinase B/Akt (PKB). This effect of THC was also exerted by the endogenous cannabinoid anandamide and the synthetic cannabinoids CP-55940 and HU-210, and was prevented by the selective CB1 antagonist SR141716. Pertussis toxin and wortmannin blocked the CB1 receptor-evoked activation of PKB, pointing to the sequential involvement of a Gi/Go protein and phosphoinositide 3ʹ-kinase. The functionality of the cannabinoid-induced stimulation of PKB was proved by the increased phosphorylation of glycogen synthase kinase-3 serine 21 observed in cannabinoid-treated cells and its prevention by SR141716 and wortmannin. Cannabinoids activated PKB in the human astrocytoma cell line U373 MG, which expresses the CB1 receptor, but not in the human promyelocytic cell line HL-60, which expresses the CB2 receptor. Data indicate that activation of PKB may be responsible for some of the effects of cannabinoids in cells expressing the CB1 receptor.

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48

Lazorchak, Adam S., Mark S. Schlissel та Yuan Zhuang. "E2A and IRF-4/Pip Promote Chromatin Modification and Transcription of the Immunoglobulin κ Locus in Pre-B Cells". Molecular and Cellular Biology 26, № 3 (1 лютого 2006): 810–21. http://dx.doi.org/10.1128/mcb.26.3.810-821.2006.

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ABSTRACT The immunoglobulin kappa light chain (Igκ) locus is regulated in a lineage- and stage-specific manner during B-cell development. The highly restricted timing of V to J gene recombination at the pre-B-cell stage is under the control of two enhancers, the intronic enhancer (κEi) and the 3′ enhancer (κE3′), flanking the constant exon. E2A transcription factors have been indicated to be directly involved in the regulation of Igκ locus activation. In this study, we utilize E2A-deficient pre-B cells to directly investigate the mechanism of E2A-mediated Igκ activation. We demonstrate that Igκ germ line transcription is severely impaired and recombination is blocked in the absence of E2A. Reconstitution of E2A −/− pre-B cells with inducible human E2A (E47R) is sufficient to promote chromatin modification of Igκ and rescue Igκ germ line transcription and Jκ gene recombinase accessibility. Furthermore, we show that increased E2A recruitment to κEi and κE3′ correlates with activation of Igκ in pre-B cells and that recruitment of E2A to κE3′ is in part dependent on the transcription factor IRF-4. Inhibition of IRF-4 expression in pre-B cells leads to a significant reduction of Igκ germ line transcription and enhancer acetylation. In the absence of E2A, increased IRF-4 expression is not sufficient to promote Igκ enhancer chromatin modification or transcription, suggesting that the sequential involvement of IRF-4 and E2A is necessary for the activation of the Igκ locus. Finally, we provide genetic evidence in the mouse that E2A gene dosage can influence the development of pre-B cells during the phase of Igκ gene activation.

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Khan, Abrar Ul Haq, Saeedah Musaed Almutairi, Alaa Kassim Ali, Rosalba Salcedo, C. Andrew Stewart, and Seung-Hwan Lee. "Expression of Nutrient Transporters on NK Cells during Murine Cytomegalovirus Infection is MyD88-Dependent." Journal of Immunology 206, no. 1_Supplement (May 1, 2021): 20.07. http://dx.doi.org/10.4049/jimmunol.206.supp.20.07.

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Abstract Natural killer (NK) cells are the predominant innate lymphocytes that provide the first line of defence without prior sensitization. In the inflammatory milieu, NK cells modify their metabolism to cope with the high energy demand required to support their proliferation, activation, and functional plasticity. This metabolic reprogramming is usually accompanied by the upregulated expression of nutrient transporters on the cell surface, leading to increased nutrient uptake needed for robust proliferation. The members of interleukin-1 family of inflammatory cytokines are critical in activating NK cells during infection; however, their function in NK cell metabolism is not fully elucidated. Previously, IL-18 has been shown to upregulate the expression of solute carrier transmembrane proteins and thereby induces a robust metabolic boost in NK cells. However, we demonstrated that IL-18 signaling is dispensable during viral infection in vivo whereas the expression of nutrient transporters on NK cells is primarily regulated by MyD88-pathway since NK cells from Myd88−/− mice displayed significantly reduced expression of nutrient receptors. Moreover, we identified that IL-33, another cytokine employing MyD88 signaling, can induce the expression of nutrient receptor but requires a sequential exposure to IL-12. Furthermore, signaling through NK cells activating receptor, Ly49H, can also promote the expression of nutrient transporters. In summary, our findings revealed multiple pathways that induce the expression of nutrient transporters on NK cells while highlighting the imperative role of MyD88 in NK cell metabolism during infection.

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50

Li, Yue, Hongxiang Hu, and Roger G. O’Neil. "Caveolae facilitate TRPV4-mediated Ca2+ signaling and the hierarchical activation of Ca2+-activated K+ channels in K+-secreting renal collecting duct cells." American Journal of Physiology-Renal Physiology 315, no. 6 (December 1, 2018): F1626—F1636. http://dx.doi.org/10.1152/ajprenal.00076.2018.

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Transient receptor potential cation channel subfamily V member 4 (TRPV4)-mediated Ca2+ signaling induces early activation of small/intermediate Ca2+-activated K+ channels, SK3 (KCNN3) and IK1 (KCNN4), which leads to membrane hyperpolarization and enhanced Ca2+ influx, which is critical for subsequent activation of the large conductance Ca2+-activated K+ channel BK (KCNMA1) and K+ secretion in kidney cortical collecting duct (CCD) cells. The focus of the present study was to determine if such coordinated hierarchical/sequential activation of these channels in CCD was orchestrated within caveolae, a known microcompartment underlying selective Ca2+-signaling events in other cells. In K+-secreting mouse principal cell (PC) line, mCCDcl1 cells, knockdown of caveolae caveolin-1 (CAV-1) depressed TRPV4-mediated Ca2+ signaling and activation of SK3, intermediate conductance channel (IK1), and BK. Immunofluorescence colocalization analysis and coimmunoprecipitation assays demonstrated direct coupling of TRPV4 with each of the KCa channels in both mCCDcl1 and whole mouse kidney homogenates. Likewise, extending this analysis to CAV-1 demonstrates colocalization and direct coupling of CAV-1 with TRPV4, SK3, IK1, and BK, providing strong support for coupling of the channels in caveolae microdomains. Furthermore, differential expression of CAV-1 along the CCD was apparent where CAV-1 was strongly expressed within and along the cell borders of kidney PCs and intercalated cells (ICs), although significantly less in ICs. It is concluded that caveolae provide a key microdomain in PCs and ICs for coupling of TRPV4 with SK3, IK1, and BK that directly contributes to TRPV4-mediated Ca2+ signaling in these domains leading to rapid and sequential coupling of TRPV4-SK3/IK1-BK that may play a central role in mediating Ca2+-dependent regulation of BK and K+ secretion.

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