Cardiac imaging in RASopathies/mitogen activated protein kinase syndromes

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Rita Gravino

2014-07-01

Full Text Available RASopathies include a spectrum of disorders due to dysregulation of RAS/mitogen activated protein kinase pathway that plays an essential role in the control of the cell cycle and differentiation. As a consequence, its dysregulation has profound developmental consequences, in particular cardiac malformations. RASopathies with cardiac features are: Noonan syndrome, multiple lentigines syndrome, cardio-faciocutaneous syndrome, Costello syndrome, neurofibromatosis- 1, Legius syndrome, neurofibromatosis- Noonan syndrome. The former syndromes are associated with a high rate of cardiac involvement (60-85% and 12 genes: PTPN11, SOS1, RAF1, KRAS, HRAS, BRAF, MEK1/MAP2K1, MEK2/MAP2K2, NRAS, SHOC2, CBL and SPRED1. Although the majority of these diseases are readily distinguishable in clinical terms, an integrated imaging study of the cardiac condition associated to RASopathies helps to better define risk assessment, surveillance, and management of these patients.

Recruitment of focal adhesion kinase and paxillin to β1 integrin promotes cancer cell migration via mitogen activated protein kinase activation

International Nuclear Information System (INIS)

Crowe, David L; Ohannessian, Arthur

2004-01-01

Integrin-extracellular matrix interactions activate signaling cascades such as mitogen activated protein kinases (MAPK). Integrin binding to extracellular matrix increases tyrosine phosphorylation of focal adhesion kinase (FAK). Inhibition of FAK activity by expression of its carboxyl terminus decreases cell motility, and cells from FAK deficient mice also show reduced migration. Paxillin is a focal adhesion protein which is also phosphorylated on tyrosine. FAK recruitment of paxillin to the cell membrane correlates with Shc phosphorylation and activation of MAPK. Decreased FAK expression inhibits papilloma formation in a mouse skin carcinogenesis model. We previously demonstrated that MAPK activation was required for growth factor induced in vitro migration and invasion by human squamous cell carcinoma (SCC) lines. Adapter protein recruitment to integrin subunits was examined by co-immunoprecipitation in SCC cells attached to type IV collagen or plastic. Stable clones overexpressing FAK or paxillin were created using the lipofection technique. Modified Boyden chambers were used for invasion assays. In the present study, we showed that FAK and paxillin but not Shc are recruited to the β1 integrin cytoplasmic domain following attachment of SCC cells to type IV collagen. Overexpression of either FAK or paxillin stimulated cancer cell migration on type IV collagen and invasion through reconstituted basement membrane which was dependent on MAPK activity. We concluded that recruitment of focal adhesion kinase and paxillin to β1 integrin promoted cancer cell migration via the mitogen activated protein kinase pathway

Recruitment of focal adhesion kinase and paxillin to β1 integrin promotes cancer cell migration via mitogen activated protein kinase activation

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Ohannessian Arthur

2004-05-01

Full Text Available Abstract Background Integrin-extracellular matrix interactions activate signaling cascades such as mitogen activated protein kinases (MAPK. Integrin binding to extracellular matrix increases tyrosine phosphorylation of focal adhesion kinase (FAK. Inhibition of FAK activity by expression of its carboxyl terminus decreases cell motility, and cells from FAK deficient mice also show reduced migration. Paxillin is a focal adhesion protein which is also phosphorylated on tyrosine. FAK recruitment of paxillin to the cell membrane correlates with Shc phosphorylation and activation of MAPK. Decreased FAK expression inhibits papilloma formation in a mouse skin carcinogenesis model. We previously demonstrated that MAPK activation was required for growth factor induced in vitro migration and invasion by human squamous cell carcinoma (SCC lines. Methods Adapter protein recruitment to integrin subunits was examined by co-immunoprecipitation in SCC cells attached to type IV collagen or plastic. Stable clones overexpressing FAK or paxillin were created using the lipofection technique. Modified Boyden chambers were used for invasion assays. Results In the present study, we showed that FAK and paxillin but not Shc are recruited to the β1 integrin cytoplasmic domain following attachment of SCC cells to type IV collagen. Overexpression of either FAK or paxillin stimulated cancer cell migration on type IV collagen and invasion through reconstituted basement membrane which was dependent on MAPK activity. Conclusions We concluded that recruitment of focal adhesion kinase and paxillin to β1 integrin promoted cancer cell migration via the mitogen activated protein kinase pathway.

Mitogen-Activated Protein Kinase Phosphatase-2 Deletion Impairs Synaptic Plasticity and Hippocampal-Dependent Memory.

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Abdul Rahman, Nor Zaihana; Greenwood, Sam M; Brett, Ros R; Tossell, Kyoko; Ungless, Mark A; Plevin, Robin; Bushell, Trevor J

2016-02-24

Mitogen-activated protein kinases (MAPKs) regulate brain function and their dysfunction is implicated in a number of brain disorders, including Alzheimer's disease. Thus, there is great interest in understanding the signaling systems that control MAPK function. One family of proteins that contribute to this process, the mitogen-activated protein kinase phosphatases (MKPs), directly inactivate MAPKs through dephosphorylation. Recent studies have identified novel functions of MKPs in development, the immune system, and cancer. However, a significant gap in our knowledge remains in relation to their role in brain functioning. Here, using transgenic mice where the Dusp4 gene encoding MKP-2 has been knocked out (MKP-2(-/-) mice), we show that long-term potentiation is impaired in MKP-2(-/-) mice compared with MKP-2(+/+) controls whereas neuronal excitability, evoked synaptic transmission, and paired-pulse facilitation remain unaltered. Furthermore, spontaneous EPSC (sEPSC) frequency was increased in acute slices and primary hippocampal cultures prepared from MKP-2(-/-) mice with no effect on EPSC amplitude observed. An increase in synapse number was evident in primary hippocampal cultures, which may account for the increase in sEPSC frequency. In addition, no change in ERK activity was detected in both brain tissue and primary hippocampal cultures, suggesting that the effects of MKP-2 deletion were MAPK independent. Consistent with these alterations in hippocampal function, MKP-2(-/-) mice show deficits in spatial reference and working memory when investigated using the Morris water maze. These data show that MKP-2 plays a role in regulating hippocampal function and that this effect may be independent of MAPK signaling. Copyright © 2016 Abdul Rahman et al.

Heat Shock Proteins and Mitogen-activated Protein Kinases in Steatotic Livers Undergoing Ischemia-Reperfusion: Some Answers

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Massip-Salcedo, Marta; Casillas-Ramirez, Araní; Franco-Gou, Rosah; Bartrons, Ramón; Ben Mosbah, Ismail; Serafin, Anna; Roselló-Catafau, Joan; Peralta, Carmen

2006-01-01

Ischemic preconditioning protects steatotic livers against ischemia-reperfusion (I/R) injury, but just how this is achieved is poorly understood. Here, I/R or preconditioning plus I/R was induced in steatotic and nonsteatotic livers followed by investigating the effect of pharmacological treatments that modulate heat shock proteins (HSPs) and mitogen-activated protein kinases (MAPKs). MAPKs, HSPs, protein kinase C, and transaminase levels were measured after reperfusion. We report that preconditioning increased HSP72 and heme-oxygenase-1 (HO-1) at 6 and 24 hours of reperfusion, respectively. Unlike nonsteatotic livers, steatotic livers benefited from HSP72 activators (geranylgeranylacetone) throughout reperfusion. This protection seemed attributable to HO-1 induction. In steatotic livers, preconditioning and geranylgeranylacetone treatment (which are responsible for HO-1 induction) increased protein kinase C activity. HO-1 activators (cobalt(III) protoporphyrin IX) protected both liver types. Preconditioning reduced p38 MAPK and c-Jun N-terminal kinase (JNK), resulting in HSP72 induction though HO-1 remained unmodified. Like HSP72, both p38 and JNK appeared not to be crucial in preconditioning, and inhibitors of p38 (SB203580) and JNK (SP600125) were less effective against hepatic injury than HO-1 activators. These results provide new data regarding the mechanisms of preconditioning and may pave the way to the development of new pharmacological strategies in liver surgery. PMID:16651615

Angiotensin II regulation of neuromodulation: downstream signaling mechanism from activation of mitogen-activated protein kinase.

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Lu, D; Yang, H; Raizada, M K

1996-12-01

Angiotensin II (Ang II) stimulates expression of tyrosine hydroxylase and norepinephrine transporter genes in brain neurons; however, the signal-transduction mechanism is not clearly defined. This study was conducted to determine the involvement of the mitogen-activated protein (MAP) kinase signaling pathway in Ang II stimulation of these genes. MAP kinase was localized in the perinuclear region of the neuronal soma. Ang II caused activation of MAP kinase and its subsequent translocation from the cytoplasmic to nuclear compartment, both effects being mediated by AT1 receptor subtype. Ang II also stimulated SRE- and AP1-binding activities and fos gene expression and its translocation in a MAP kinase-dependent process. These observations are the first demonstration of a downstream signaling pathway involving MAP kinase in Ang II-mediated neuromodulation in noradrenergic neurons.

LmxMPK4, an essential mitogen-activated protein kinase of Leishmania mexicana is phosphorylated and activated by the STE7-like protein kinase LmxMKK5

DEFF Research Database (Denmark)

John von Freyend, Simona; Rosenqvist, Heidi; Fink, Annette

2010-01-01

The essential mitogen-activated protein kinase (MAP kinase), LmxMPK4, of Leishmania mexicana is minimally active when purified following recombinant expression in Escherichia coli and was therefore unsuitable for drug screening until now. Using an E. coli protein co-expression system we identified...... LmxMKK5, a STE7-like protein kinase from L. mexicana, which phosphorylates and activates recombinant LmxMPK4 in vitro. LmxMKK5 is comprised of 525 amino acids and has a calculated molecular mass of 55.9kDa. The co-expressed, purified LmxMPK4 showed strong phosphotransferase activity in radiometric...... kinase assays and was confirmed by immunoblot and tandem mass spectrometry analyses to be phosphorylated on threonine 190 and tyrosine 192 of the typical TXY MAP kinase activation motif. The universal protein kinase inhibitor staurosporine reduced the phosphotransferase activity of co...

Sustained mitogen-activated protein kinase activation reprograms defense metabolism and phosphoprotein profile in Arabidopsis thaliana.

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Ines eLassowskat

2014-10-01

Full Text Available Mitogen-activated protein kinases (MAPKs target a variety of protein substrates to regulate cellular signaling processes in eukaryotes. In plants, the number of identified MAPK substrates that control plant defense responses is still limited. Here, we generated transgenic Arabidopsis thaliana plants with an inducible system to simulate in vivo activation of two stress-activated MAPKs, MPK3 and MPK6. Metabolome analysis revealed that this artificial MPK3/6 activation (without any exposure to pathogens or other stresses is sufficient to drive the production of major defense-related metabolites, including various camalexin, indole glucosinolate and agmatine derivatives. An accompanying (phosphoproteome analysis led to detection of hundreds of potential phosphoproteins downstream of MPK3/6 activation. Besides known MAPK substrates, many candidates on this list possess typical MAPK-targeted phosphosites and in many cases, the corresponding phosphopeptides were detected by mass spectrometry. Notably, several of these putative phosphoproteins have been reported to be associated with the biosynthesis of antimicrobial defense substances (e.g. WRKY transcription factors and proteins encoded by the genes from the PEN pathway required for penetration resistance to filamentous pathogens. Thus, this work provides an inventory of candidate phosphoproteins, including putative direct MAPK substrates, for future analysis of MAPK-mediated defense control. (Proteomics data are available with the identifier PXD001252 via ProteomeXchange, http://proteomecentral.proteomexchange.org.

Systems biology analysis of mitogen activated protein kinase inhibitor resistance in malignant melanoma.

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Zecena, Helma; Tveit, Daniel; Wang, Zi; Farhat, Ahmed; Panchal, Parvita; Liu, Jing; Singh, Simar J; Sanghera, Amandeep; Bainiwal, Ajay; Teo, Shuan Y; Meyskens, Frank L; Liu-Smith, Feng; Filipp, Fabian V

2018-04-04

Kinase inhibition in the mitogen activated protein kinase (MAPK) pathway is a standard therapy for cancer patients with activating BRAF mutations. However, the anti-tumorigenic effect and clinical benefit are only transient, and tumors are prone to treatment resistance and relapse. To elucidate mechanistic insights into drug resistance, we have established an in vitro cellular model of MAPK inhibitor resistance in malignant melanoma. The cellular model evolved in response to clinical dosage of the BRAF inhibitor, vemurafenib, PLX4032. We conducted transcriptomic expression profiling using RNA-Seq and RT-qPCR arrays. Pathways of melanogenesis, MAPK signaling, cell cycle, and metabolism were significantly enriched among the set of differentially expressed genes of vemurafenib-resistant cells vs control. The underlying mechanism of treatment resistance and pathway rewiring was uncovered to be based on non-genomic adaptation and validated in two distinct melanoma models, SK-MEL-28 and A375. Both cell lines have activating BRAF mutations and display metastatic potential. Downregulation of dual specific phosphatases, tumor suppressors, and negative MAPK regulators reengages mitogenic signaling. Upregulation of growth factors, cytokines, and cognate receptors triggers signaling pathways circumventing BRAF blockage. Further, changes in amino acid and one-carbon metabolism support cellular proliferation despite MAPK inhibitor treatment. In addition, treatment-resistant cells upregulate pigmentation and melanogenesis, pathways which partially overlap with MAPK signaling. Upstream regulator analysis discovered significant perturbation in oncogenic forkhead box and hypoxia inducible factor family transcription factors. The established cellular models offer mechanistic insight into cellular changes and therapeutic targets under inhibitor resistance in malignant melanoma. At a systems biology level, the MAPK pathway undergoes major rewiring while acquiring inhibitor resistance

Functional Roles of p38 Mitogen-Activated Protein Kinase in Macrophage-Mediated Inflammatory Responses

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Yanyan Yang

2014-01-01

Full Text Available Inflammation is a natural host defensive process that is largely regulated by macrophages during the innate immune response. Mitogen-activated protein kinases (MAPKs are proline-directed serine and threonine protein kinases that regulate many physiological and pathophysiological cell responses. p38 MAPKs are key MAPKs involved in the production of inflammatory mediators, including tumor necrosis factor-α (TNF-α and cyclooxygenase-2 (COX-2. p38 MAPK signaling plays an essential role in regulating cellular processes, especially inflammation. In this paper, we summarize the characteristics of p38 signaling in macrophage-mediated inflammation. In addition, we discuss the potential of using inhibitors targeting p38 expression in macrophages to treat inflammatory diseases.

The Mitogen-Activated Protein Kinase p38 alpha Regulates Tubular Damage in Murine Anti-Glomerular Basement Membrane Nephritis

NARCIS (Netherlands)

Mueller, Ralf; Daniel, Christoph; Hugo, Christian; Amann, Kerstin; Mielenz, Dirk; Endlich, Karlhans; Braun, Tobias; van der Veen, Betty; Heeringa, Peter; Schett, Georg; Zwerina, Jochen

2013-01-01

p38 mitogen-activated protein kinase (MAPK) is thought to play a central role in acute and chronic inflammatory responses. Whether p38MAPK plays a pathogenic role in crescentic GN (GN) and which of its four isoforms is preferentially involved in kidney inflammation is not definitely known. We thus

The mitogen-activated protein kinase (MAPK pathway: role in immune evasion by trypanosomatids

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Mercedes Carolina Soares-Silva

2016-02-01

Full Text Available Leishmania spp and Trypanosoma cruzi are the causative agents of leishmaniasis and Chagas' disease, respectively, two neglected tropical diseases that affect about 25 million people worldwide. These parasites belong to the family Trypanosomatidae and are both obligate intracellular parasites that manipulate host signaling pathways to establish the infection, and also subvert the host innate immune system. Mitogen-activated protein kinases (MAPKs are serine and threonine protein kinases, highly conserved in eukaryotes, and are involved in signal transduction pathways that are related to modulation of physiological and pathophysiological cell responses. This mini-review highlights the current knowledge about the mechanisms that Leishmania spp and T. cruzi have evolved to target host MAPK signaling pathway, highjack immune response, and in this manner, promote parasite maintenance in the host.

Cyclic nucleotides and mitogen-activated protein kinases: regulation of simvastatin in platelet activation

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Hou Ssu-Yu

2010-06-01

Full Text Available Abstract Background 3-Hydroxy-3-methyl-glutaryl coenzyme A (HMG-CoA reductase inhibitors (statins have been widely used to reduce cardiovascular risk. These statins (i.e., simvastatin may exert other effects besides from their cholesterol-lowering actions, including inhibition of platelet activation. Platelet activation is relevant to a variety of coronary heart diseases. Although the inhibitory effect of simvastatin in platelet activation has been studied; the detailed signal transductions by which simvastatin inhibit platelet activation has not yet been completely resolved. Methods The aim of this study was to systematically examine the detailed mechanisms of simvastatin in preventing platelet activation. Platelet aggregation, flow cytometric analysis, immunoblotting, and electron spin resonance studies were used to assess the antiplatelet activity of simvastatin. Results Simvastatin (20-50 μM exhibited more-potent activity of inhibiting platelet aggregation stimulated by collagen than other agonists (i.e., thrombin. Simvastatin inhibited collagen-stimulated platelet activation accompanied by [Ca2+]i mobilization, thromboxane A2 (TxA2 formation, and phospholipase C (PLCγ2, protein kinase C (PKC, and mitogen-activated protein kinases (i.e., p38 MAPK, JNKs phosphorylation in washed platelets. Simvastatin obviously increased both cyclic AMP and cyclic GMP levels. Simvastatin markedly increased NO release, vasodilator-stimulated phosphoprotein (VASP phosphorylation, and endothelial nitric oxide synthase (eNOS expression. SQ22536, an inhibitor of adenylate cyclase, markedly reversed the simvastatin-mediated inhibitory effects on platelet aggregation, PLCγ2 and p38 MAPK phosphorylation, and simvastatin-mediated stimulatory effects on VASP and eNOS phosphorylation. Conclusion The most important findings of this study demonstrate for the first time that inhibitory effect of simvastatin in platelet activation may involve activation of the cyclic AMP

Ménage à trois: the complex relationships between mitogen-activated protein kinases, WRKY transcription factors, and VQ-motif-containing proteins.

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Weyhe, Martin; Eschen-Lippold, Lennart; Pecher, Pascal; Scheel, Dierk; Lee, Justin

2014-01-01

Out of the 34 members of the VQ-motif-containing protein (VQP) family, 10 are phosphorylated by the mitogen-activated protein kinases (MAPKs), MPK3 and MPK6. Most of these MPK3/6-targeted VQPs (MVQs) interacted with specific sub-groups of WRKY transcription factors in a VQ-motif-dependent manner. In some cases, the MAPK appears to phosphorylate either the MVQ or the WRKY, while in other cases, both proteins have been reported to act as MAPK substrates. We propose a network of dynamic interactions between members from the MAPK, MVQ and WRKY families - either as binary or as tripartite interactions. The compositions of the WRKY-MVQ transcriptional protein complexes may change - for instance, through MPK3/6-mediated modulation of protein stability - and therefore control defense gene transcription.

Involvement of protein kinase B and mitogen-activated protein kinases in experimental normothermic liver ischaemia-reperfusion injury.

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Cursio, R; Filippa, N; Miele, C; Van Obberghen, E; Gugenheim, J

2006-06-01

This study evaluated the role of protein kinase B (PKB), phosphatidylinositol 3-kinase (PI3-K), Bcl-2-associated death protein (BAD) and mitogen-activated protein kinases (MAPKs) in normothermic ischaemia-reperfusion (IR)-induced apoptosis in rat liver. Rats were divided into two groups that received either phosphate-buffered saline (control) or the caspase inhibitor Z-Asp-2,6-dichorobenzoyloxymethylketone (Z-Asp-cmk), injected intravenously 2 min before the induction of 120 min of normothermic liver ischaemia. Liver apoptosis was assessed by the terminal deoxyribonucleotidyltransferase-mediated dUTP nick end labelling (TUNEL) method. PI3-K, PKB, BAD and MAPK activities were measured in ischaemic and non-ischaemic lobes at various times after reperfusion. The number of TUNEL-positive cells was significantly decreased after pretreatment with Z-Asp-cmk. In controls, PI3-K and PKB activities and BAD phosphorylation were inhibited in ischaemic liver lobes. The MAPKs (extracellular signal-regulated kinases, c-Jun N-terminal kinase and p38) showed different patterns of activation during IR. PKB activity was not modified by pretreatment with Z-Asp-cmk. Induction of apoptosis during IR liver injury might be triggered by inactivation of the antiapoptotic PI3-K-PKB pathway and activation of the proapoptotic MAPKs. Copyright (c) 2006 British Journal of Surgery Society Ltd. Published by John Wiley & Sons, Ltd.

Toward a Comprehensive Phylogenetic Reconstruction of the Evolutionary History of Mitogen-Activated Protein Kinases in the Plant Kingdom

OpenAIRE

Janitza, Philipp; Ullrich, Kristian Karsten; Quint, Marcel

2012-01-01

The mitogen-activated protein kinase (MAPK) pathway is a three-tier signaling cascade that transmits cellular information from the plasma membrane to the cytoplasm where it triggers downstream responses. The MAPKs represent the last step in this cascade and are activated when both tyrosine and threonine residues in a conserved TxY motif are phosphorylated by MAPK kinases, which in turn are themselves activated by phosphorylation by MAPK kinase kinases. To understand the molecular evolution of...

Cocoa Procyanidins Suppress Transformation by Inhibiting Mitogen-activated Protein Kinase Kinase*S⃞

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Kang, Nam Joo; Lee, Ki Won; Lee, Dong Eun; Rogozin, Evgeny A.; Bode, Ann M.; Lee, Hyong Joo; Dong, Zigang

2008-01-01

Cocoa was shown to inhibit chemically induced carcinogenesis in animals and exert antioxidant activity in humans. However, the molecular mechanisms of the chemopreventive potential of cocoa and its active ingredient(s) remain unknown. Here we report that cocoa procyanidins inhibit neoplastic cell transformation by suppressing the kinase activity of mitogen-activated protein kinase kinase (MEK). A cocoa procyanidin fraction (CPF) and procyanidin B2 at 5 μg/ml and 40 μm, respectively, inhibited 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced neoplastic transformation of JB6 P+ mouse epidermal (JB6 P+) cells by 47 and 93%, respectively. The TPA-induced promoter activity and expression of cyclooxygenase-2, which is involved in tumor promotion and inflammation, were dose-dependently inhibited by CPF or procyanidin B2. The activation of activator protein-1 and nuclear factor-κB induced by TPA was also attenuated by CPF or procyanidin B2. The TPA-induced phosphorylation of MEK, extracellular signal-regulated kinase, and p90 ribosomal s6 kinase was suppressed by CPF or procyanidin B2. In vitro and ex vivo kinase assay data demonstrated that CPF or procyanidin B2 inhibited the kinase activity of MEK1 and directly bound with MEK1. CPF or procyanidin B2 suppressed JB6 P+ cell transformation induced by epidermal growth factor or H-Ras, both of which are known to be involved in MEK/ERK signal activation. In contrast, theobromine (up to 80 μm) had no effect on TPA-induced transformation, cyclooxygenase-2 expression, the transactivation of activator protein-1 or nuclear factor-κB, or MEK. Notably, procyanidin B2 exerted stronger inhibitory effects compared with PD098059 (a well known pharmacological inhibitor of MEK) on MEK1 activity and neoplastic cell transformation. PMID:18519570

Blockades of mitogen-activated protein kinase and calcineurin both change fibre-type markers in skeletal muscle culture

DEFF Research Database (Denmark)

Higginson, James; Wackerhage, Henning; Woods, Niall

2002-01-01

A and mitogen-activated protein kinase kinase (MEK1/2) blockade with U0126 upon myosin heavy chain (MHC) isoform mRNA levels and activities of metabolic enzymes after 1 day, 3 days and 7 days of treatment in primary cultures of spontaneously twitching rat skeletal muscle. U0126 treatment significantly decreased......Activation of either the calcineurin or the extracellular signal-regulated kinase (ERK1/2) pathway increases the percentage of slow fibres in vivo suggesting that both pathways can regulate fibre phenotypes in skeletal muscle. We investigated the effect of calcineurin blockade with cyclosporin...

Mitigation of direct neurotoxic effects of lidocaine and amitriptyline by inhibition of p38 mitogen-activated protein kinase in vitro and in vivo

NARCIS (Netherlands)

Lirk, Philipp; Haller, Ingrid; Myers, Robert R.; Klimaschewski, Lars; Kau, Yi-Chuan; Hung, Yu-Chun; Gerner, Peter

2006-01-01

Local anesthetic-induced direct neurotoxicity (paresthesia, failure to regain normal sensory and motor function) is a potentially devastating complication of regional anesthesia. Local anesthetics activate the p38 mitogen-activated protein kinase (MAPK) system, which is involved in apoptotic cell

Review: Mitogen-Activated Protein kinases in nutritional signaling in Arabidopsis

KAUST Repository

Chardin, Camille; Schenk, Sebastian T.; Hirt, Heribert; Colcombet, Jean; Krapp, Anne

2017-01-01

Mitogen-Activated Protein Kinase (MAPK) cascades are functional modules widespread among eukaryotic organisms. In plants, these modules are encoded by large multigenic families and are involved in many biological processes ranging from stress responses to cellular differentiation and organ development. Furthermore, MAPK pathways are involved in the perception of environmental and physiological modifications. Interestingly, some MAPKs play a role in several signaling networks and could have an integrative function for the response of plants to their environment. In this review, we describe the classification of MAPKs and highlight some of their biochemical actions. We performed an in silico analysis of MAPK gene expression in response to nutrients supporting their involvement in nutritional signaling. While several MAPKs have been identified as players in sugar, nitrogen, phosphate, iron and potassium-related signaling pathways, their biochemical functions are yet mainly unknown. The integration of these regulatory cascades in the current understanding of nutrient signaling is discussed and potential new avenues for approaches toward plants with higher nutrient use efficiencies are evoked.

Review: Mitogen-Activated Protein kinases in nutritional signaling in Arabidopsis

KAUST Repository

Chardin, Camille

2017-04-14

Mitogen-Activated Protein Kinase (MAPK) cascades are functional modules widespread among eukaryotic organisms. In plants, these modules are encoded by large multigenic families and are involved in many biological processes ranging from stress responses to cellular differentiation and organ development. Furthermore, MAPK pathways are involved in the perception of environmental and physiological modifications. Interestingly, some MAPKs play a role in several signaling networks and could have an integrative function for the response of plants to their environment. In this review, we describe the classification of MAPKs and highlight some of their biochemical actions. We performed an in silico analysis of MAPK gene expression in response to nutrients supporting their involvement in nutritional signaling. While several MAPKs have been identified as players in sugar, nitrogen, phosphate, iron and potassium-related signaling pathways, their biochemical functions are yet mainly unknown. The integration of these regulatory cascades in the current understanding of nutrient signaling is discussed and potential new avenues for approaches toward plants with higher nutrient use efficiencies are evoked.

Regulation of WRKY46 transcription factor function by mitogen-activated protein kinases in Arabidopsis thaliana.

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Arsheed Hussain Sheikh

2016-02-01

Full Text Available AbstractMitogen-activated protein kinase (MAPK cascades are central signalling pathways activated in plants after sensing internal developmental and external stress cues. Knowledge about the downstream substrate proteins of MAPKs is still limited in plants. We screened Arabidopsis WRKY transcription factors as potential targets downstream of MAPKs, and concentrated on characterizing WRKY46 as a substrate of the MAPK, MPK3. Mass spectrometry revealed in vitro phosphorylation of WRKY46 at amino acid position S168 by MPK3. However, mutagenesis studies showed that a second phosphosite, S250, can also be phosphorylated. Elicitation with pathogen-associated molecular patterns (PAMPs, such as the bacterial flagellin-derived flg22 peptide led to in vivo destabilization of WRKY46 in Arabidopsis protoplasts. Mutation of either phosphorylation site reduced the PAMP-induced degradation of WRKY46. Furthermore, the protein for the double phosphosite mutant is expressed at higher levels compared to wild-type proteins or single phosphosite mutants. In line with its nuclear localization and predicted function as a transcriptional activator, overexpression of WRKY46 in protoplasts raised basal plant defence as reflected by the increase in promoter activity of the PAMP-responsive gene, NHL10, in a MAPK-dependent manner. Thus, MAPK-mediated regulation of WRKY46 is a mechanism to control plant defence.

Context Specificity of Stress-activated Mitogen-activated Protein (MAP) Kinase Signaling: The Story as Told by Caenorhabditis elegans*

Science.gov (United States)

Andrusiak, Matthew G.; Jin, Yishi

2016-01-01

Stress-associated p38 and JNK mitogen-activated protein (MAP) kinase signaling cascades trigger specific cellular responses and are involved in multiple disease states. At the root of MAP kinase signaling complexity is the differential use of common components on a context-specific basis. The roundworm Caenorhabditis elegans was developed as a system to study genes required for development and nervous system function. The powerful genetics of C. elegans in combination with molecular and cellular dissections has led to a greater understanding of how p38 and JNK signaling affects many biological processes under normal and stress conditions. This review focuses on the studies revealing context specificity of different stress-activated MAPK components in C. elegans. PMID:26907690

Eotaxin induces degranulation and chemotaxis of eosinophils through the activation of ERK2 and p38 mitogen-activated protein kinases

DEFF Research Database (Denmark)

Kampen, G T; Stafford, S; Adachi, T

2000-01-01

Eotaxin and other CC chemokines acting via CC chemokine receptor-3 (CCR3) are believed to play an integral role in the development of eosinophilic inflammation in asthma and allergic inflammatory diseases. However, little is known about the intracellular events following agonist binding to CCR3...... and the relationship of these events to the functional response of the cell. The objectives of this study were to investigate CCR3-mediated activation of the mitogen-activated protein (MAP) kinases extracellular signal-regulated kinase-2 (ERK2), p38, and c-jun N-terminal kinase (JNK) in eosinophils and to assess...... the requirement for MAP kinases in eotaxin-induced eosinophil cationic protein (ECP) release and chemotaxis. MAP kinase activation was studied in eotaxin-stimulated eosinophils (more than 97% purity) by Western blotting and immune-complex kinase assays. ECP release was measured by radioimmunoassay. Chemotaxis...

Expression of MMPs is dependent on the activity of mitogen-activated protein kinase in chondrosarcoma.

Science.gov (United States)

Yao, Min; Wang, Xiaomei; Zhao, Yufeng; Wang, Xiaomeng; Gao, Feng

2017-02-01

Matrix metalloproteinases (MMPs) and tissue inhibitors of matrix metalloproteinases (TIMPs) serve an important role in chondrosarcoma. The present study investigated whether the expression of MMPs was dependent on the activity of mitogen-activated protein kinase (MAPK) in chondrosarcoma. Surgical pathological specimens were collected to detect MMP-1, MMP-13, TIMP-1, type II collagen and phosphorylated MAPK levels in normal cartilage, enchondroma and chondrosarcoma tissues. The expression of MMP‑1, MMP‑13, TIMP‑1 and type II collagen was investigated utilizing MAPK inhibitors in chondrosarcoma cells. It was noted that the expression levels of MMP‑1, MMP‑13 and TIMP‑1 were increased in chondrosarcoma with the activity of MAPK. After chondrosarcoma cells were pretreated with MAPK inhibitors, the levels of MMP‑1, MMP‑13 and TIMP‑1 were inhibited. Furthermore, MMP‑1 and MMP‑13 are essential in regulating the degradation of type II collagen and decomposing cartilage matrix major. The high expression levels of MMP‑1 and MMP‑13 in chondrosarcoma expedite the invasion by chondrosarcoma cells and their expression can be depressed by MAPK inhibitors.

Aspergillus nidulans Natural Product Biosynthesis Is Regulated by MpkB, a Putative Pheromone Response Mitogen-Activated Protein Kinase

International Nuclear Information System (INIS)

Atoui, A.; Bao, D.; Kaur, N.; Grayburn, W.S.; Calvo, A.M.

2008-01-01

The Aspergillus nidulans putative mitogen-activated protein kinase encoded by mpkB has a role in natural product biosynthesis. An mpkB mutant exhibited a decrease in sterigmatocystin gene expression and low mycotoxin levels. The mutation also affected the expression of genes involved in penicillin and terrequinone A synthesis. mpkB was necessary for normal expression of laeA, which has been found to regulate secondary metabolism gene clusters. (author)

Context Specificity of Stress-activated Mitogen-activated Protein (MAP) Kinase Signaling: The Story as Told by Caenorhabditis elegans.

Science.gov (United States)

Andrusiak, Matthew G; Jin, Yishi

2016-04-08

Stress-associated p38 and JNK mitogen-activated protein (MAP) kinase signaling cascades trigger specific cellular responses and are involved in multiple disease states. At the root of MAP kinase signaling complexity is the differential use of common components on a context-specific basis. The roundwormCaenorhabditis eleganswas developed as a system to study genes required for development and nervous system function. The powerful genetics ofC. elegansin combination with molecular and cellular dissections has led to a greater understanding of how p38 and JNK signaling affects many biological processes under normal and stress conditions. This review focuses on the studies revealing context specificity of different stress-activated MAPK components inC. elegans. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

Noise exposure immediately activates cochlear mitogen-activated protein kinase signaling

Directory of Open Access Journals (Sweden)

Kumar N Alagramam

2014-01-01

Full Text Available Noise-induced hearing loss (NIHL is a major public health issue worldwide. Uncovering the early molecular events associated with NIHL would reveal mechanisms leading to the hearing loss. Our aim is to investigate the immediate molecular responses after different levels of noise exposure and identify the common and distinct pathways that mediate NIHL. Previous work showed mice exposed to 116 decibels sound pressure level (dB SPL broadband noise for 1 h had greater threshold shifts than the mice exposed to 110 dB SPL broadband noise, hence we used these two noise levels in this study. Groups of 4-8-week-old CBA/CaJ mice were exposed to no noise (control or to broadband noise for 1 h, followed by transcriptome analysis of total cochlear RNA isolated immediately after noise exposure. Previously identified and novel genes were found in all data sets. Following exposure to noise at 116 dB SPL, the earliest responses included up-regulation of 243 genes and down-regulation of 61 genes, while a similar exposure at 110 dB SPL up-regulated 155 genes and down-regulated 221 genes. Bioinformatics analysis indicated that mitogen-activated protein kinase (MAPK signaling was the major pathway in both levels of noise exposure. Nevertheless, both qualitative and quantitative differences were noticed in some MAPK signaling genes, after exposure to different noise levels. Cacna1b , Cacna1g , and Pla2g6 , related to calcium signaling were down-regulated after 110 dB SPL exposure, while the fold increase in the expression of Fos was relatively lower than what was observed after 116 dB SPL exposure. These subtle variations provide insight on the factors that may contribute to the differences in NIHL despite the activation of a common pathway.

Ebselen impairs cellular oxidative state and induces endoplasmic reticulum stress and activation of crucial mitogen-activated protein kinases in pancreatic tumour AR42J cells.

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Santofimia-Castaño, Patricia; Izquierdo-Alvarez, Alicia; Plaza-Davila, María; Martinez-Ruiz, Antonio; Fernandez-Bermejo, Miguel; Mateos-Rodriguez, Jose M; Salido, Gines M; Gonzalez, Antonio

2018-01-01

Ebselen (2-phenyl-1,2-benzisoselenazol-3(2H)-one) is an organoselenium radical scavenger compound, which has strong antioxidant and anti-inflammatory effects. However, evidence suggests that this compound could exert deleterious actions on cell physiology. In this study, we have analyzed the effect of ebselen on rat pancreatic AR42J cells. Cytosolic free-Ca 2+ concentration ([Ca 2+ ] c ), cellular oxidative status, setting of endoplasmic reticulum stress, and phosphorylation of major mitogen-activated protein kinases were analyzed. Our results show that ebselen evoked a concentration-dependent increase in [Ca 2+ ] c . The compound induced an increase in the generation of reactive oxygen species in the mitochondria. We also observed an increase in global cysteine oxidation in the presence of ebselen. In the presence of ebselen an impairment of cholecystokinin-evoked amylase release was noted. Moreover, involvement of the unfolded protein response markers, ER chaperone and signaling regulator GRP78/BiP, eukaryotic translation initiation factor 2α and X-box binding protein 1 was detected. Finally, increases in the phosphorylation of SAPK/JNK, p38 MAPK, and p44/42 MAPK in the presence of ebselen were also observed. Our results provide evidences for an impairment of cellular oxidative state and enzyme secretion, the induction of endoplasmic reticulum stress and the activation of crucial mitogen-activated protein kinases in the presence of ebselen. As a consequence ebselen exerts a potential toxic effect on AR42J cells. © 2017 Wiley Periodicals, Inc.

Evidence of a New Role for the High-Osmolarity Glycerol Mitogen-Activated Protein Kinase Pathway in Yeast: Regulating Adaptation to Citric Acid Stress†

OpenAIRE

Lawrence, Clare L.; Botting, Catherine H.; Antrobus, Robin; Coote, Peter J.

2004-01-01

Screening the Saccharomyces cerevisiae disruptome, profiling transcripts, and determining changes in protein expression have identified an important new role for the high-osmolarity glycerol (HOG) mitogen-activated protein kinase (MAPK) pathway in the regulation of adaptation to citric acid stress. Deletion of HOG1, SSK1, PBS2, PTC2, PTP2, and PTP3 resulted in sensitivity to citric acid. Furthermore, citric acid resulted in the dual phosphorylation, and thus activation, of Hog1p. Despite mino...

Stress and vascular responses: atheroprotective effect of laminar fluid shear stress in endothelial cells: possible role of mitogen-activated protein kinases.

Science.gov (United States)

Yoshizumi, Masanori; Abe, Jun-Ichi; Tsuchiya, Koichiro; Berk, Bradford C; Tamaki, Toshiaki

2003-03-01

Atherosclerosis preferentially occurs in areas of turbulent blood flow and low fluid shear stress, whereas laminar blood flow and high shear stress are atheroprotective. Inflammatory cytokines, such as tumor necrosis factor-alpha (TNF-alpha), stimulate expression of endothelial cell (EC) genes that may promote atherosclerosis. Recent findings suggest a steady laminar blood flow decreases EC apoptosis and inhibits TNF-mediated EC activation. EC apoptosis or activation is suggested to be involved in plaque erosion, which may lead to platelet aggregation. TNF-alpha regulates gene expression in ECs, in part, by stimulating mitogen-activated protein (MAP) kinases, which phosphorylate transcription factors. We hypothesized that steady laminar flow inhibits cytokine-mediated activation of MAP kinases in ECs. To test this hypothesis, we determined the effects of steady laminar flow (shear stress = 12 dynes/cm(2)) on TNF-alpha-stimulated activity of three MAP kinases in human umbilical vein ECs (HUVEC): extracellular signal-regulated kinase (ERK1/2), c-Jun N-terminal kinase (JNK), and p38. TNF-alpha activated ERK1/2, JNK, and p38 maximally at 15 min in HUVEC. Pre-exposing HUVEC for 10 min to flow inhibited TNF-alpha activation of JNK, but showed no significant effect on ERK1/2 or p38 activation. Incubation of HUVEC with PD98059, a specific ERK1/2 inhibitor, blocked the flow-mediated inhibition of TNF activation of JNK. Transfection studies with dominant-negative constructs of the protein kinase MEK5 suggested an important role for big mitogen-activated protein kinase 1 (BMK1) in flow-mediated regulation of EC activation by TNF-alpha. Understanding the mechanisms by which steady laminar flow regulates JNK activation by cytokines may provide insight into the atheroprotective mechanisms induced by laminar blood flow.

Phosphorylation of paxillin via the ERK mitogen-activated protein kinase cascade in EL4 thymoma cells.

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Ku, H; Meier, K E

2000-04-14

Intracellular signals can regulate cell adhesion via several mechanisms in a process referred to as "inside-out" signaling. In phorbol ester-sensitive EL4 thymoma cells, phorbol-12-myristate 13-acetate (PMA) induces activation of extracellular signal-regulated kinase (ERK) mitogen-activated protein kinases and promotes cell adhesion. In this study, clonal EL4 cell lines with varying abilities to activate ERKs in response to PMA were used to examine signaling events occurring downstream of ERK activation. Paxillin, a multifunctional docking protein involved in cell adhesion, was phosphorylated on serine/threonine residues in response to PMA treatment. This response was correlated with the extent and time course of ERK activation. PMA-induced phosphorylation of paxillin was inhibited by compounds that block the ERK activation pathway in EL4 cells, primary murine thymocytes, and primary murine splenocytes. Paxillin was phosphorylated in vitro by purified active ERK2. Two-dimensional electrophoresis revealed that PMA treatment generated a complex pattern of phosphorylated paxillin species in intact cells, some of which were generated by ERK-mediated phosphorylation in vitro. An ERK pathway inhibitor interfered with PMA-induced adhesion of sensitive EL4 cells to substrate. These findings describe a novel inside-out signaling pathway by which the ERK cascade may regulate events involved in adhesion.

Human prostatic cancer cells, PC3, elaborate mitogenic activity which selectively stimulates human bone cells

International Nuclear Information System (INIS)

Perkel, V.S.; Mohan, S.; Herring, S.J.; Baylink, D.J.; Linkhart, T.A.

1990-01-01

Prostatic cancer typically produces osteoblastic metastases which are not attended by marrow fibrosis. In the present study we sought to test the hypothesis that prostatic cancer cells produce factor(s) which act selectively on human osteoblasts. Such a paracrine mechanism would explain the observed increase in osteoblasts, unaccompanied by an increase in marrow fibroblasts. To test this hypothesis we investigated the mitogenic activity released by the human prostatic tumor cell line, PC3. PC3 cells have been reported previously to produce mitogenic activity for cells that was relatively specific for rat osteoblasts compared to rat fibroblasts. However, the effects of this activity on human cells has not been examined previously. PC3-conditioned medium (CM) (5-50 micrograms CM protein/ml) stimulated human osteoblast proliferation by 200-950% yet did not stimulate human fibroblast proliferation ([3H]thymidine incorporation). PC3 CM also increased cell numbers in human osteoblast but not fibroblast cell cultures. To determine whether the osteoblast-specific mitogenic activity could be attributed to known bone growth factors, specific assays for these growth factors were performed. PC3 CM contained 10 pg insulin-like growth factor (IGF) I, less than 2 pg IGF II, 54 pg basic fibroblast growth factor, and 16 pg transforming growth factor beta/microgram CM protein. None of these growth factors alone or in combination could account for the observed osteoblast-specific PC3 cell-derived mitogenic activity. Furthermore, when 5 micrograms/ml PC3 CM was tested in combination with maximally effective concentrations of either basic fibroblast growth factor, IGF I, IGF II, or transforming growth factor beta, it produced an additive effect suggesting that PC3 CM stimulates osteoblast proliferation by a mechanism independent of these bone mitogens

The Na+/H+ exchanger, NHE1, differentially regulates mitogen-activated protein kinase subfamilies after osmotic shrinkage in Ehrlich Lettre Ascites cells

DEFF Research Database (Denmark)

Petersen, Stine Helene Falsig; Rasmussen, Maria; Darborg, Barbara Vasek

2007-01-01

Osmotic stress modulates mitogen activated protein kinase (MAPK) activities, leading to altered gene transcription and cell death/survival balance, however, the mechanisms involved are incompletely elucidated. Here, we show, using a combination of biochemical and molecular biology approaches...... by human (h) NHE1 expression in cells lacking endogenous NHE1 activity. The effect of NHE1 on ERK1/2 was pH(i)-independent and upstream of MEK1/2. Shrinkage-activation of JNK1/2 was attenuated by EIPA, augmented by hNHE1 expression, and abolished in the presence of HCO(3)(-). Basal JNK activity...

Regulation of Schistosoma mansoni development and reproduction by the mitogen-activated protein kinase signaling pathway.

Science.gov (United States)

Andrade, Luiza Freire de; Mourão, Marina de Moraes; Geraldo, Juliana Assis; Coelho, Fernanda Sales; Silva, Larissa Lopes; Neves, Renata Heisler; Volpini, Angela; Machado-Silva, José Roberto; Araujo, Neusa; Nacif-Pimenta, Rafael; Caffrey, Conor R; Oliveira, Guilherme

2014-06-01

Protein kinases are proven targets for drug development with an increasing number of eukaryotic Protein Kinase (ePK) inhibitors now approved as drugs. Mitogen-activated protein kinase (MAPK) family members connect cell-surface receptors to regulatory targets within cells and influence a number of tissue-specific biological activities such as cell proliferation, differentiation and survival. However, the contributions of members of the MAPK pathway to schistosome development and survival are unclear. We employed RNA interference (RNAi) to elucidate the functional roles of five S. mansoni genes (SmCaMK2, SmJNK, SmERK1, SmERK2 and SmRas) involved in MAPK signaling pathway. Mice were injected with post-infective larvae (schistosomula) subsequent to RNAi and the development of adult worms observed. The data demonstrate that SmJNK participates in parasite maturation and survival of the parasites, whereas SmERK are involved in egg production as infected mice had significantly lower egg burdens with female worms presenting underdeveloped ovaries. Furthermore, it was shown that the c-fos transcription factor was overexpressed in parasites submitted to RNAi of SmERK1, SmJNK and SmCaMK2 indicating its putative involvement in gene regulation in this parasite's MAPK signaling cascade. We conclude that MAPKs proteins play important roles in the parasite in vivo survival, being essential for normal development and successful survival and reproduction of the schistosome parasite. Moreover SmERK and SmJNK are potential targets for drug development.

Modulation of Cyclins, p53 and Mitogen-Activated Protein Kinases Signaling in Breast Cancer Cell Lines by 4-(3,4,5-Trimethoxyphenoxybenzoic Acid

Directory of Open Access Journals (Sweden)

Kuan-Han Lee

2014-01-01

Full Text Available Despite the advances in cancer therapy and early detection, breast cancer remains a leading cause of cancer-related deaths among females worldwide. The aim of the current study was to investigate the antitumor activity of a novel compound, 4-(3,4,5-trimethoxyphenoxybenzoic acid (TMPBA and its mechanism of action, in breast cancer. Results indicated the relatively high sensitivity of human breast cancer cell-7 and MDA-468 cells towards TMPBA with IC50 values of 5.9 and 7.9 µM, respectively compared to hepatocarcinoma cell line Huh-7, hepatocarcinoma cell line HepG2, and cervical cancer cell line Hela cells. Mechanistically, TMPBA induced apoptotic cell death in MCF-7 cells as indicated by 4',6-diamidino-2-phenylindole (DAPI nuclear staining, cell cycle analysis and the activation of caspase-3. Western blot analysis revealed the ability of TMPBA to target pathways mediated by mitogen-activated protein (MAP kinases, 5' adenosine monophosphate-activated protein kinase (AMPK, and p53, of which the concerted action underlined its antitumor efficacy. In addition, TMPBA induced alteration of cyclin proteins’ expression and consequently modulated the cell cycle. Taken together, the current study underscores evidence that TMPBA induces apoptosis in breast cancer cells via the modulation of cyclins and p53 expression as well as the modulation of AMPK and mitogen-activated protein kinases (MAPK signaling. These findings support TMPBA’s clinical promise as a potential candidate for breast cancer therapy.

Induction of viral, 7-methyl-guanosine cap-independent translation and oncolysis by mitogen-activated protein kinase-interacting kinase-mediated effects on the serine/arginine-rich protein kinase.

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Brown, Michael C; Bryant, Jeffrey D; Dobrikova, Elena Y; Shveygert, Mayya; Bradrick, Shelton S; Chandramohan, Vidyalakshmi; Bigner, Darell D; Gromeier, Matthias

2014-11-01

Protein synthesis, the most energy-consuming process in cells, responds to changing physiologic priorities, e.g., upon mitogen- or stress-induced adaptations signaled through the mitogen-activated protein kinases (MAPKs). The prevailing status of protein synthesis machinery is a viral pathogenesis factor, particularly for plus-strand RNA viruses, where immediate translation of incoming viral RNAs shapes host-virus interactions. In this study, we unraveled signaling pathways centered on the ERK1/2 and p38α MAPK-interacting kinases MNK1/2 and their role in controlling 7-methyl-guanosine (m(7)G) "cap"-independent translation at enterovirus type 1 internal ribosomal entry sites (IRESs). Activation of Raf-MEK-ERK1/2 signals induced viral IRES-mediated translation in a manner dependent on MNK1/2. This effect was not due to MNK's known functions as eukaryotic initiation factor (eIF) 4G binding partner or eIF4E(S209) kinase. Rather, MNK catalytic activity enabled viral IRES-mediated translation/host cell cytotoxicity through negative regulation of the Ser/Arg (SR)-rich protein kinase (SRPK). Our investigations suggest that SRPK activity is a major determinant of type 1 IRES competency, host cell cytotoxicity, and viral proliferation in infected cells. We are targeting unfettered enterovirus IRES activity in cancer with PVSRIPO, the type 1 live-attenuated poliovirus (PV) (Sabin) vaccine containing a human rhinovirus type 2 (HRV2) IRES. A phase I clinical trial of PVSRIPO with intratumoral inoculation in patients with recurrent glioblastoma (GBM) is showing early promise. Viral translation proficiency in infected GBM cells is a core requirement for the antineoplastic efficacy of PVSRIPO. Therefore, it is critically important to understand the mechanisms controlling viral cap-independent translation in infected host cells. Copyright © 2014, American Society for Microbiology. All Rights Reserved.

Mitogen activated protein kinases selectively regulate palytoxin-stimulated gene expression in mouse keratinocytes

International Nuclear Information System (INIS)

Zeliadt, Nicholette A.; Warmka, Janel K.; Wattenberg, Elizabeth V.

2003-01-01

We have been investigating how the novel skin tumor promoter palytoxin transmits signals through mitogen activated protein kinases (MAPKs). Palytoxin activates three major MAPKs, extracellular signal-regulated kinase (ERK), c-Jun N-terminal kinase (JNK), and p38, in a keratinocyte cell line derived from initiated mouse skin (308). We previously showed that palytoxin requires ERK to increase matrix metalloproteinase-13 (MMP-13) gene expression, an enzyme implicated in carcinogenesis. Diverse stimuli require JNK and p38 to increase MMP-13 gene expression, however. We therefore used the JNK and p38 inhibitors SP 600125 and SB 202190, respectively, to investigate the role of these MAPKs in palytoxin-induced MMP-13 gene expression. Surprisingly, palytoxin does not require JNK and p38 to increase MMP-13 gene expression. Accordingly, ERK activation, independent of palytoxin and in the absence of JNK and p38 activation, is sufficient to induce MMP-13 gene expression in 308 keratinocytes. Dexamethasone, a synthetic glucocorticoid that inhibits activator protein-1 (AP-1), blocked palytoxin-stimulated MMP-13 gene expression. Therefore, the AP-1 site present in the promoter of the MMP-13 gene appears to be functional and to play a key role in palytoxin-stimulated gene expression. Previous studies showed that palytoxin simulates an ERK-dependent selective increase in the c-Fos content of AP-1 complexes that bind to the promoter of the MMP-13 gene. JNK and p38 can also modulate c-Fos. Palytoxin does not require JNK or p38 to increase c-Fos binding, however. Altogether, these studies indicate that ERK plays a distinctly essential role in transmitting palytoxin-stimulated signals to specific nuclear targets in keratinocytes derived from initiated mouse skin

Molecular Cloning and Characterization of a P38-Like Mitogen-Activated Protein Kinase from Echinococcus granulosus.

Science.gov (United States)

Lü, Guodong; Li, Jing; Zhang, Chuanshan; Li, Liang; Bi, Xiaojuan; Li, Chaowang; Fan, Jinliang; Lu, Xiaomei; Vuitton, Dominique A; Wen, Hao; Lin, Renyong

2016-12-01

Cystic echinococcosis (CE) treatment urgently requires a novel drug. The p38 mitogen-activated protein kinases (MAPKs) are a family of Ser/Thr protein kinases, but still have to be characterized in Echinococcus granulosus . We identified a 1,107 bp cDNA encoding a 368 amino acid MAPK protein (Egp38) in E. granulosus . Egp38 exhibits 2 distinguishing features of p38-like kinases: a highly conserved T-X-Y motif and an activation loop segment. Structural homology modeling indicated a conserved structure among Egp38, EmMPK2, and H. sapiens p38α, implying a common binding mechanism for the ligand domain and downstream signal transduction processing similar to that described for p38α. Egp38 and its phosphorylated form are expressed in the E. granulosus larval stages vesicle and protoscolices during intermediate host infection of an intermediate host. Treatment of in vitro cultivated protoscolices with the p38-MAPK inhibitor ML3403 effectively suppressed Egp38 activity and led to significant protoscolices death within 5 days. Treatment of in vitro-cultivated protoscolices with TGF-β1 effectively induced Egp38 phosphorylation. In summary, the MAPK, Egp38, was identified in E. granulosus , as an anti-CE drug target and participates in the interplay between the host and E. granulosus via human TGF-β1.

Mitogen-activated protein kinase phosphatase (MKP)-1 as a neuroprotective agent: promotion of the morphological development of midbrain dopaminergic neurons.

Science.gov (United States)

Collins, Louise M; O'Keeffe, Gerard W; Long-Smith, Caitriona M; Wyatt, Sean L; Sullivan, Aideen M; Toulouse, André; Nolan, Yvonne M

2013-06-01

A greater understanding of the mechanisms that promote the survival and growth of dopaminergic neurons is essential for the advancement of cell replacement therapies for Parkinson's disease (PD). Evidence supports a role for the mitogen-activated protein kinase p38 in the demise of dopaminergic neurons, while mitogen-activated protein kinase phosphatase-1 (MKP-1), which negatively regulates p38 activity, has not yet been investigated in this context. Here, we show that MKP-1 is expressed in dopaminergic neurons cultured from E14 rat ventral mesencephalon (VM). When dopaminergic neurons were transfected to overexpress MKP-1, they displayed a more complex morphology than their control counterparts in vitro. Specifically, MKP-1-transfection induced significant increases in neurite length and branching with a maximum increase observed in primary branches. We demonstrate that inhibition of dopaminergic neurite growth induced by treatment of rat VM neurons with the dopaminergic neurotoxin 6-hydroxydopamine (6-OHDA) in vitro is mediated by p38 and is concomitant with a significant and selective decrease in MKP-1 expression in these neurons. We further show that overexpression of MKP-1 in dopaminergic neurons contributes to neuroprotection against the effects of 6-OHDA. Collectively, we report that MKP-1 can promote the growth and elaboration of dopaminergic neuronal processes and can help protect them from the neurotoxic effects of 6-OHDA. Thus, we propose that strategies aimed at augmenting MKP-1 expression or activity may be beneficial in protecting dopaminergic neurons and may provide potential therapeutic approaches for PD.

Repeated pulses of serotonin required for long-term facilitation activate mitogen-activated protein kinase in sensory neurons of Aplysia

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Michael, Dan; Martin, Kelsey C.; Seger, Rony; Ning, Ming-Ming; Baston, Rene; Kandel, Eric R.

1998-01-01

Long-term facilitation of the connections between the sensory and motor neurons of the gill-withdrawal reflex in Aplysia requires five repeated pulses of serotonin (5-HT). The repeated pulses of 5-HT initiate a cascade of gene activation that leads ultimately to the growth of new synaptic connections. Several genes in this process have been identified, including the transcriptional regulators apCREB-1, apCREB-2, apC/EBP, and the cell adhesion molecule apCAM, which is thought to be involved in the formation of new synaptic connections. Here we report that the transcriptional regulators apCREB-2 and apC/EBP, as well as a peptide derived from the cytoplasmic domain of apCAM, are phosphorylated in vitro by Aplysia mitogen-activated protein kinase (apMAPK). We have cloned the cDNA encoding apMAPK and show that apMAPK activity is increased in sensory neurons treated with repeated pulses of 5-HT and by the cAMP pathway. These results suggest that apMAPK may participate with cAMP-dependent protein kinase during long-term facilitation in sensory cells by modifying some of the key elements involved in the consolidation of short- to long-lasting changes in synaptic strength. PMID:9465108

The atypical mitogen-activated protein kinase ERK3 is essential for establishment of epithelial architecture.

Science.gov (United States)

Takahashi, Chika; Miyatake, Koichi; Kusakabe, Morioh; Nishida, Eisuke

2018-06-01

Epithelia contribute to physical barriers that protect internal tissues from the external environment and also support organ structure. Accordingly, establishment and maintenance of epithelial architecture are essential for both embryonic development and adult physiology. Here, using gene knockout and knockdown techniques along with gene profiling, we show that extracellular signal-regulated kinase 3 (ERK3), a poorly characterized atypical mitogen-activated protein kinase (MAPK), regulates the epithelial architecture in vertebrates. We found that in Xenopus embryonic epidermal epithelia, ERK3 knockdown impairs adherens and tight-junction protein distribution, as well as tight-junction barrier function, resulting in epidermal breakdown. Moreover, in human epithelial breast cancer cells, inhibition of ERK3 expression induced thickened epithelia with aberrant adherens and tight junctions. Results from microarray analyses suggested that transcription factor AP-2α (TFAP2A), a transcriptional regulator important for epithelial gene expression, is involved in ERK3-dependent changes in gene expression. Of note, TFAP2A knockdown phenocopied ERK3 knockdown in both Xenopus embryos and human cells, and ERK3 was required for full activation of TFAP2A-dependent transcription. Our findings reveal that ERK3 regulates epithelial architecture, possibly together with TFAP2A. © 2018 by The American Society for Biochemistry and Molecular Biology, Inc.

Ibuprofen abates cypermethrin-induced expression of pro-inflammatory mediators and mitogen-activated protein kinases and averts the nigrostriatal dopaminergic neurodegeneration.

Science.gov (United States)

Singh, Ashish; Tripathi, Pratibha; Prakash, Om; Singh, Mahendra Pratap

2016-12-01

Cypermethrin induces oxidative stress, microglial activation, inflammation and apoptosis leading to Parkinsonism in rats. While ibuprofen, a non-steroidal anti-inflammatory drug, relieves from inflammation, its efficacy against cypermethrin-induced Parkinsonism has not yet been investigated. The study aimed to explore the protective role of ibuprofen in cypermethrin-induced Parkinsonism, an environmentally relevant model of Parkinson's disease (PD), along with its underlying mechanism. Animals were treated with/without cypermethrin in the presence/absence of ibuprofen. Behavioural, immunohistochemical and biochemical parameters of Parkinsonism and expression of pro-inflammatory and pro-apoptotic proteins along with mitogen-activated protein kinases (MAPKs) were determined. Ibuprofen resisted cypermethrin-induced behavioural impairments, striatal dopamine depletion, oxidative stress in the nigrostriatal tissues and loss of the nigral dopamine producing cells and increase in microglial activation along with atypical expression of pro-inflammatory and apoptotic proteins that include cyclooxygenase-2, tumour necrosis factor-α, MAPKs (c-Jun N-terminal kinase, p38 and extracellular signal-regulated kinase), B cell lymphoma 2-associated protein X, tumour suppressor protein p53, cytochrome c and caspase-3 in the nigrostriatal tissue. The results obtained thus demonstrate that ibuprofen lessens inflammation and regulates MAPKs expression thereby averts cypermethrin-induced Parkinsonism.

Glucose, other secretagogues, and nerve growth factor stimulate mitogen-activated protein kinase in the insulin-secreting beta-cell line, INS-1

DEFF Research Database (Denmark)

Frödin, M; Sekine, N; Roche, E

1995-01-01

The signaling pathways whereby glucose and hormonal secretagogues regulate insulin-secretory function, gene transcription, and proliferation of pancreatic beta-cells are not well defined. We show that in the glucose-responsive beta-cell line INS-1, major secretagogue-stimulated signaling pathways...... converge to activate 44-kDa mitogen-activated protein (MAP) kinase. Thus, glucose-induced insulin secretion was found to be associated with a small stimulatory effect on 44-kDa MAP kinase, which was synergistically enhanced by increased levels of intracellular cAMP and by the hormonal secretagogues......-1. Phorbol ester, an activator of protein kinase C, stimulated 44-kDa MAP kinase by both Ca(2+)-dependent and -independent pathways. Nerve growth factor, independently of changes in cytosolic Ca2+, efficiently stimulated 44-kDa MAP kinase without causing insulin release, indicating that activation...

p38gamma and p38delta mitogen activated protein kinases (MAPKs, new stars in the MAPK galaxy

Directory of Open Access Journals (Sweden)

Alejandra eEscós

2016-04-01

Full Text Available The protein kinases p38γ and p38δ belong to the p38 mitogen-activated protein kinase (MAPK family. p38MAPK signalling controls many cellular processes and is one of the most conserved mechanisms in eukaryotes for the cellular response to environmental stress and inflammation. Although p38γ and p38δ are widely expressed, it is likely that they perform specific functions in different tissues. Their involvement in human pathologies such as inflammation-related diseases or cancer is starting to be uncovered. In this article we give a general overview and highlight recent advances made in defining the functions of p38γ and p38δ, focusing in innate immunity and inflammation. We consider the potential of the pharmacological targeting of MAPK pathways to treat autoimmune and inflammatory diseases and cancer

Activation of Extracellular Signal-Regulated Kinase but Not of p38 Mitogen-Activated Protein Kinase Pathways in Lymphocytes Requires Allosteric Activation of SOS

Science.gov (United States)

Jun, Jesse E.; Yang, Ming; Chen, Hang; Chakraborty, Arup K.

2013-01-01

Thymocytes convert graded T cell receptor (TCR) signals into positive selection or deletion, and activation of extracellular signal-related kinase (ERK), p38, and Jun N-terminal protein kinase (JNK) mitogen-activated protein kinases (MAPKs) has been postulated to play a discriminatory role. Two families of Ras guanine nucleotide exchange factors (RasGEFs), SOS and RasGRP, activate Ras and the downstream RAF-MEK-ERK pathway. The pathways leading to lymphocyte p38 and JNK activation are less well defined. We previously described how RasGRP alone induces analog Ras-ERK activation while SOS and RasGRP cooperate to establish bimodal ERK activation. Here we employed computational modeling and biochemical experiments with model cell lines and thymocytes to show that TCR-induced ERK activation grows exponentially in thymocytes and that a W729E allosteric pocket mutant, SOS1, can only reconstitute analog ERK signaling. In agreement with RasGRP allosterically priming SOS, exponential ERK activation is severely decreased by pharmacological or genetic perturbation of the phospholipase Cγ (PLCγ)-diacylglycerol-RasGRP1 pathway. In contrast, p38 activation is not sharply thresholded and requires high-level TCR signal input. Rac and p38 activation depends on SOS1 expression but not allosteric activation. Based on computational predictions and experiments exploring whether SOS functions as a RacGEF or adaptor in Rac-p38 activation, we established that the presence of SOS1, but not its enzymatic activity, is critical for p38 activation. PMID:23589333

p38 mitogen-activated protein kinase mediates IL-8 induction by the ribotoxin deoxynivalenol in human monocytes

International Nuclear Information System (INIS)

Islam, Zahidul; Gray, Jennifer S.; Pestka, James J.

2006-01-01

The effects of the ribotoxic trichothecene deoxynivalenol (DON) on mitogen-activated protein kinase (MAPK)-mediated IL-8 expression were investigated in cloned human monocytes and peripheral blood mononuclear cells (PBMC). DON (250 to 1000 ng/ml) induced both IL-8 mRNA and IL-8 heteronuclear RNA (hnRNA), an indicator of IL-8 transcription, in the human U937 monocytic cell line in a concentration-dependent manner. Expression of IL-8 hnRNA, mRNA and protein correlated with p38 phosphorylation and was completely abrogated by the p38 MAPK inhibitor SB203580. DON at 500 ng/ml similarly induced p38-dependent IL-8 protein and mRNA expression in PBMC cultures from healthy volunteers. Significantly increased IL-6 and IL-1β intracellular protein and mRNA expression was also observed in PBMC treated with DON (500 ng/ml) which were also partially p38-dependent. Flow cytometry of PBMC revealed that DON-induced p38 phosphorylation varied among individuals relative to both threshold toxin concentrations (25-100 ng/ml) and relative increases in percentages of phospho-p38 + cells. DON-induced p38 activation occurred exclusively in the CD14 + monocyte population. DON was devoid of agonist activity for human Toll-like receptors 2, 3, 4, 5, 7, 8 and 9. However, two other ribotoxins, emetine and anisomycin, induced p38 phosphorylation in PBMC similarly to DON. Taken together, these data suggest that (1) p38 activation was required for induction of IL-8 and proinflammatory gene expression in the monocyte and (2) DON induced p38 activation in human monocytes via the ribotoxic stress response

Mitogen-activated protein kinase signaling in plants

DEFF Research Database (Denmark)

Rodriguez, Maria Cristina Suarez; Petersen, Morten; Mundy, John

2010-01-01

crossinhibition, feedback control, and scaffolding. Plant MAPK cascades regulate numerous processes, including stress and hormonal responses, innate immunity, and developmental programs. Genetic analyses have uncovered several predominant MAPK components shared by several of these processes including...... of substrate proteins, whose altered activities mediate a wide array of responses, including changes in gene expression. Cascades may share kinase components, but their signaling specificity is maintained by spaciotemporal constraints and dynamic protein-protein interactions and by mechanisms that include...

Adult-onset hyperthyroidism impairs spatial learning: possible involvement of mitogen-activated protein kinase signaling pathways.

Science.gov (United States)

Bitiktaş, Soner; Kandemir, Başak; Tan, Burak; Kavraal, Şehrazat; Liman, Narin; Dursun, Nurcan; Dönmez-Altuntaş, Hamiyet; Aksan-Kurnaz, Işil; Suer, Cem

2016-08-03

Given evidence that mitogen-activated protein kinase (MAPK) activation is part of the nongenomic actions of thyroid hormones, we investigated the possible consequences of hyperthyroidism for the cognitive functioning of adult rats. Young adult rats were treated with L-thyroxine or saline. Twenty rats in each group were exposed to Morris water maze testing, measuring their performance in a hidden-platform spatial task. In a separate set of rats not exposed to Morris water maze testing (untrained rats), the expression and phosphorylated levels of p38-MAPK and of its two downstream effectors, Elk-1 and cAMP response element-binding protein, were evaluated using quantitative reverse transcriptase-PCR and western blotting. Rats with hyperthyroidism showed delayed acquisition of learning compared with their wild-type counterparts, as shown by increased escape latencies and distance moved on the last two trials of daily training in the water maze. The hyperthyroid rats, however, showed no difference during probe trials. Western blot analyses of the hippocampus showed that hyperthyroidism increased phosphorylated p38-MAPK levels in untrained rats. Although our study is correlative in nature and does not exclude the contribution of other molecular targets, our findings suggest that the observed impairments in acquisition during actual learning in rats with hyperthyroidism may result from the increased phosphorylation of p38-MAPK.

The Role of Unfolded Protein Response and Mitogen-Activated Protein Kinase Signaling in Neurodegenerative Diseases with Special Focus on Prion Diseases

Directory of Open Access Journals (Sweden)

Lifeng Yang

2017-05-01

Full Text Available Prion diseases are neurodegenerative pathologies characterized by the accumulation of a protease-resistant form of the cellular prion protein named prion protein scrapie (PrPSc in the brain. PrPSc accumulation in the endoplasmic reticulum (ER result in a dysregulated calcium (Ca2+ homeostasis and subsequent initiation of unfolded protein response (UPR leading to neuronal dysfunction and apoptosis. The molecular mechanisms for the transition between adaptation to ER stress and ER stress-induced apoptosis are still unclear. Mitogen-activated protein kinases (MAPKs are serine/threonine protein kinases that rule the signaling of many extracellular stimuli from plasma membrane to the nucleus. However the identification of numerous points of cross talk between the UPR and MAPK signaling pathways may contribute to our understanding of the consequences of ER stress in prion diseases. Indeed the MAPK signaling network is known to regulate cell cycle progression and cell survival or death responses following a variety of stresses including misfolded protein response stress. In this article, we review the UPR signaling in prion diseases and discuss the triad of MAPK signaling pathways. We also describe the role played by MAPK signaling cascades in Alzheimer’s (AD and Parkinson’s disease (PD. We will also overview the mechanisms of cell death and the role of MAPK signaling in prion disease progression and highlight potential avenues for therapeutic intervention.

Mitogen-activated protein kinase (MAPK) dynamics determine cell fate in the yeast mating response.

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Li, Yang; Roberts, Julie; AkhavanAghdam, Zohreh; Hao, Nan

2017-12-15

In the yeast Saccharomyces cerevisiae , the exposure to mating pheromone activates a prototypic mitogen-activated protein kinase (MAPK) cascade and triggers a dose-dependent differentiation response. Whereas a high pheromone dose induces growth arrest and formation of a shmoo-like morphology in yeast cells, lower pheromone doses elicit elongated cell growth. Previous population-level analysis has revealed that the MAPK Fus3 plays an important role in mediating this differentiation switch. To further investigate how Fus3 controls the fate decision process at the single-cell level, we developed a specific translocation-based reporter for monitoring Fus3 activity in individual live cells. Using this reporter, we observed strikingly different dynamic patterns of Fus3 activation in single cells differentiated into distinct fates. Cells committed to growth arrest and shmoo formation exhibited sustained Fus3 activation. In contrast, most cells undergoing elongated growth showed either a delayed gradual increase or pulsatile dynamics of Fus3 activity. Furthermore, we found that chemically perturbing Fus3 dynamics with a specific inhibitor could effectively redirect the mating differentiation, confirming the causative role of Fus3 dynamics in driving cell fate decisions. MAPKs mediate proliferation and differentiation signals in mammals and are therapeutic targets in many cancers. Our results highlight the importance of MAPK dynamics in regulating single-cell responses and open up the possibility that MAPK signaling dynamics could be a pharmacological target in therapeutic interventions. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

Mercuric ions inhibit mitogen-activated protein kinase dephosphorylation by inducing reactive oxygen species

International Nuclear Information System (INIS)

Haase, Hajo; Engelhardt, Gabriela; Hebel, Silke; Rink, Lothar

2011-01-01

Mercury intoxication profoundly affects the immune system, in particular, signal transduction of immune cells. However, the mechanism of the interaction of mercury with cellular signaling pathways, such as mitogen activated protein kinases (MAPK), remains elusive. Therefore, the objective of this study is to investigate three potential ways in which Hg 2+ ions could inhibit MAPK dephosphorylation in the human T-cell line Jurkat: (1) by direct binding to phosphatases; (2) by releasing cellular zinc (Zn 2+ ); and (3) by inducing reactive oxygen species (ROS). Hg 2+ causes production of ROS, measured by dihydrorhodamine 123, and triggers ROS-mediated Zn 2+ release, detected with FluoZin-3. Yet, phosphatase-inhibition is not mediated by binding of Zn 2+ or Hg 2+ . Rather, phosphatases are inactivated by at least two forms of thiol oxidation; initial inhibition is reversible with reducing agents such as Tris(2-carboxyethyl)phosphine. Prolonged inhibition leads to non-reversible phosphatase oxidation, presumably oxidizing the cysteine thiol to sulfinic- or sulfonic acid. Notably, phosphatases are a particularly sensitive target for Hg 2+ -induced oxidation, because phosphatase activity is inhibited at concentrations of Hg 2+ that have only minor impact on over all thiol oxidation. This phosphatase inhibition results in augmented, ROS-dependent MAPK phosphorylation. MAPK are important regulators of T-cell function, and MAPK-activation by inhibition of phosphatases seems to be one of the molecular mechanisms by which mercury affects the immune system.

Involvement of p38 mitogen-activated protein kinase in acquired gemcitabine-resistant human urothelial carcinoma sublines

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Yu-Ting Kao

2014-07-01

Full Text Available Resistance to chemotherapeutic drugs is one of the major challenges in the treatment of cancer. A better understanding of how resistance arises and what molecular alterations correlate with resistance is the key to developing novel effective therapeutic strategies. To investigate the underlying mechanisms of gemcitabine (Gem resistance and provide possible therapeutic options, three Gem-resistant urothelial carcinoma sublines were established (NG0.6, NG0.8, and NG1.0. These cells were cross-resistant to arabinofuranosyl cytidine and cisplatin, but sensitive to 5-fluorouracil. The resistant cells expressed lower values of [hENT1 × dCK/RRM1 × RRM2] mRNA ratio. Two adenosine triphosphate-binding cassette proteins ABCD1 as well as multidrug resistance protein 1 were elevated. Moreover, cyclin D1, cyclin-dependent kinases 2 and 4 were upregulated, whereas extracellular signal-regulated kinase 1/2 and p38 mitogen-activated protein kinase (MAPK activity were repressed significantly. Administration of p38 MAPK inhibitor significantly reduced the Gem sensitivity in NTUB1 cells, whereas that of an extracellular signal-regulated kinase MAPK inhibitor did not. Furthermore, the Gem-resistant sublines also exhibited higher migration ability. Forced expression of p38 MAPK impaired the cell migration activity and augmented Gem sensitivity in NG1.0 cells. Taken together, these results demonstrate that complex mechanisms were merged in acquiring Gem resistance and provide information that can be important for developing therapeutic targets for treating Gem-resistant tumors.

Excessive L-cysteine induces vacuole-like cell death by activating endoplasmic reticulum stress and mitogen-activated protein kinase signaling in intestinal porcine epithelial cells.

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Ji, Yun; Wu, Zhenlong; Dai, Zhaolai; Sun, Kaiji; Zhang, Qing; Wu, Guoyao

2016-01-01

High intake of dietary cysteine is extremely toxic to animals and the underlying mechanism remains largely unknown. This study was conducted to test the hypothesis that excessive L-cysteine induces cell death by activating endoplasmic reticulum (ER) stress and mitogen-activated protein kinase (MAPK) signaling in intestinal porcine epithelial cells. Jejunal enterocytes were cultured in the presence of 0-10 mmol/L L-cysteine. Cell viability, morphologic alterations, mRNA levels for genes involved in ER stress, protein abundances for glucose-regulated protein 78, C/EBP homologous protein (CHOP), alpha subunit of eukaryotic initiation factor-2 (eIF2α), extracellular signal-regulated kinase (ERK1/2), p38 MAPK, and c-Jun N-terminal protein kinase (JNK1/2) were determined. The results showed that L-cysteine (5-10 mmol/L) reduced cell viability (P L-cysteine were not affected by the autophagy inhibitor 3-methyladenine. The protein abundances for CHOP, phosphorylated (p)-eIF2α, p-JNK1/2, p-p38 MAPK, and the spliced form of XBP-1 mRNA were enhanced (P L-cysteine induces vacuole-like cell death via the activation of ER stress and MAPK signaling in small intestinal epithelial cells. These signaling pathways may be potential targets for developing effective strategies to prevent the toxicity of dietary cysteine.

p38 mitogen-activated protein kinase plays a key role in regulating MAPKAPK2 expression

International Nuclear Information System (INIS)

Sudo, Tatsuhiko; Kawai, Kayoko; Matsuzaki, Hiroshi; Osada, Hiroyuki

2005-01-01

One of three major families of the mitogen-activated kinases (MAPK), p38 as well as JNK, has been shown to transduce extracellular stress stimuli into cellular responses by phospho-relay cascades. Among p38 families, p38α is a widely characterized isoform and the biological phenomena are explained by its kinase activity regulating functions of its downstream substrates. However, its specific contributions to each phenomenon are yet not fully elucidated. For better understanding of the role of MAPKs, especially p38α, we utilized newly established mouse fibroblast cell lines originated from a p38α null mouse, namely, a parental cell line without p38α gene locus, knockout of p38α (KOP), Zeosin-resistant (ZKOP), revertant of p38α (RKOP), and Exip revertant (EKOP). EKOP is smaller in size but grows faster than the others. Although comparable amounts of ERK and JNK are expressed in each cell line, ERK is highly phosphorylated in EKOP even in normal culture conditions. Serum stimulation after serum starvation led to ERK phosphorylation in RKOP and ZKOP, but not in EKOP as much. On the contrary, relative phosphorylation level of JNK to total JNK in response to UV was low in RKOP. And its phosphorylation as well as total JNK is slightly lower in EKOP. RKOP is less sensitive to UV irradiation as judged by the survival rate. Stress response upon UV or sorbitol stimuli, leading to mitogen activate protein kinase activated kinase 2 (MAPKAPK2) phosphorylation, was only observed in RKOP. Further experiments reveal that MAPKAPK2 expression is largely suppressed in ZKOP and EKOP. Its expression was recovered by re-introduction of p38α. The loss of MAPKAPK2 expression accompanied by the defect of p38α is confirmed in an embryonic extract prepared from p38α null mice. These data demonstrate that p38 signal pathway is regulated not only by phosphorylation but also by modulation of the expression of its component. Together, we have established cell lines that can be used in

SMALL GRAIN 1, which encodes a mitogen-activated protein kinase kinase 4, influences grain size in rice.

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Duan, Penggen; Rao, Yuchun; Zeng, Dali; Yang, Yaolong; Xu, Ran; Zhang, Baolan; Dong, Guojun; Qian, Qian; Li, Yunhai

2014-02-01

Although grain size is one of the most important components of grain yield, little information is known about the mechanisms that determine final grain size in crops. Here we characterize rice small grain1 (smg1) mutants, which exhibit small and light grains, dense and erect panicles and comparatively slightly shorter plants. The short grain and panicle phenotypes of smg1 mutants are caused by a defect in cell proliferation. The smg1 mutations were identified, using a map-based cloning approach, in mitogen-activated protein kinase kinase 4 (OsMKK4). Relatively higher expression of OsMKK4/SMG1 was detected in younger organs than in older ones, consistent with its role in cell proliferation. Green fluorescent protein (GFP)-OsMKK4/SMG1 fusion proteins appear to be distributed ubiquitously in plant cells. Further results revealed that OsMKK4 influenced brassinosteroid (BR) responses and the expression of BR-related genes. Thus, our findings have identified OsMKK4 as a factor for grain size, and suggest a possible link between the MAPK pathways and BRs in grain growth. © 2013 The Authors The Plant Journal © 2013 John Wiley & Sons Ltd.

Mitogen-Activated Protein Kinase Kinase 3 Regulates Seed Dormancy in Barley.

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Nakamura, Shingo; Pourkheirandish, Mohammad; Morishige, Hiromi; Kubo, Yuta; Nakamura, Masako; Ichimura, Kazuya; Seo, Shigemi; Kanamori, Hiroyuki; Wu, Jianzhong; Ando, Tsuyu; Hensel, Goetz; Sameri, Mohammad; Stein, Nils; Sato, Kazuhiro; Matsumoto, Takashi; Yano, Masahiro; Komatsuda, Takao

2016-03-21

Seed dormancy has fundamental importance in plant survival and crop production; however, the mechanisms regulating dormancy remain unclear [1-3]. Seed dormancy levels generally decrease during domestication to ensure that crops successfully germinate in the field. However, reduction of seed dormancy can cause devastating losses in cereals like wheat (Triticum aestivum L.) and barley (Hordeum vulgare L.) due to pre-harvest sprouting, the germination of mature seed (grain) on the mother plant when rain occurs before harvest. Understanding the mechanisms of dormancy can facilitate breeding of crop varieties with the appropriate levels of seed dormancy [4-8]. Barley is a model crop [9, 10] and has two major seed dormancy quantitative trait loci (QTLs), SD1 and SD2, on chromosome 5H [11-19]. We detected a QTL designated Qsd2-AK at SD2 as the single major determinant explaining the difference in seed dormancy between the dormant cultivar "Azumamugi" (Az) and the non-dormant cultivar "Kanto Nakate Gold" (KNG). Using map-based cloning, we identified the causal gene for Qsd2-AK as Mitogen-activated Protein Kinase Kinase 3 (MKK3). The dormant Az allele of MKK3 is recessive; the N260T substitution in this allele decreases MKK3 kinase activity and appears to be causal for Qsd2-AK. The N260T substitution occurred in the immediate ancestor allele of the dormant allele, and the established dormant allele became prevalent in barley cultivars grown in East Asia, where the rainy season and harvest season often overlap. Our findings show fine-tuning of seed dormancy during domestication and provide key information for improving pre-harvest sprouting tolerance in barley and wheat. Copyright © 2016 Elsevier Ltd. All rights reserved.

Specific and differential activation of mitogen-activated protein kinase cascades by unfamiliar taste in the insular cortex of the behaving rat.

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Berman, D E; Hazvi, S; Rosenblum, K; Seger, R; Dudai, Y

1998-12-01

Rats were given to drink an unfamiliar taste solution under conditions that result in long-term memory of that taste. The insular cortex, which contains the taste cortex, was then removed and assayed for activation of mitogen-activated protein kinase (MAPK) cascades by using antibodies to the activated forms of various MAPKs. Extracellular responsive kinase 1-2 (ERK1-2) in the cortical homogenate was significantly activated within taste solution, without alteration in the total level of the ERK1-2 proteins. The activity subsided to basal levels within ERK1-2 was not activated when the taste was made familiar. The effect of the unfamiliar taste was specific to the insular cortex. Jun N-terminal kinase 1-2 (JNK1-2) was activated by drinking the taste but with a delayed time course, whereas the activity of Akt kinase and p38MAPK remained unchanged. Elk-1, a member of the ternary complex factor and an ERK/JNK downstream substrate, was activated with a time course similar to that of ERK1-2. Microinjection of a reversible inhibitor of MAPK/ERK kinase into the insular cortex shortly before exposure to the novel taste in a conditioned taste aversion training paradigm attenuated long-term taste aversion memory without significantly affecting short-term memory or the sensory, motor, and motivational faculties required to express long-term taste aversion memory. It was concluded that ERK and JNK are specifically and differentially activated in the insular cortex after exposure to a novel taste, and that this activation is required for consolidation of long-term taste memory.

Induction of glutathione synthesis in human hepatocytes by acute and chronic arsenic exposure: Differential roles of mitogen-activated protein kinases

International Nuclear Information System (INIS)

Hou, Yongyong; Wang, Yi; Wang, Huihui; Xu, Yuanyuan

2014-01-01

Highlights: • Arsenic exposure increased intracellular levels of glutathione. • Mitogen-activated protein kinases were involved in glutathione homeostasis. • ERK contributed to glutathione synthesis during acute arsenic exposure. • Glutathione synthesis was regulated by p38 at least in part independent of NRF2 during chronic arsenic exposure. - Abstract: Glutathione (GSH) is a vital component of antioxidant defense which protects cells from toxic insults. Previously we found intracellular GSH was involved in cell resistance against arsenic-induced cytotoxicity. However, molecular mechanisms of GSH homeostasis during arsenic exposure are largely undefined. Here, we investigated roles of mitogen-activated protein kinases (MAPKs) in GSH synthesis pathway with two arsenic exposure strategies by using Chang human hepatocytes. In one strategy, acute arsenic exposure (20 μM, 24 h) was applied, as MAPK signaling is generally considered to be transient. In the other one, chronic arsenic exposure (500 nM, 20 weeks) was applied, which mimicked the general human exposure to arsenic. We found that acute arsenic exposure activated extracellular signal-regulated 1/2 kinases (ERK1/2) and c-Jun N-terminal kinase (JNK) in parallel with increased transcription and nuclear translocation of factor-erythroid 2-related factor 2 (NRF2) and enhanced expression of γ-glutamyl cysteine ligase catalytic subunit (GCLC), resulting in elevated intracellular GSH levels. Specific ERK inhibitor abolished arsenic-induced NRF2 nuclear translocation and GSH synthesis. During chronic arsenic exposure which induced a malignant cellular phenotype, continuous p38 activation and NRF2 nuclear translocation were observed with enhanced GSH synthesis. Specific p38 inhibitor attenuated arsenic-enhanced GSH synthesis without changing NRF2 nuclear translocation. Taken together, our results indicate MAPK pathways play an important role in cellular GSH homeostasis in response to arsenic. However, the

An Ime2-like mitogen-activated protein kinase is involved in cellulase expression in the filamentous fungus Trichoderma reesei.

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Chen, Fei; Chen, Xiu-Zhen; Su, Xiao-Yun; Qin, Li-Na; Huang, Zhen-Bang; Tao, Yong; Dong, Zhi-Yang

2015-10-01

Eukaryotic mitogen-activated protein kinases (MAPKs) play crucial roles in transducing environmental and developmental signals inside the cell and regulating gene expression, however, the roles of MAPKs remain largely unknown in Trichoderma reesei. T. reesei ime2 (TrIme2) encodes an Ime2-like MAPK in T. reesei. The deletion of the TrIme2 gene led to 90% increase in cellulase activity against filter paper during earlier period time of cellulase induction as well as the extracellular protein production. Compared to the parent strain, the transcriptional levels of the three major cellulase genes cbh1,cbh2, egl1 were increased by about 9 times, 4 times, 2 times, respectively, at 8 h after cellulase induction in the ΔTrIme2 mutant. In addition, the disruption of TrIme2 caused over 50% reduction of the transcript levels of cellulase transcriptional regulators cre1 and xyr1. TrIme2 functions in regulation of the expression of cellulase gene in T.reesei, and is a good candidate for genetically engineering of T. reesei for higher cellulase production.

Effects of Butyltins (BTs) on Mitogen-Activated-Protein Kinase Kinase Kinase (MAP3K) and Ras Activity in Human Natural Killer Cells

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Celada, Lindsay J.; Whalen, Margaret M.

2013-01-01

Butyltins (BTs) contaminate the environment and are found in human blood. BTs, tributyltin (TBT) and dibutyltin (DBT), diminish the cytotoxic function and levels of key proteins of human natural killer (NK) cells. NK cells are an initial immune defense against tumors, virally-infected cells and antibody-coated cells and thus critical to human health. The signaling pathways that regulate NK cell functions include mitogen-activated protein kinases (MAPKs). Studies have shown that exposure to BTs leads to the activation of specific MAPKs and MAPK kinases (MAP2Ks) in human NK cells. MAP2K kinases (MAP3Ks) are upstream activators of MAP2Ks, which then activate MAPKs. The current study examined if BT-induced activation of MAP3Ks was responsible for MAP2K and thus, MAPK activation. This study examines the effects of TBT and DBT on the total levels of two MAP3Ks, c-Raf and ASK1, as well as activating and inhibitory phosphorylation sites on these MAP3Ks. In addition, the immediate upstream activator of c-Raf, Ras, was examined for BT-induced alterations. Our results show significant activation of the MAP3K, c-Raf, in human NK cells within 10 minutes of TBT exposure and the MAP3K, ASK1, after one hour exposures to TBT. In addition, our results suggest that both TBT and DBT are impacting the regulation of c-Raf. PMID:24038145

A Role for Protein Phosphatase 2A in Regulating p38 Mitogen Activated Protein Kinase Activation and Tumor Necrosis Factor-Alpha Expression during Influenza Virus Infection

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Anna H. Y. Law

2013-04-01

Full Text Available Influenza viruses of avian origin continue to pose pandemic threats to human health. Some of the H5N1 and H9N2 virus subtypes induce markedly elevated cytokine levels when compared with the seasonal H1N1 virus. We previously showed that H5N1/97 hyperinduces tumor necrosis factor (TNF-alpha through p38 mitogen activated protein kinase (MAPK. However, the detailed mechanisms of p38MAPK activation and TNF-alpha hyperinduction following influenza virus infections are not known. Negative feedback regulations of cytokine expression play important roles in avoiding overwhelming production of proinflammatory cytokines. Here we hypothesize that protein phosphatases are involved in the regulation of cytokine expressions during influenza virus infection. We investigated the roles of protein phosphatases including MAPK phosphatase-1 (MKP-1 and protein phosphatase type 2A (PP2A in modulating p38MAPK activation and downstream TNF-alpha expressions in primary human monocyte-derived macrophages (PBMac infected with H9N2/G1 or H1N1 influenza virus. We demonstrate that H9N2/G1 virus activated p38MAPK and hyperinduced TNF-alpha production in PBMac when compared with H1N1 virus. H9N2/G1 induced PP2A activity in PBMac and, with the treatment of a PP2A inhibitor, p38MAPK phosphorylation and TNF-alpha production were further increased in the virus-infected macrophages. However, H9N2/G1 did not induce the expression of PP2A indicating that the activation of PP2A is not mediated by p38MAPK in virus-infected PBMac. On the other hand, PP2A may not be the targets of H9N2/G1 in the upstream of p38MAPK signaling pathways since H1N1 also induced PP2A activation in primary macrophages. Our results may provide new insights into the control of cytokine dysregulation.

TRAF6 promotes myogenic differentiation via the TAK1/p38 mitogen-activated protein kinase and Akt pathways.

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Fang Xiao

Full Text Available p38 mitogen-activated protein kinase (MAPK is an essential kinase involved in myogenic differentiation. Although many substrates of p38 MAPK have been identified, little is known about its upstream activators during myogenic differentiation. TRAF6 is known to function in cytokine signaling during inflammatory responses. However, not much is known about its role in myogenic differentiation and muscle regeneration. We showed here that TRAF6 and its intrinsic ubiquitin E3 ligase activity are required for myogenic differentiation. In mouse myoblasts, knockdown of TRAF6 compromised the p38 MAPK and Akt pathways, while deliberate activation of either pathway rescued the differentiation defect caused by TRAF6 knockdown. TAK1 acted as a key signal transducer downstream of TRAF6 in myogenic differentiation. In vivo, knockdown of TRAF6 in mouse muscles compromised the injury-induced muscle regeneration without impairing macrophage infiltration and myoblast proliferation. Collectively, we demonstrated that TRAF6 promotes myogenic differentiation and muscle regeneration via the TAK1/p38 MAPK and Akt pathways.

An inhibition of p38 mitogen activated protein kinase delays the platelet storage lesion.

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Andrey Skripchenko

Full Text Available BACKGROUND AND OBJECTIVES: Platelets during storage undergo diverse alterations collectively known as the platelet storage lesion, including metabolic, morphological, functional and structural changes. Some changes correlate with activation of p38 mitogen activated protein kinase (p38 MAPK. Another MAPK, extracellular signal-related kinase (ERK, is involved in PLT activation. The aim of this study was to compare the properties of platelets stored in plasma in the presence or absence of p38 and ERK MAPK inhibitors. MATERIALS AND METHODS: A single Trima apheresis platelet unit (n = 12 was aliquoted into five CLX storage bags. Two aliquots were continuously agitated with or without MAPK inhibitors. Two aliquots were subjected to 48 hours of interruption of agitation with or without MAPK inhibitors. One aliquot contained the same amount of solvent vehicle used to deliver the inhibitor. Platelets were stored at 20-24°C for 7 days and sampled on Days 1, 4, and 7 for 18 in vitro parameters. RESULTS: Inhibition of p38 MAPK by VX-702 leads to better maintenance of all platelet in vitro storage parameters including platelet mitochondrial function. Accelerated by interruption of agitation, the platelet storage lesion of units stored with VX-702 was diminished to that of platelets stored with continuous agitation. Inhibition of ERK MAPK did not ameliorate decrements in any in vitro platelet properties. CONCLUSION: Signaling through p38 MAPK, but not ERK, is associated with platelet deterioration during storage.

The F-box protein Fbp1 functions in the invasive growth and cell wall integrity mitogen-activated protein kinase (MAPK) pathways in Fusarium oxysporum.

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Miguel-Rojas, Cristina; Hera, Concepcion

2016-01-01

F-box proteins determine substrate specificity of the ubiquitin-proteasome system. Previous work has demonstrated that the F-box protein Fbp1, a component of the SCF(Fbp1) E3 ligase complex, is essential for invasive growth and virulence of the fungal plant pathogen Fusarium oxysporum. Here, we show that, in addition to invasive growth, Fbp1 also contributes to vegetative hyphal fusion and fungal adhesion to tomato roots. All of these functions have been shown previously to require the mitogen-activated protein kinase (MAPK) Fmk1. We found that Fbp1 is required for full phosphorylation of Fmk1, indicating that Fbp1 regulates virulence and invasive growth via the Fmk1 pathway. Moreover, the Δfbp1 mutant is hypersensitive to sodium dodecylsulfate (SDS) and calcofluor white (CFW) and shows reduced phosphorylation levels of the cell wall integrity MAPK Mpk1 after SDS treatment. Collectively, these results suggest that Fbp1 contributes to both the invasive growth and cell wall integrity MAPK pathways of F. oxysporum. © 2015 BSPP AND JOHN WILEY & SONS LTD.

p75 Neurotrophin Receptor Signaling Activates Sterol Regulatory Element-binding Protein-2 in Hepatocyte Cells via p38 Mitogen-activated Protein Kinase and Caspase-3.

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Pham, Dan Duc; Do, Hai Thi; Bruelle, Céline; Kukkonen, Jyrki P; Eriksson, Ove; Mogollón, Isabel; Korhonen, Laura T; Arumäe, Urmas; Lindholm, Dan

2016-05-13

Nerve growth factor (NGF) influences the survival and differentiation of a specific population of neurons during development, but its role in non-neuronal cells has been less studied. We observed here that NGF and its pro-form, pro-NGF, are elevated in fatty livers from leptin-deficient mice compared with controls, concomitant with an increase in low density lipoprotein receptors (LDLRs). Stimulation of mouse primary hepatocytes with NGF or pro-NGF increased LDLR expression through the p75 neurotrophin receptor (p75NTR). Studies using Huh7 human hepatocyte cells showed that the neurotrophins activate the sterol regulatory element-binding protein-2 (SREBP2) that regulates genes involved in lipid metabolism. The mechanisms for this were related to stimulation of p38 mitogen-activated protein kinase (p38 MAPK) and activation of caspase-3 and SREBP2 cleavage following NGF and pro-NGF stimulations. Cell fractionation experiments showed that caspase-3 activity was increased particularly in the membrane fraction that harbors SREBP2 and caspase-2. Experiments showed further that caspase-2 interacts with pro-caspase-3 and that p38 MAPK reduced this interaction and caused caspase-3 activation. Because of the increased caspase-3 activity, the cells did not undergo cell death following p75NTR stimulation, possibly due to concomitant activation of nuclear factor-κB (NF-κB) pathway by the neurotrophins. These results identify a novel signaling pathway triggered by ligand-activated p75NTR that via p38 MAPK and caspase-3 mediate the activation of SREBP2. This pathway may regulate LDLRs and lipid uptake particularly after injury or during tissue inflammation accompanied by an increased production of growth factors, including NGF and pro-NGF. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

Uncaria rhynchophylla inhibits the production of nitric oxide and interleukin-1β through blocking nuclear factor κB, Akt, and mitogen-activated protein kinase activation in macrophages.

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Kim, Ji-Hee; Bae, Chang Hwan; Park, Sun Young; Lee, Sang Joon; Kim, YoungHee

2010-10-01

The stems with hook of Uncaria rhynchophylla have been used in traditional medicine as an antipyretic, antihypertensive, and anticonvulsant in China and Korea. In this study, we investigated the mechanism responsible for anti-inflammatory effects of U. rhynchophylla in RAW 264.7 macrophages. The aqueous extract of U. rhynchophylla inhibited lipopolysaccharide (LPS)-induced nitric oxide (NO) and interleukin (IL)-1β secretion as well as inducible NO synthase (iNOS) expression, without affecting cell viability. Furthermore, U. rhynchophylla suppressed LPS-induced nuclear factor κB (NF-κB) activation, phosphorylation, and degradation of inhibitory protein IκB (IκB)-α, phosphorylation of Akt, extracellular signal-regulated kinase 1/2, p38 kinase, and c-Jun N-terminal kinase. These results suggest that U. rhynchophylla has the inhibitory effects on LPS-induced NO and IL-1β production in macrophages through blockade in the phosphorylation of Akt and mitogen-activated protein kinases, following IκB-α degradation and NF-κB activation.

Nitric oxide mediates the indole acetic acid induction activation of a mitogen-activated protein kinase cascade involved in adventitious root development.

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Pagnussat, Gabriela Carolina; Lanteri, María Luciana; Lombardo, María Cristina; Lamattina, Lorenzo

2004-05-01

Recently, it was demonstrated that nitric oxide (NO) and cGMP are involved in the auxin response during the adventitious rooting process in cucumber (Cucumis sativus; Pagnussat et al., 2002, 2003). However, not much is known about the complex molecular network operating during the cell proliferation and morphogenesis triggered by auxins and NO in that process. Anatomical studies showed that formation of adventitious root primordia was clearly detected in indole acetic acid (IAA)- and NO-treated cucumber explants, while neither cell proliferation nor differentiation into root primordia could be observed in control explants 3 d after primary root was removed. In order to go further with signal transduction mechanisms that operate during IAA- and NO-induced adventitious root formation, experiments were designed to test the involvement of a mitogen-activated protein kinase (MAPK) cascade in that process. Cucumber explants were treated with the NO-donor sodium nitroprusside (SNP) or with SNP plus the specific NO-scavenger cPTIO. Protein extracts from those explants were assayed for protein kinase (PK) activity by using myelin basic protein (MBP) as substrate in both in vitro and in-gel assays. The activation of a PK of approximately 48 kD could be detected 1 d after NO treatment with a maximal activation after 3 d of treatment. In control explants, a PK activity was detected only after 4 d of treatment. The MBP-kinase activity was also detected in extracts from IAA-treated explants, while no signal was observed in IAA + cPTIO treatments. The PK activity could be inhibited by the cell-permeable MAPK kinase inhibitor PD098059, suggesting that the NO-dependent MBP-kinase activity is a MAPK. Furthermore, when PD098059 was administered to explants treated with SNP or IAA, it produced a delay in root emergence and a dose-dependent reduction in root number. Altogether, our results suggest that a MAPK signaling cascade is activated during the adventitious rooting process

Effects of phorbol ester on mitogen-activated protein kinase kinase activity in wild-type and phorbol ester-resistant EL4 thymoma cells.

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Gause, K C; Homma, M K; Licciardi, K A; Seger, R; Ahn, N G; Peterson, M J; Krebs, E G; Meier, K E

1993-08-05

Phorbol ester-sensitive and -resistant EL4 thymoma cell lines differ in their ability to activate mitogen-activated protein kinase (MAPK) in response to phorbol ester. Treatment of wild-type EL4 cells with phorbol ester results in the rapid activations of MAPK and pp90rsk kinase, a substrate for MAPK, while neither kinase is activated in response to phorbol ester in variant EL4 cells. This study examines the activation of MAPK kinase (MAPKK), an activator of MAPK, in wild-type and variant EL4 cells. Phosphorylation of a 40-kDa substrate, identified as MAPK, was observed following in vitro phosphorylation reactions using cytosolic extracts or Mono Q column fractions prepared from phorbol ester-treated wild-type EL4 cells. MAPKK activity coeluted with a portion of the inactive MAPK upon Mono Q anion-exchange chromatography, permitting detection of the MAPKK activity in fractions containing both kinases. This MAPKK activity was present in phorbol ester-treated wild-type cells, but not in phorbol ester-treated variant cells or in untreated wild-type or variant cells. The MAPKK from wild-type cells was able to activate MAPK prepared from either wild-type or variant cells. MAPKK activity could be stimulated in both wildtype and variant EL4 cells in response to treatment of cells with okadaic acid. These results indicate that the failure of variant EL4 cells to activate MAP kinase in response to phorbol ester is due to a failure to activate MAPKK. Therefore, the step that confers phorbol ester resistance to variant EL4 cells lies between the activation of protein kinase C and the activation of MAPKK.

Constitutive Activation of the Fission Yeast Pheromone-Responsive Pathway Induces Ectopic Meiosis and Reveals Ste11 as a Mitogen-Activated Protein Kinase Target

DEFF Research Database (Denmark)

Kjærulff, Søren; Lautrup-Larsen, I.; Truelsen, S.

2005-01-01

In the fission yeast Schizosaccharomyces pombe, meiosis normally takes place in diploid zygotes resulting from conjugation of haploid cells. In the present study, we report that the expression of a constitutively activated version of the pheromone-responsive mitogen-activated protein kinase kinase...... found that haploid meiosis was dramatically reduced when Ste11 was mutated to mimic phosphorylation by Pat1. The mutation of two putative MAPK sites in Ste11 also dramatically reduced the level of haploid meiosis, suggesting that Ste11 is a direct target of Spk1. Supporting this, we show that Spk1 can...... interact physically with Ste11 and also phosphorylate the transcription factor in vitro. Finally, we demonstrate that ste11 is required for pheromone-induced G1 arrest. Interestingly, when we mutated Ste11 in the sites for Pat1 and Spk1 phosphorylation simultaneously, the cells could still arrest in G1...

The cis decoy against the estrogen response element suppresses breast cancer cells via target disrupting c-fos not mitogen-activated protein kinase activity.

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Wang, Li Hua; Yang, Xiao Yi; Zhang, Xiaohu; Mihalic, Kelly; Xiao, Weihua; Farrar, William L

2003-05-01

Breast cancer, the most common malignancy in women, has been demonstrated to be associated with the steroid hormone estrogen and its receptor (ER), a ligand-activated transcription factor. Therefore, we developed a phosphorothiolate cis-element decoy against the estrogen response element (ERE decoy) to target disruption of ER DNA binding and transcriptional activity. Here, we showed that the ERE decoy potently ablated the 17beta-estrogen-inducible cell proliferation and induced apoptosis of human breast carcinoma cells by functionally affecting expression of c-fos gene and AP-1 luciferase gene reporter activity. Specificity of the decoy was demonstrated by its ability to directly block ER binding to a cis-element probe and transactivation. Moreover, the decoy failed to inhibit ER-mediated mitogen-activated protein kinase signaling pathways and cell growth of ER-negative breast cancer cells. Taken together, these data suggest that estrogen-mediated cell growth of breast cancer cells can be preferentially restricted via targeted disruption of ER at the level of DNA binding by a novel and specific decoy strategy applied to steroid nuclear receptors.

Protein Kinase Mitogen-activated Protein Kinase Kinase Kinase Kinase 4 (MAP4K4) Promotes Obesity-induced Hyperinsulinemia.

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Roth Flach, Rachel J; Danai, Laura V; DiStefano, Marina T; Kelly, Mark; Menendez, Lorena Garcia; Jurczyk, Agata; Sharma, Rohit B; Jung, Dae Young; Kim, Jong Hun; Kim, Jason K; Bortell, Rita; Alonso, Laura C; Czech, Michael P

2016-07-29

Previous studies revealed a paradox whereby mitogen-activated protein kinase kinase kinase kinase 4 (Map4k4) acted as a negative regulator of insulin sensitivity in chronically obese mice, yet systemic deletion of Map4k4 did not improve glucose tolerance. Here, we report markedly reduced glucose-responsive plasma insulin and C-peptide levels in whole body Map4k4-depleted mice (M4K4 iKO) as well as an impaired first phase of insulin secretion from islets derived from M4K4 iKO mice ex vivo After long-term high fat diet (HFD), M4K4 iKO mice pancreata also displayed reduced β cell mass, fewer proliferating β cells and reduced islet-specific gene mRNA expression compared with controls, although insulin content was normal. Interestingly, the reduced plasma insulin in M4K4 iKO mice exposed to chronic (16 weeks) HFD was not observed in response to acute HFD challenge or short term treatment with the insulin receptor antagonist S961. Furthermore, the improved insulin sensitivity in obese M4K4 iKO mice was abrogated by high exogenous insulin over the course of a euglycemic clamp study, indicating that hypoinsulinemia promotes insulin sensitivity in chronically obese M4K4 iKO mice. These results demonstrate that protein kinase Map4k4 drives obesity-induced hyperinsulinemia and insulin resistance in part by promoting insulin secretion from β cells in mice. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

Mesothelioma Cells Escape Heat Stress by Upregulating Hsp40/Hsp70 Expression via Mitogen-Activated Protein Kinases

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Michael Roth

2009-01-01

Full Text Available Therapy with hyperthermal chemotherapy in pleural diffuse malignant mesothelioma had limited benefits for patients. Here we investigated the effect of heat stress on heat shock proteins (HSP, which rescue tumour cells from apoptosis. In human mesothelioma and mesothelial cells heat stress (39–42°C induced the phosphorylation of two mitogen activated kinases (MAPK Erk1/2 and p38, and increased Hsp40, and Hsp70 expression. Mesothelioma cells expressed more Hsp40 and were less sensitive to heat stress compared to mesothelial cells. Inhibition of Erk1/2 MAPK by PD98059 or by Erk1 siRNA down-regulated heat stress-induced Hsp40 and Hsp70 expression and reduced mesothelioma cell survival. Inhibition of p38MAPK by SB203580 or siRNA reduced Hsp40, but not Hsp70, expression and also increased mesothelioma cell death. Thus hyperthermia combined with suppression of p38 MAPK or Hsp40 may represent a novel approach to improve mesothelioma therapy.

Brominated flame retardants, tetrabromobisphenol A and hexabromocyclododecane, activate mitogen-activated protein kinases (MAPKs) in human natural killer cells.

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Cato, Anita; Celada, Lindsay; Kibakaya, Esther Caroline; Simmons, Nadia; Whalen, Margaret M

2014-12-01

Natural killer (NK) cells provide a vital surveillance against virally infected cells, tumor cells, and antibody-coated cells through the release of cytolytic mediators and gamma interferon (IFN-γ). Hexabromocyclododecane (HBCD) is a brominated flame retardant used primarily in expanded (EPS) and extruded (XPS) polystyrene foams for thermal insulation in the building and construction industry. Tetrabromobisphenol A (TBBPA) is used both as a reactive and an additive flame retardant in a variety of materials. HBCD and TBBPA contaminate the environment and are found in human blood samples. In previous studies, we have shown that other environmental contaminants, such as the dibutyltin (DBT) and tributyltin (TBT), decrease NK lytic function by activating mitogen-activated protein kinases (MAPKs) in the NK cells. HBCD and TBBPA also interfere with NK cell(s) lytic function. The current study evaluates whether HBCD and/or TBBPA have the capacity to activate MAPKs and MAPK kinases (MAP2Ks). The effects of concentrations of HBCD and TBBPA that inhibited lytic function on the phosphorylation state and total levels of the MAPKs (p44/42, p38, and JNK) and the phosphorylation and total levels of the MAP2Ks (MEK1/2 and MKK3/6) were examined. Results indicate that exposure of human NK cells to 10-0.5 μM HBCD or TBBPA activate MAPKs and MAP2Ks. This HBCD and TBBPA-induced activation of MAPKs may leave them unavailable for activation by virally infected or tumor target cells and thus contributes to the observed decreases in lytic function seen in NK cells exposed to HBCD and TBBPA.

Loss of mitogen-activated protein kinase kinase kinase 4 (MAP3K4 reveals a requirement for MAPK signalling in mouse sex determination.

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Debora Bogani

2009-09-01

Full Text Available Sex determination in mammals is controlled by the presence or absence of the Y-linked gene SRY. In the developing male (XY gonad, sex-determining region of the Y (SRY protein acts to up-regulate expression of the related gene, SOX9, a transcriptional regulator that in turn initiates a downstream pathway of testis development, whilst also suppressing ovary development. Despite the requirement for a number of transcription factors and secreted signalling molecules in sex determination, intracellular signalling components functioning in this process have not been defined. Here we report a role for the phylogenetically ancient mitogen-activated protein kinase (MAPK signalling pathway in mouse sex determination. Using a forward genetic screen, we identified the recessive boygirl (byg mutation. On the C57BL/6J background, embryos homozygous for byg exhibit consistent XY gonadal sex reversal. The byg mutation is an A to T transversion causing a premature stop codon in the gene encoding MAP3K4 (also known as MEKK4, a mitogen-activated protein kinase kinase kinase. Analysis of XY byg/byg gonads at 11.5 d post coitum reveals a growth deficit and a failure to support mesonephric cell migration, both early cellular processes normally associated with testis development. Expression analysis of mutant XY gonads at the same stage also reveals a dramatic reduction in Sox9 and, crucially, Sry at the transcript and protein levels. Moreover, we describe experiments showing the presence of activated MKK4, a direct target of MAP3K4, and activated p38 in the coelomic region of the XY gonad at 11.5 d post coitum, establishing a link between MAPK signalling in proliferating gonadal somatic cells and regulation of Sry expression. Finally, we provide evidence that haploinsufficiency for Map3k4 accounts for T-associated sex reversal (Tas. These data demonstrate that MAP3K4-dependent signalling events are required for normal expression of Sry during testis development, and

Loss of Mitogen-Activated Protein Kinase Kinase Kinase 4 (MAP3K4) Reveals a Requirement for MAPK Signalling in Mouse Sex Determination

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Bogani, Debora; Siggers, Pam; Brixey, Rachel; Warr, Nick; Beddow, Sarah; Edwards, Jessica; Williams, Debbie; Wilhelm, Dagmar; Koopman, Peter; Flavell, Richard A.; Chi, Hongbo; Ostrer, Harry; Wells, Sara; Cheeseman, Michael; Greenfield, Andy

2009-01-01

Sex determination in mammals is controlled by the presence or absence of the Y-linked gene SRY. In the developing male (XY) gonad, sex-determining region of the Y (SRY) protein acts to up-regulate expression of the related gene, SOX9, a transcriptional regulator that in turn initiates a downstream pathway of testis development, whilst also suppressing ovary development. Despite the requirement for a number of transcription factors and secreted signalling molecules in sex determination, intracellular signalling components functioning in this process have not been defined. Here we report a role for the phylogenetically ancient mitogen-activated protein kinase (MAPK) signalling pathway in mouse sex determination. Using a forward genetic screen, we identified the recessive boygirl (byg) mutation. On the C57BL/6J background, embryos homozygous for byg exhibit consistent XY gonadal sex reversal. The byg mutation is an A to T transversion causing a premature stop codon in the gene encoding MAP3K4 (also known as MEKK4), a mitogen-activated protein kinase kinase kinase. Analysis of XY byg/byg gonads at 11.5 d post coitum reveals a growth deficit and a failure to support mesonephric cell migration, both early cellular processes normally associated with testis development. Expression analysis of mutant XY gonads at the same stage also reveals a dramatic reduction in Sox9 and, crucially, Sry at the transcript and protein levels. Moreover, we describe experiments showing the presence of activated MKK4, a direct target of MAP3K4, and activated p38 in the coelomic region of the XY gonad at 11.5 d post coitum, establishing a link between MAPK signalling in proliferating gonadal somatic cells and regulation of Sry expression. Finally, we provide evidence that haploinsufficiency for Map3k4 accounts for T-associated sex reversal (Tas). These data demonstrate that MAP3K4-dependent signalling events are required for normal expression of Sry during testis development, and create a novel

The p38 mitogen-activated protein kinase signaling pathway is involved in regulating low-density lipoprotein receptor-related protein 1-mediated β-amyloid protein internalization in mouse brain.

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Ma, Kai-Ge; Lv, Jia; Hu, Xiao-Dan; Shi, Li-Li; Chang, Ke-Wei; Chen, Xin-Lin; Qian, Yi-Hua; Yang, Wei-Na; Qu, Qiu-Min

2016-07-01

Alzheimer's disease (AD) is one of the most common neurodegenerative diseases. Recently, increasing evidence suggests that intracellular β-amyloid protein (Aβ) alone plays a pivotal role in the progression of AD. Therefore, understanding the signaling pathway and proteins that control Aβ internalization may provide new insight for regulating Aβ levels. In the present study, the regulation of Aβ internalization by p38 mitogen-activated protein kinases (MAPK) through low-density lipoprotein receptor-related protein 1 (LRP1) was analyzed in vivo. The data derived from this investigation revealed that Aβ1-42 were internalized by neurons and astrocytes in mouse brain, and were largely deposited in mitochondria and lysosomes, with some also being found in the endoplasmic reticulum. Aβ1-42-LRP1 complex was formed during Aβ1-42 internalization, and the p38 MAPK signaling pathway was activated by Aβ1-42 via LRP1. Aβ1-42 and LRP1 were co- localized in the cells of parietal cortex and hippocampus. Furthermore, the level of LRP1-mRNA and LRP1 protein involved in Aβ1-42 internalization in mouse brain. The results of this investigation demonstrated that Aβ1-42 induced an LRP1-dependent pathway that related to the activation of p38 MAPK resulting in internalization of Aβ1-42. These results provide evidence supporting a key role for the p38 MAPK signaling pathway which is involved in the regulation of Aβ1-42 internalization in the parietal cortex and hippocampus of mouse through LRP1 in vivo. Copyright © 2016 Elsevier Ltd. All rights reserved.

Nodularin induces tumor necrosis factor-alpha and mitogen-activated protein kinases (MAPK) and leads to induction of endoplasmic reticulum stress

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Meili, Nicole; Christen, Verena [University of Applied Sciences and Arts Northwestern Switzerland (FHNW), Gründenstrasse 40, CH-4132 Muttenz (Switzerland); Fent, Karl, E-mail: karl.fent@fhnw.ch [University of Applied Sciences and Arts Northwestern Switzerland (FHNW), Gründenstrasse 40, CH-4132 Muttenz (Switzerland); Swiss Federal Institute of Technology Zürich (ETH Zürich), Department of Environmental Systems Science, CH-8092 Zürich (Switzerland)

2016-06-01

Nodularin is produced by the cyanobacterium Nodularia spumigena. It is of concern due to hepatotoxicity in humans and animals. Here we investigated unexplored molecular mechanisms by transcription analysis in human liver cells, focusing on induction of pro-inflammatory cytokines, the tumor necrosis factor α (TNF-α), endoplasmic reticulum (ER) stress and components of the activator protein-1 complex in human hepatoma cells (Huh7) exposed to non-cytotoxic (0.1 and 1 μM) and toxic concentrations (5 μM) for 24, 48, and 72 h. Transcripts of TNF-α and ER stress marker genes were strongly induced at 1 and 5 μM at all time-points. TNF-α led to induction of mitogen-activated protein kinases (MAPK), as demonstrated by induction of CJUN and CFOS, which form the AP-1 complex. Human primary liver cells reacted more sensitive than Huh7 cells. They showed higher cytotoxicity and induction of TNF-α and ER stress at 2.5 nM, while HepG2 cells were insensitive up to 10 μM due to low expression of organic anion transporting polypeptides. Furthermore, nodularin led to induction of TNF-α protein, and CCAAT/enhancer-binding protein-homologous (CHOP) protein. Our data indicate that nodularin induces inflammation and ER stress and leads to activation of MAPK in liver cells. All of these activated pathways, which were analysed here for the first time in detail, may contribute to the hepatotoxic, and tumorigenic action of nodularin. - Highlights: • Toxicity of nodularin and its mechanisms of action are poorly understood. • We investigated mechanisms of nodularin toxicity in human liver cell lines and human hepatocytes. • We identified several pathways involved in nodularin toxicity. • Nodularin induces TNF-α, MAPK pathway and ER stress • These activated pathways may contribute to the hepatotoxic and tumorigenic action of nodularin.

Nodularin induces tumor necrosis factor-alpha and mitogen-activated protein kinases (MAPK) and leads to induction of endoplasmic reticulum stress

International Nuclear Information System (INIS)

Meili, Nicole; Christen, Verena; Fent, Karl

2016-01-01

Nodularin is produced by the cyanobacterium Nodularia spumigena. It is of concern due to hepatotoxicity in humans and animals. Here we investigated unexplored molecular mechanisms by transcription analysis in human liver cells, focusing on induction of pro-inflammatory cytokines, the tumor necrosis factor α (TNF-α), endoplasmic reticulum (ER) stress and components of the activator protein-1 complex in human hepatoma cells (Huh7) exposed to non-cytotoxic (0.1 and 1 μM) and toxic concentrations (5 μM) for 24, 48, and 72 h. Transcripts of TNF-α and ER stress marker genes were strongly induced at 1 and 5 μM at all time-points. TNF-α led to induction of mitogen-activated protein kinases (MAPK), as demonstrated by induction of CJUN and CFOS, which form the AP-1 complex. Human primary liver cells reacted more sensitive than Huh7 cells. They showed higher cytotoxicity and induction of TNF-α and ER stress at 2.5 nM, while HepG2 cells were insensitive up to 10 μM due to low expression of organic anion transporting polypeptides. Furthermore, nodularin led to induction of TNF-α protein, and CCAAT/enhancer-binding protein-homologous (CHOP) protein. Our data indicate that nodularin induces inflammation and ER stress and leads to activation of MAPK in liver cells. All of these activated pathways, which were analysed here for the first time in detail, may contribute to the hepatotoxic, and tumorigenic action of nodularin. - Highlights: • Toxicity of nodularin and its mechanisms of action are poorly understood. • We investigated mechanisms of nodularin toxicity in human liver cell lines and human hepatocytes. • We identified several pathways involved in nodularin toxicity. • Nodularin induces TNF-α, MAPK pathway and ER stress • These activated pathways may contribute to the hepatotoxic and tumorigenic action of nodularin.

Riboflavin-Induced Disease Resistance Requires the Mitogen-Activated Protein Kinases 3 and 6 in Arabidopsis thaliana.

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Nie, Shengjun; Xu, Huilian

2016-01-01

As a resistance elicitor, riboflavin (vitamin B2) protects plants against a wide range of pathogens. At molecular biological levels, it is important to elucidate the signaling pathways underlying the disease resistance induced by riboflavin. Here, riboflavin was tested to induce resistance against virulent Pseudomonas syringae pv. Tomato DC3000 (Pst DC3000) in Arabidopsis. Results showed that riboflavin induced disease resistance based on MAPK-dependent priming for the expression of PR1 gene. Riboflavin induced transient expression of PR1 gene. However, following Pst DC3000 inoculation, riboflavin potentiated stronger PR1 gene transcription. Further was suggested that the transcript levels of mitogen-activated protein kinases, MPK3 and MPK6, were primed under riboflavin. Upon infection by Pst DC3000, these two enzymes were more strongly activated. The elevated activation of both MPK3 and MPK6 was responsible for enhanced defense gene expression and resistance after riboflavin treatment. Moreover, riboflavin significantly reduced the transcript levels of MPK3 and MPK6 by application of AsA and BAPTA, an H2O2 scavenger and a calcium (Ca2+) scavenger, respectively. In conclusion, MPK3 and MPK6 were responsible for riboflavin-induced resistance, and played an important role in H2O2- and Ca2+-related signaling pathways, and this study could provide a new insight into the mechanistic study of riboflavin-induced defense responses.

Genome-wide identification and expression analysis of the mitogen-activated protein kinase gene family in cassava

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Yan Yan

2016-08-01

Full Text Available Mitogen-activated protein kinases (MAPKs play central roles in plant developmental processes, hormone signaling transduction, and responses to abiotic stress. However, no data are currently available about the MAPK family in cassava, an important tropical crop. Herein, 21 MeMAPK genes were identified from cassava. Phylogenetic analysis indicated that MeMAPKs could be classified into four subfamilies. Gene structure analysis demonstrated that the number of introns in MeMAPK genes ranged from 1 to 10, suggesting large variation among cassava MAPK genes. Conserved motif analysis indicated that all MeMAPKs had typical protein kinase domains. Transcriptomic analysis suggested that MeMAPK genes showed differential expression patterns in distinct tissues and in response to drought stress between wild subspecies and cultivated varieties. Interaction networks and co-expression analyses revealed that crucial pathways controlled by MeMAPK networks may be involved in the differential response to drought stress in different accessions of cassava. Expression of nine selected MAPK genes showed that these genes could comprehensively respond to osmotic, salt, cold, oxidative stressors, and abscisic acid (ABA signaling. These findings yield new insights into the transcriptional control of MAPK gene expression, provide an improved understanding of abiotic stress responses and signaling transduction in cassava, and lead to potential applications in the genetic improvement of cassava cultivars.

Expression of the Grb2-related protein of the lymphoid system in B cell subsets enhances B cell antigen receptor signaling through mitogen-activated protein kinase pathways.

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Yankee, Thomas M; Solow, Sasha A; Draves, Kevin D; Clark, Edward A

2003-01-01

Adapter proteins play a critical role in regulating signals triggered by Ag receptor cross-linking. These small molecules link receptor proximal events with downstream signaling pathways. In this study, we explore the expression and function of the Grb2-related protein of the lymphoid system (GrpL)/Grb2-related adaptor downstream of Shc adapter protein in human B cells. GrpL is expressed in naive B cells and is down-regulated following B cell Ag receptor ligation. By contrast, germinal center and memory B cells express little or no GrpL. Using human B cell lines, we detected constitutive interactions between GrpL and B cell linker protein, Src homology (SH)2 domain-containing leukocyte protein of 76 kDa, hemopoietic progenitor kinase 1, and c-Cbl. The N-terminal SH3 domain of GrpL binds c-Cbl while the C-terminal SH3 domain binds B cell linker protein and SH2 domain-containing leukocyte protein of 76 kDa. Exogenous expression of GrpL in a GrpL-negative B cell line leads to enhanced Ag receptor-induced extracellular signal-related kinase and p38 mitogen-activated protein kinase phosphorylation. Thus, GrpL expression in human B cell subsets appears to regulate Ag receptor-mediated signaling events.

Parathyroid mitogenic activity in plasma from patients with familial multiple endocrine neoplasia type 1

International Nuclear Information System (INIS)

Brandi, M.L.; Aurbach, G.D.; Fitzpatrick, L.A.; Quarto, R.; Spiegel, A.M.; Bliziotes, M.M.; Norton, J.A.; Doppman, J.L.; Marx, S.J.

1986-01-01

Hyperplasia of the parathyroid glands is a central feature of familial multiple endocrine neoplasia type 1. We used cultured bovine parathyroid cells to test for mitogenic activity in plasma from patients with this disorder. Normal plasma stimulated [ 3 H]thymidine incorporation, on the average, to the same extent as it was stimulated in a plasma-free control culture. This contrasted with the results of the tests with plasma from patients with familial multiple endocrine neoplasia type 1, in which parathyroid mitogenic activity increased 2400 percent over the control value (P less than 0.001). Plasma from these patients also stimulated the proliferation of bovine parathyroid cells in culture, whereas plasma from normal subjects inhibited it. Parathyroid mitogenic activity in plasma from the patients with familial multiple endocrine neoplasia type 1 was greater than that in plasma from patients with various other disorders, including sporadic primary hyperparathyroidism (with adenoma, hyperplasia, or cancer of the parathyroid), sporadic primary hypergastrinemia, sporadic pituitary tumor, familial hypocalciuric hypercalcemia, and multiple endocrine neoplasia type 2 (P less than 0.05). Parathyroid mitogenic activity in the plasma of patients with familial multiple endocrine neoplasia type 1 persisted for up to four years after total parathyroidectomy. The plasma also had far more mitogenic activity in cultures of parathyroid cells than did optimal concentrations of known growth factors or of any parathyroid secretagogue. This mitogenic activity had an apparent molecular weight of 50,000 to 55,000. We conclude that primary hyperparathyroidism in familial multiple endocrine neoplasia type 1 may have a humoral cause

Inhibition of gap-junctional intercellular communication and activation of mitogen-activated protein kinases by cyanobacterial extracts--indications of novel tumor-promoting cyanotoxins?

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Bláha, Ludĕk; Babica, Pavel; Hilscherová, Klára; Upham, Brad L

2010-01-01

Toxicity and liver tumor promotion of cyanotoxins microcystins have been extensively studied. However, recent studies document that other metabolites present in the complex cyanobacterial water blooms may also have adverse health effects. In this study we used rat liver epithelial stem-like cells (WB-F344) to examine the effects of cyanobacterial extracts on two established markers of tumor promotion, inhibition of gap-junctional intercellular communication (GJIC) and activation of mitogen-activated protein kinases (MAPKs) - ERK1/2. Extracts of cyanobacteria (laboratory cultures of Microcystis aeruginosa and Aphanizomenon flos-aquae and water blooms dominated by these species) inhibited GJIC and activated MAPKs in a dose-dependent manner (effective concentrations ranging 0.5-5mgd.w./mL). Effects were independent of the microcystin content and the strongest responses were elicited by the extracts of Aphanizomenon sp. Neither pure microcystin-LR nor cylindrospermopsin inhibited GJIC or activated MAPKs. Modulations of GJIC and MAPKs appeared to be specific to cyanobacterial extracts since extracts from green alga Chlamydomonas reinhardtii, heterotrophic bacterium Klebsiella terrigena, and isolated bacterial lipopolysaccharides had no comparable effects. Our study provides the first evidence on the existence of unknown cyanobacterial toxic metabolites that affect in vitro biomarkers of tumor promotion, i.e. inhibition of GJIC and activation of MAPKs.

Modulation of mitogen-activated protein kinase-activated protein kinase 3 by hepatitis C virus core protein

DEFF Research Database (Denmark)

Ngo, HT; Pham, Long; Kim, JW

2013-01-01

Hepatitis C virus (HCV) is highly dependent on cellular proteins for its own propagation. In order to identify the cellular factors involved in HCV propagation, we performed protein microarray assays using the HCV core protein as a probe. Of ~9,000 host proteins immobilized in a microarray...... inducers. Binding of HCV core to MAPKAPK3 was confirmed by in vitro pulldown assay and further verified by coimmunoprecipitation assay. HCV core protein interacted with MAPKAPK3 through amino acid residues 41 to 75 of core and the N-terminal half of kinase domain of MAPKAPK3. In addition, both RNA...... increased HCV IRES-mediated translation and MAPKAPK3-dependent HCV IRES activity was further increased by core protein. These data suggest that HCV core may modulate MAPKAPK3 to facilitate its own propagation....

The yeast mitogen-activated protein kinase Slt2 is involved in the cellular response to genotoxic stress

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Soriano-Carot María

2012-02-01

Full Text Available Abstract Background The maintenance of genomic integrity is essential for cell viability. Complex signalling pathways (DNA integrity checkpoints mediate the response to genotoxic stresses. Identifying new functions involved in the cellular response to DNA-damage is crucial. The Saccharomyces cerevisiae SLT2 gene encodes a member of the mitogen-activated protein kinase (MAPK cascade whose main function is the maintenance of the cell wall integrity. However, different observations suggest that SLT2 may also have a role related to DNA metabolism. Results This work consisted in a comprehensive study to connect the Slt2 protein to genome integrity maintenance in response to genotoxic stresses. The slt2 mutant strain was hypersensitive to a variety of genotoxic treatments, including incubation with hydroxyurea (HU, methylmetanosulfonate (MMS, phleomycin or UV irradiation. Furthermore, Slt2 was activated by all these treatments, which suggests that Slt2 plays a central role in the cellular response to genotoxic stresses. Activation of Slt2 was not dependent on the DNA integrity checkpoint. For MMS and UV, Slt2 activation required progression through the cell cycle. In contrast, HU also activated Slt2 in nocodazol-arrested cells, which suggests that Slt2 may respond to dNTP pools alterations. However, neither the protein level of the distinct ribonucleotide reductase subunits nor the dNTP pools were affected in a slt2 mutant strain. An analysis of the checkpoint function revealed that Slt2 was not required for either cell cycle arrest or the activation of the Rad53 checkpoint kinase in response to DNA damage. However, slt2 mutant cells showed an elongated bud and partially impaired Swe1 degradation after replicative stress, indicating that Slt2 could contribute, in parallel with Rad53, to bud morphogenesis control after genotoxic stresses. Conclusions Slt2 is activated by several genotoxic treatments and is required to properly cope with DNA damage. Slt

p38 Mitogen Activated Protein Kinase (MAPK): A New Therapeutic Target for Reducing the Risk of Adverse Pregnancy Outcomes

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Menon, Ramkumar; Papaconstantinou, John

2016-01-01

Introduction Spontaneous preterm birth (PTB) and preterm premature rupture of the membranes (pPROM) remain as a major clinical and therapeutic problem for intervention and management. Current strategies, based on our knowledge of pathways of preterm labor, have only been effective, in part, due to major gaps in our existing knowledge of risks and risk specific pathways. Areas covered Recent literature has identified physiologic aging of fetal tissues as a potential mechanistic feature of normal parturition. This process is affected by telomere dependent and p38 mitogen activated protein kinase (MAPK) induced senescence activation. Pregnancy associated risk factors can cause pathologic activation of this pathway that can cause oxidative stress induced p38 MAPK activation leading to senescence and premature aging of fetal tissues. Premature aging is associated with sterile inflammation capable of triggering preterm labor or preterm premature rupture of membranes. Preterm activation of p38MAPK can be considered as a key contributor to adverse pregnancies. Expert Opinion This review considers p38MAPK activation as a potential target for therapeutic interventions to prevent adverse pregnancy outcomes mediated by stress factors. In this review, we propose multiple strategies to prevent p38MAPK activation and its functional effects. PMID:27459026

Fisetin Ameliorated Photodamage by Suppressing the Mitogen-Activated Protein Kinase/Matrix Metalloproteinase Pathway and Nuclear Factor-κB Pathways.

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Chiang, Hsiu-Mei; Chan, Shih-Yun; Chu, Yin; Wen, Kuo-Ching

2015-05-13

Ultraviolet (UV) irradiation is one of the most important extrinsic factors contributing to skin photodamage. After UV irradiation, a series of signal transductions in the skin will be activated, leading to inflammatory response and photoaged skin. In this study, fisetin, a flavonol that exists in fruits and vegetables, was investigated for its photoprotective effects. The results revealed that 5-25 μM fisetin inhibits cyclooxygenase-2 (COX-2) and matrix metalloproteinase (MMP)-1, MMP-3, MMP-9 expression induced by ultraviolet B (UVB) irradiation in human skin fibroblasts. In addition, fisetin suppressed UVB-induced collagen degradation. With regard to its effect on upper-stream signal transduction, we found that fisetin reduced the expression of ultraviolet (UV)-induced ERK, JNK, and p38 phosphorylation in the mitogen-activated protein kinase (MAP kinase) pathway. Furthermore, fisetin reduced inhibitor κB (IκB) degradation and increased the amount of p65, which is a major subunit of nuclear factor-κB (NF-κB), in cytoplasm. It also suppressed NF-κB translocated to the nucleus and inhibited cAMP response element-binding protein (CREB) Ser-133 phosphorylation level in the phosphoinositide 3-kinase/protein kinase B/CREB (PI3K/AKT/CREB) pathway. Finally, fisetin inhibited UV-induced intracellular reactive oxygen species (ROS), prostaglandin E2 (PGE2), and nitric oxide (NO) generation. The mentioned effects and mechanisms suggest that fisetin can be used in the development of photoprotective agents.

Genome-wide identification of mitogen-activated protein kinase gene family in Gossypium raimondii and the function of their corresponding orthologs in tetraploid cultivated cotton.

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Zhang, Xueying; Wang, Liman; Xu, Xiaoyang; Cai, Caiping; Guo, Wangzhen

2014-12-10

Mitogen-activated protein kinase (MAPK) cascades play a crucial role in plant growth and development as well as biotic and abiotic stress responses. Knowledge about the MAPK gene family in cotton is limited, and systematic investigation of MAPK family proteins has not been reported. By performing a bioinformatics homology search, we identified 28 putative MAPK genes in the Gossypium raimondii genome. These MAPK members were anchored onto 11 chromosomes in G. raimondii, with uneven distribution. Phylogenetic analysis showed that the MAPK candidates could be classified into the four known A, B, C and D groups, with more MAPKs containing the TEY phosphorylation site (18 members) than the TDY motif (10 members). Furthermore, 21 cDNA sequences of MAPKs with complete open reading frames (ORFs) were identified in G. hirsutum via PCR-based approaches, including 13 novel MAPKs and eight with homologs reported previously in tetraploid cotton. The expression patterns of 23 MAPK genes reveal their important roles in diverse functions in cotton, in both various developmental stages of vegetative and reproductive growth and in the stress response. Using a reverse genetics approach based on tobacco rattle virus-induced gene silencing (TRV-VIGS), we further verified that MPK9, MPK13 and MPK25 confer resistance to defoliating isolates of Verticillium dahliae in cotton. Silencing of MPK9, MPK13 and MPK25 can significantly enhance cotton susceptibility to this pathogen. This study presents a comprehensive identification of 28 mitogen-activated protein kinase genes in G. raimondii. Their phylogenetic relationships, transcript expression patterns and responses to various stressors were verified. This study provides the first systematic analysis of MAPKs in cotton, improving our understanding of defense responses in general and laying the foundation for future crop improvement using MAPKs.

Immunomodulatory Activity of Ganoderma atrum Polysaccharide on Purified T Lymphocytes through Ca2+/CaN and Mitogen-Activated Protein Kinase Pathway Based on RNA Sequencing.

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Xiang, Quan-Dan; Yu, Qiang; Wang, Hui; Zhao, Ming-Ming; Liu, Shi-Yu; Nie, Shao-Ping; Xie, Ming-Yong

2017-07-05

Our previous study has demonstrated that Ganoderma atrum polysaccharide (PSG-1) has immunomodulatory activity on spleen lymphocytes. However, how PSG-1 exerts its effect on purified lymphocytes is still obscure. Thus, this study aimed to investigate the immunomodulatory activity of PSG-1 on purified T lymphocytes and further elucidate the underlying mechanism based on RNA sequencing (RNA-seq). Our results showed that PSG-1 promoted T lymphocytes proliferation and increased the production of IL-2, IFN-γ, and IL-12. Meanwhile, RNA-seq analysis found 394 differentially expressed genes. KEGG pathway analysis identified 20 significant canonical pathways and seven biological functions. Furthermore, PSG-1 elevated intracellular Ca 2+ concentration and calcineurin (CaN) activity and raised the p-ERK, p-JNK, and p-p38 expression levels. T lymphocytes proliferation and the production of IL-2, IFN-γ, and IL-12 were decreased by the inhibitors of calcium channel and mitogen-activated protein kinases (MAPKs). These results indicated that PSG-1 possesses immunomodulatory activity on purified T lymphocytes, in which Ca 2+ /CaN and MAPK pathways play essential roles.

Moringa oleifera fruit induce apoptosis via reactive oxygen species-dependent activation of mitogen-activated protein kinases in human melanoma A2058 cells.

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Guon, Tae Eun; Chung, Ha Sook

2017-08-01

The present study was performed to determine the effect of Moringa oleifera fruit extract on the apoptosis of human melanoma A2058 cells. A2058 cells were treated for 72 h with Moringa oleifera fruit extract at 50-100 µg/ml, and cell viability with apoptotic changes was examined. The involvement of reactive oxygen species (ROS) and mitogen-activated protein kinases (MAPKs) was examined. It was revealed that Moringa oleifera fruit extract significantly inhibited the cell viability and promoted apoptosis of A2058 cells in a concentration-dependent manner. Moringa oleifera fruit extract-treated A2058 cells exhibited increased activities of cleaved caspase-9 and caspase-3. It also caused an enhancement of MAPK phosphorylation and ROS production. The pro-apoptotic activity of Moringa oleifera fruit extract was significantly reversed by pretreatment with the c-Jun N-terminal kinase (JNK) inhibitor SP600125, extracellular-signal-regulated kinase (ERK) inhibitor PD98058 or ROS inhibitor N-acetyl-L-cysteine. Taken together, Moringa oleifera fruit extract is effective in inducing mitochondrial apoptosis of A2058 cells, which is mediated through induction of ROS formation, and JNK and ERK activation. Moringa oleifera fruit extract may thus have therapeutic benefits for human melanoma A2058 cells.

Enhanced expression of WD repeat-containing protein 35 (WDR35 stimulated by domoic acid in rat hippocampus: involvement of reactive oxygen species generation and p38 mitogen-activated protein kinase activation

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Tsunekawa Koji

2013-01-01

Full Text Available Abstract Background Domoic acid (DA is an excitatory amino acid analogue of kainic acid (KA that acts via activation of glutamate receptors to elicit a rapid and potent excitotoxic response, resulting in neuronal cell death. Recently, DA was shown to elicit reactive oxygen species (ROS production and induce apoptosis accompanied by activation of p38 mitogen-activated protein kinase (MAPK in vitro. We have reported that WDR35, a WD-repeat protein, may mediate apoptosis in several animal models. In the present study, we administered DA to rats intraperitoneally, then used liquid chromatography/ion trap tandem mass spectrometry (LC-MS/MS to identify and quantify DA in the brains of the rats and performed histological examinations of the hippocampus. We further investigated the potential involvement of glutamate receptors, ROS, p38 MAPK, and WDR35 in DA-induced toxicity in vivo. Results Our results showed that intraperitoneally administered DA was present in the brain and induced neurodegenerative changes including apoptosis in the CA1 region of the hippocampus. DA also increased the expression of WDR35 mRNA and protein in a dose- and time-dependent manner in the hippocampus. In experiments using glutamate receptor antagonists, the AMPA/KA receptor antagonist NBQX significantly attenuated the DA-induced increase in WDR35 protein expression, but the NMDA receptor antagonist MK-801 did not. In addition, the radical scavenger edaravone significantly attenuated the DA-induced increase in WDR35 protein expression. Furthermore, NBQX and edaravone significantly attenuated the DA-induced increase in p38 MAPK phosphorylation. Conclusion In summary, our results indicated that DA activated AMPA/KA receptors and induced ROS production and p38 MAPK phosphorylation, resulting in an increase in the expression of WDR35 in vivo.

Epidermal growth factor induction of front–rear polarity and migration in keratinocytes is mediated by integrin-linked kinase and ELMO2

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Ho, Ernest; Dagnino, Lina

2012-01-01

Epidermal growth factor (EGF) is a potent chemotactic and mitogenic factor for epidermal keratinocytes, and these properties are central for normal epidermal regeneration after injury. The involvement of mitogen-activated protein kinases as mediators of the proliferative effects of EGF is well established. However, the molecular mechanisms that mediate motogenic responses to this growth factor are not clearly understood. An obligatory step for forward cell migration is the development of front–rear polarity and formation of lamellipodia at the leading edge. We show that stimulation of epidermal keratinocytes with EGF, but not with other growth factors, induces development of front–rear polarity and directional migration through a pathway that requires integrin-linked kinase (ILK), Engulfment and Cell Motility-2 (ELMO2), integrin β1, and Rac1. Furthermore, EGF induction of front–rear polarity and chemotaxis require the tyrosine kinase activity of the EGF receptor and are mediated by complexes containing active RhoG, ELMO2, and ILK. Our findings reveal a novel link between EGF receptor stimulation, ILK-containing complexes, and activation of small Rho GTPases necessary for acquisition of front–rear polarity and forward movement. PMID:22160594

Epidermal growth factor induction of front-rear polarity and migration in keratinocytes is mediated by integrin-linked kinase and ELMO2.

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Ho, Ernest; Dagnino, Lina

2012-02-01

Epidermal growth factor (EGF) is a potent chemotactic and mitogenic factor for epidermal keratinocytes, and these properties are central for normal epidermal regeneration after injury. The involvement of mitogen-activated protein kinases as mediators of the proliferative effects of EGF is well established. However, the molecular mechanisms that mediate motogenic responses to this growth factor are not clearly understood. An obligatory step for forward cell migration is the development of front-rear polarity and formation of lamellipodia at the leading edge. We show that stimulation of epidermal keratinocytes with EGF, but not with other growth factors, induces development of front-rear polarity and directional migration through a pathway that requires integrin-linked kinase (ILK), Engulfment and Cell Motility-2 (ELMO2), integrin β1, and Rac1. Furthermore, EGF induction of front-rear polarity and chemotaxis require the tyrosine kinase activity of the EGF receptor and are mediated by complexes containing active RhoG, ELMO2, and ILK. Our findings reveal a novel link between EGF receptor stimulation, ILK-containing complexes, and activation of small Rho GTPases necessary for acquisition of front-rear polarity and forward movement.

The p38 mitogen activated protein kinase regulates β-amyloid protein internalization through the α7 nicotinic acetylcholine receptor in mouse brain.

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Ma, Kai-Ge; Lv, Jia; Yang, Wei-Na; Chang, Ke-Wei; Hu, Xiao-Dan; Shi, Li-Li; Zhai, Wan-Ying; Zong, Hang-Fan; Qian, Yi-Hua

2018-03-01

Alzheimer's disease (AD) is one of the most devastating neurodegenerative disorders. Intracellular β-amyloid protein (Aβ) is an early event in AD. It induces the formation of amyloid plaques and neuron damage. The α7 nicotinic acetylcholine receptor (α7nAChR) has been suggested to play an important role in Aβ caused cognition. It has high affinity with Aβ and could mediate Aβ internalization in vitro. However, whether in mouse brain the p38 MAPK signaling pathway is involved in the regulation of the α7nAChR mediated Aβ internalization and their role in mitochondria remains little known. Therefore, in this study, we revealed that Aβ is internalized by cholinergic and GABAergic neurons. The internalized Aβ were found deposits in lysosomes/endosomes and mitochondria. Aβ could form Aβ-α7nAChR complex with α7nAChR, activates the p38 mitogen activated protein kinase (MAPK). And the increasing of α7nAChR could in return mediate Aβ internalization in the cortex and hippocampus. In addition, by using the α7nAChR agonist PNU282987, the p38 phosphorylation level decreases, rescues the biochemical changes which are tightly associated with Aβ-induced apoptosis, such as Bcl2/Bax level, cytochrome c (Cyt c) release. Collectively, the p38 MAPK signaling pathway could regulate the α7nAChR-mediated internalization of Aβ. The activation of α7nAChR or the inhibition of p38 MAPK signaling pathway may be a beneficial therapy to AD. Copyright © 2017 Elsevier Inc. All rights reserved.

Rearrangement of a polar core provides a conserved mechanism for constitutive activation of class B G protein-coupled receptors

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Yin, Yanting; de Waal, Parker W.; He, Yuanzheng; Zhao, Li-Hua; Yang, Dehua; Cai, Xiaoqing; Jiang, Yi; Melcher, Karsten; Wang, Ming-Wei; Xu, H. Eric

2017-01-01

The glucagon receptor (GCGR) belongs to the secretin-like (class B) family of G protein-coupled receptors (GPCRs) and is activated by the peptide hormone glucagon. The structures of an activated class B GPCR have remained unsolved, preventing a mechanistic understanding of how these receptors are activated. Using a combination of structural modeling and mutagenesis studies, we present here two modes of ligand-independent activation of GCGR. First, we identified a GCGR-specific hydrophobic lock comprising Met-338 and Phe-345 within the IC3 loop and transmembrane helix 6 (TM6) and found that this lock stabilizes the TM6 helix in the inactive conformation. Disruption of this hydrophobic lock led to constitutive G protein and arrestin signaling. Second, we discovered a polar core comprising conserved residues in TM2, TM3, TM6, and TM7, and mutations that disrupt this polar core led to constitutive GCGR activity. On the basis of these results, we propose a mechanistic model of GCGR activation in which TM6 is held in an inactive conformation by the conserved polar core and the hydrophobic lock. Mutations that disrupt these inhibitory elements allow TM6 to swing outward to adopt an active TM6 conformation similar to that of the canonical β2-adrenergic receptor complexed with G protein and to that of rhodopsin complexed with arrestin. Importantly, mutations in the corresponding polar core of several other members of class B GPCRs, including PTH1R, PAC1R, VIP1R, and CRFR1, also induce constitutive G protein signaling, suggesting that the rearrangement of the polar core is a conserved mechanism for class B GPCR activation. PMID:28356352

Detection of phosphorylated mitogen-activated protein kinase in the developing spinal cord of the mouse embryo

International Nuclear Information System (INIS)

Teraishi, Toshiya; Miura, Kenji

2011-01-01

Highlights: → We detected physiologically phosphorylated MAPKs in developing spinal cord. → We detected physiologically phosphorylated MAPKs by an improved method. → p-ERK1/2 and p-JNK1/2 were detected in the marginal layer and the dorsal horn. → p-ERK1/2 and p-JNK1/2 might play critical roles in the developing spinal cord. → Constructing phosphoprotein atlases will be possible if expanding this work. -- Abstract: Global understanding of the proteome is a major research topic. The comprehensive visualization of the distribution of proteins in vivo or the construction of in situ protein atlases may be a valuable strategy for proteomic researchers. Information about the distribution of various proteins under physiological and pathological conditions should be extremely valuable for the basic and clinical sciences. The mitogen-activated protein kinase (MAPK) cascade plays an essential role in intracellular signaling in organisms. This cascade also regulates biological processes involving development, differentiation, and proliferation. Phosphorylation and dephosphorylation are integral reactions in regulating the activity of MAPKs. Changes in the phosphorylation state of MAPKs are rapid and reversible; therefore, the localizations of physiologically phosphorylated MAPKs in vivo are difficult to accurately detect. Furthermore, phosphorylated MAPKs are likely to change phosphorylated states through commonly used experimental manipulations. In the present study, as a step toward the construction of in situ phosphoprotein atlases, we attempted to detect physiologically phosphorylated MAPKs in vivo in developing spinal cords of mice. We previously reported an improved immunohistochemical method for detecting unstable phosphorylated MAPKs. The distribution patterns of phosphorylated MAPKs in the spinal cords of embryonic mice from embryonic day 13 (E13) to E17 were observed with an improved immunohistochemical method. Phosphorylated extracellular signal

Involvement of mitogen-activated protein kinases and NFκB in LPS-induced CD40 expression on human monocytic cells

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Wu Weidong; Alexis, Neil E.; Chen Xian; Bromberg, Philip A.; Peden, David B.

2008-01-01

CD40 is a costimulatory molecule linking innate and adaptive immune responses to bacterial stimuli, as well as a critical regulator of functions of other costimulatory molecules. The mechanisms regulating lipopolysaccharide (LPS)-induced CD40 expression have not been adequately characterized in human monocytic cells. In this study we used a human monocytic cell line, THP-1, to investigate the possible mechanisms of CD40 expression following LPS exposure. Exposure to LPS resulted in a dose- and time-dependent increase in CD40 expression. Further studies using immunoblotting and pharmacological inhibitors revealed that mitogen-activated protein kinases (MAPKs) and NFκB were activated by LPS exposure and involved in LPS-induced CD40 expression. Activation of MAPKs was not responsible for LPS-induced NFκB activation. TLR4 was expressed on THP-1 cells and pretreatment of cells with a Toll-like receptor 4 (TLR4) neutralizing antibody (HTA125) significantly blunted LPS-induced MAPK and NFκB activation and ensuing CD40 expression. Additional studies with murine macrophages expressing wild type and mutated TLR4 showed that TLR4 was implicated in LPS-induced ERK and NFκB activation, and CD40 expression. Moreover, blockage of MAPK and NFκB activation inhibited LPS-induced TLR4 expression. In summary, LPS-induced CD40 expression in monocytic cells involves MAPKs and NFκB

Mitogen-activated protein kinase phosphatase-1 modulates regional effects of injurious mechanical ventilation in rodent lungs.

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Park, Moo Suk; He, Qianbin; Edwards, Michael G; Sergew, Amen; Riches, David W H; Albert, Richard K; Douglas, Ivor S

2012-07-01

Mechanical ventilation induces heterogeneous lung injury by mitogen-activated protein kinase (MAPK) and nuclear factor-κB. Mechanisms regulating regional injury and protective effects of prone positioning are unclear. To determine the key regulators of the lung regional protective effects of prone positioning in rodent lungs exposed to injurious ventilation. Adult rats were ventilated with high (18 ml/kg, positive end-expiratory pressure [PEEP] 0) or low Vt (6 ml/kg; PEEP 3 cm H(2)O; 3 h) in supine or prone position. Dorsal-caudal lung mRNA was analyzed by microarray and MAPK phosphatases (MKP)-1 quantitative polymerase chain reaction. MKP-1(-/-) or wild-type mice were ventilated with very high (24 ml/kg; PEEP 0) or low Vt (6-7 ml/kg; PEEP 3 cm H(2)O). The MKP-1 regulator PG490-88 (MRx-108; 0.75 mg/kg) or phosphate-buffered saline was administered preventilation. Injury was assessed by lung mechanics, bronchioalveolar lavage cell counts, protein content, and lung injury scoring. Immunoblotting for MKP-1, and IκBα and cytokine ELISAs were performed on lung lysates. Prone positioning was protective against injurious ventilation in rats. Expression profiling demonstrated MKP-1 20-fold higher in rats ventilated prone rather than supine and regional reduction in p38 and c-jun N-terminal kinase activation. MKP-1(-/-) mice experienced amplified injury. PG490-88 improved static lung compliance and injury scores, reduced bronchioalveolar lavage cell counts and cytokine levels, and induced MKP-1 and IκBα. Injurious ventilation induces MAPK in an MKP-1-dependent fashion. Prone positioning is protective and induces MKP-1. PG490-88 induced MKP-1 and was protective against high Vt in a nuclear factor-κB-dependent manner. MKP-1 is a potential target for modulating regional effects of injurious ventilation.

Receptor trafficking via the perinuclear recycling compartment accompanied by cell division is necessary for permanent neurotensin cell sensitization and leads to chronic mitogen-activated protein kinase activation.

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Toy-Miou-Leong, Mireille; Cortes, Catherine Llorens; Beaudet, Alain; Rostène, William; Forgez, Patricia

2004-03-26

Most G protein-coupled receptors are internalized after interaction with their respective ligand, a process that subsequently contributes to cell desensitization, receptor endocytosis, trafficking, and finally cell resensitization. Although cellular mechanisms leading to cell desensitization have been widely studied, those responsible for cell resensitization are still poorly understood. We examined here the traffic of the high affinity neurotensin receptor (NT1 receptor) following prolonged exposure to high agonist concentration. Fluorescence and confocal microscopy of Chinese hamster ovary, human neuroblastoma (CHP 212), and murine neuroblastoma (N1E-115) cells expressing green fluorescent protein-tagged NT1 receptor revealed that under prolonged treatment with saturating concentrations of neurotensin (NT) agonist, NT1 receptor and NT transiently accumulated in the perinuclear recycling compartment (PNRC). During this cellular event, cell surface receptors remained markedly depleted as detected by both confocal microscopy and (125)I-NT binding assays. In dividing cells, we observed that following prolonged NT agonist stimulation, NT1 receptors were removed from the PNRC, accumulated in dispersed vesicles inside the cytoplasm, and subsequently reappeared at the cell surface. This NT binding recovery allowed for constant cell sensitization and led to a chronic activation of mitogen-activated protein kinases p42 and p44. Under these conditions, the constant activation of NT1 receptor generates an oncogenic regulation. These observations support the potent role for neuropeptides, such as NT, in cancer progression.

Rearrangement of a polar core provides a conserved mechanism for constitutive activation of class B G protein-coupled receptors.

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Yin, Yanting; de Waal, Parker W; He, Yuanzheng; Zhao, Li-Hua; Yang, Dehua; Cai, Xiaoqing; Jiang, Yi; Melcher, Karsten; Wang, Ming-Wei; Xu, H Eric

2017-06-16

The glucagon receptor (GCGR) belongs to the secretin-like (class B) family of G protein-coupled receptors (GPCRs) and is activated by the peptide hormone glucagon. The structures of an activated class B GPCR have remained unsolved, preventing a mechanistic understanding of how these receptors are activated. Using a combination of structural modeling and mutagenesis studies, we present here two modes of ligand-independent activation of GCGR. First, we identified a GCGR-specific hydrophobic lock comprising Met-338 and Phe-345 within the IC3 loop and transmembrane helix 6 (TM6) and found that this lock stabilizes the TM6 helix in the inactive conformation. Disruption of this hydrophobic lock led to constitutive G protein and arrestin signaling. Second, we discovered a polar core comprising conserved residues in TM2, TM3, TM6, and TM7, and mutations that disrupt this polar core led to constitutive GCGR activity. On the basis of these results, we propose a mechanistic model of GCGR activation in which TM6 is held in an inactive conformation by the conserved polar core and the hydrophobic lock. Mutations that disrupt these inhibitory elements allow TM6 to swing outward to adopt an active TM6 conformation similar to that of the canonical β 2 -adrenergic receptor complexed with G protein and to that of rhodopsin complexed with arrestin. Importantly, mutations in the corresponding polar core of several other members of class B GPCRs, including PTH1R, PAC1R, VIP1R, and CRFR1, also induce constitutive G protein signaling, suggesting that the rearrangement of the polar core is a conserved mechanism for class B GPCR activation. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

AMP N1-Oxide, a Unique Compound of Royal Jelly, Induces Neurite Outgrowth from PC12 Vells via Signaling by Protein Kinase A Independent of that by Mitogen-Activated Protein Kinase

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Noriko Hattori

2010-01-01

Full Text Available Earlier we identified adenosine monophosphate (AMP N1-oxide as a unique compound of royal jelly (RJ that induces neurite outgrowth (neuritegenesis from cultured rat pheochromocytoma PC12 cells via the adenosine A2A receptor. Now, we found that AMP N1-oxide stimulated the phosphorylation of not only mitogen-activated protein kinase (MAPK but also that of cAMP/calcium-response element-binding protein (CREB in a dose-dependent manner. Inhibition of MAPK activation by a MEK inhibitor, PD98059, did not influence the AMP N1-oxide-induced neuritegenesis, whereas that of protein kinase A (PKA by a selective inhibitor, KT5720, significantly reduced neurite outgrowth. AMP N1-oxide also had the activity of suppressing the growth of PC12 cells, which correlated well with the neurite outgrowth-promoting activity. KT5720 restored the growth of AMP N1-oxide-treated PC12 cells. It is well known that nerve growth factor suppresses proliferation of PC12 cells before causing stimulation of neuronal differentiation. Thus, AMP N1-oxide elicited neuronal differentiation of PC12 cells, as evidenced by generation of neurites, and inhibited cell growth through adenosine A2A receptor-mediated PKA signaling, which may be responsible for characteristic actions of RJ.

Role of polarized G protein signaling in tracking pheromone gradients

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McClure, Allison W.; Minakova, Maria; Dyer, Jayme M.; Zyla, Trevin R.; Elston, Timothy C.; Lew, Daniel J.

2015-01-01

Summary Yeast cells track gradients of pheromones to locate mating partners. Intuition suggests that uniform distribution of pheromone receptors over the cell surface would yield optimal gradient sensing. However, yeast cells display polarized receptors. The benefit of such polarization was unknown. During gradient tracking, cell growth is directed by a patch of polarity regulators that wanders around the cortex. Patch movement is sensitive to pheromone dose, with wandering reduced on the up-gradient side of the cell, resulting in net growth in that direction. Mathematical modeling suggests that active receptors and associated G proteins lag behind the polarity patch and act as an effective drag on patch movement. In vivo, the polarity patch is trailed by a G protein-rich domain, and this polarized distribution of G proteins is required to constrain patch wandering. Our findings explain why G protein polarization is beneficial, and illuminate a novel mechanism for gradient tracking. PMID:26609960

p38 mitogen-activated protein kinase is involved in arginase-II-mediated eNOS-uncoupling in obesity.

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Yu, Yi; Rajapakse, Angana G; Montani, Jean-Pierre; Yang, Zhihong; Ming, Xiu-Fen

2014-07-18

Endothelial nitric oxide synthase (eNOS)-uncoupling links obesity-associated insulin resistance and type-II diabetes to the increased incidence of cardiovascular disease. Studies have indicated that increased arginase is involved in eNOS-uncoupling through competing with the substrate L-arginine. Given that arginase-II (Arg-II) exerts some of its biological functions through crosstalk with signal transduction pathways, and that p38 mitogen-activated protein kinase (p38mapk) is involved in eNOS-uncoupling, we investigated here whether p38mapk is involved in Arg-II-mediated eNOS-uncoupling in a high fat diet (HFD)-induced obesity mouse model. Obesity was induced in wild type (WT) and Arg-II-deficient (Arg-II(-/-)) mice on C57BL/6 J background by high-fat diet (HFD, 55% fat) for 14 weeks starting from age of 7 weeks. The entire aortas were isolated and subjected to 1) immunoblotting analysis of the protein level of eNOS, Arg-II and p38mapk activation; 2) arginase activity assay; 3) endothelium-dependent and independent vasomotor responses; 4) en face staining of superoxide anion and NO production with Dihydroethidium and 4,5-Diaminofluorescein Diacetate, respectively, to assess eNOS-uncoupling. To evaluate the role of p38mapk, isolated aortas were treated with p38mapk inhibitor SB203580 (10 μmol/L, 1 h) prior to the analysis. In addition, the role of p38mapk in Arg-II-induced eNOS-uncoupling was investigated in cultured human endothelial cells overexpressing Arg-II in the absence or presence of shRNA against p38mapk. HFD enhanced Arg-II expression/activity and p38mapk activity, which was associated with eNOS-uncoupling as revealed by decreased NO and enhanced L-NAME-inhibitable superoxide in aortas of WT obese mice. In accordance, WT obese mice revealed decreased endothelium-dependent relaxations to acetylcholine despite of higher eNOS protein level, whereas Arg-II(-/-) obese mice were protected from HFD-induced eNOS-uncoupling and endothelial dysfunction, which

Regulation of p53, nuclear factor κB and cyclooxygenase-2 expression by bromelain through targeting mitogen-activated protein kinase pathway in mouse skin

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Kalra, Neetu; Bhui, Kulpreet; Roy, Preeti; Srivastava, Smita; George, Jasmine; Prasad, Sahdeo; Shukla, Yogeshwer

2008-01-01

Bromelain is a pharmacologically active compound, present in stems and immature fruits of pineapples (Ananas cosmosus), which has been shown to have anti-edematous, anti-inflammatory, anti-thrombotic and anti-metastatic properties. In the present study, antitumorigenic activity of bromelain was recorded in 7,12-dimethylbenz(a)anthracene (DMBA)-initiated and 12-O-tetradecanoylphorbol-13-acetate (TPA)-promoted 2-stage mouse skin model. Results showed that bromelain application delayed the onset of tumorigenesis and reduced the cumulative number of tumors, tumor volume and the average number of tumors/mouse. To establish a cause and effect relationship, we targeted the proteins involved in the cell death pathway. Bromelain treatment resulted in upregulation of p53 and Bax and subsequent activation of caspase 3 and caspase 9 with concomitant decrease in antiapoptotic protein Bcl-2 in mouse skin. Since persistent induction of cyclooxygenase-2 (Cox-2) is frequently implicated in tumorigenesis and is regulated by nuclear factor-kappa B (NF-κB), we also investigated the effect of bromelain on Cox-2 and NF-κB expression. Results showed that bromelain application significantly inhibited Cox-2 and inactivated NF-κB by blocking phosphorylation and subsequent degradation of IκBα. In addition, bromelain treatment attenuated DMBA-TPA-induced phosphorylation of extracellular signal-regulated protein kinase (ERK1/2), mitogen-activated protein kinase (MAPK) and Akt. Taken together, we conclude that bromelain induces apoptosis-related proteins along with inhibition of NF-κB-driven Cox-2 expression by blocking the MAPK and Akt/protein kinase B signaling in DMBA-TPA-induced mouse skin tumors, which may account for its anti-tumorigenic effects

Amitriptyline induces early growth response-1 gene expression via ERK and JNK mitogen-activated protein kinase pathways in rat C6 glial cells.

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Chung, Eun Young; Shin, Soon Young; Lee, Young Han

2007-07-05

Astrocytes play important roles in guiding the construction of the nervous system, controlling extracellular ions and neurotransmitters, and regulating CNS synaptogenesis. Egr-1 is a transcription factor involved in neuronal differentiation and astrocyte cell proliferation. In this study, we investigated whether the tricyclic antidepressant (TCA) amitriptyline induces Egr-1 expression in astrocytes using rat C6 glioma cells as a model. We found that amitriptyline increased the expression of Egr-1 in a dose- and time-dependent manner. The amitriptyline-induced Egr-1 expression was mediated through serum response elements (SREs) in the Egr-1 promoter. SREs were activated by the Ets-domain transcription factor Elk-1 through the ERK and JNK mitogen-activated protein (MAP) kinase pathways. The inhibition of the ERK and JNK MAP kinase signals attenuated amitriptyline-induced transactivation of Gal4-Elk-1 and Egr-1 promoter activity. Our findings suggest that the induction of Egr-1 expression in astrocytes may be required to attain the therapeutic effects of antidepressant drugs.

Electrical stimulation with periodic alternating intervals stimulates neuronal cells to produce neurotrophins and cytokines through activation of mitogen-activated protein kinase pathways.

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Yamamoto, Kenta; Yamamoto, Toshiro; Honjo, Kenichi; Ichioka, Hiroaki; Oseko, Fumishige; Kishida, Tsunao; Mazda, Osam; Kanamura, Narisato

2015-12-01

Peripheral neuropathy is a representative complication of dental surgery. Electrical therapy, based on electrical stimulation with periodic alternating intervals (ES-PAI), may promote nerve regeneration after peripheral nerve injury in a non-invasive manner, potentially providing an effective therapy for neuropathy. This study aimed to analyze the molecular mechanisms underlying the nerve recovery stimulated by ES-PAI. In brief, ES-PAI was applied to a neuronal cell line, Neuro2A, at various intensities using the pulse generator apparatus, FREUDE. Cell viability, neurotrophin mRNA expression, and cytokine production were examined using a tetrazolium-based assay, real-time RT-PCR, and ELISA, respectively. Mitogen-activated protein kinase (MAPK) signaling was assessed using flow cytometry. It was found that ES-PAI increased the viability of cells and elevated expression of nerve growth factor (NGF) and neurotrophin-3 (NT-3); ESPAI also augmented vascular endothelial growth factor (VEGF) and platelet-derived growth factor (PDGF) expression, which was restored by addition of p38 inhibitors. Phosphorylation of p38 and extracellular signal-regulated kinase 1/2 (ERK-1/2) was augmented by ES-PAI. Hence, ES-PAI may ameliorate peripheral neuropathy by promoting neuronal cell proliferation and production of neurogenic factors by activating p38 and ERK-1/2 pathways. © 2015 Eur J Oral Sci.

Scaffold Protein Ahk1, Which Associates with Hkr1, Sho1, Ste11, and Pbs2, Inhibits Cross Talk Signaling from the Hkr1 Osmosensor to the Kss1 Mitogen-Activated Protein Kinase

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Nishimura, Akiko; Yamamoto, Katsuyoshi; Oyama, Masaaki; Kozuka-Hata, Hiroko

2016-01-01

In the budding yeast Saccharomyces cerevisiae, osmostress activates the Hog1 mitogen-activated protein kinase (MAPK), which regulates diverse osmoadaptive responses. Hkr1 is a large, highly glycosylated, single-path transmembrane protein that is a putative osmosensor in one of the Hog1 upstream pathways termed the HKR1 subbranch. The extracellular region of Hkr1 contains both a positive and a negative regulatory domain. However, the function of the cytoplasmic domain of Hkr1 (Hkr1-cyto) is unknown. Here, using a mass spectrometric method, we identified a protein, termed Ahk1 (Associated with Hkr1), that binds to Hkr1-cyto. Deletion of the AHK1 gene (in the absence of other Hog1 upstream branches) only partially inhibited osmostress-induced Hog1 activation. In contrast, Hog1 could not be activated by constitutively active mutants of the Hog1 pathway signaling molecules Opy2 or Ste50 in ahk1Δ cells, whereas robust Hog1 activation occurred in AHK1+ cells. In addition to Hkr1-cyto binding, Ahk1 also bound to other signaling molecules in the HKR1 subbranch, including Sho1, Ste11, and Pbs2. Although osmotic stimulation of Hkr1 does not activate the Kss1 MAPK, deletion of AHK1 allowed Hkr1 to activate Kss1 by cross talk. Thus, Ahk1 is a scaffold protein in the HKR1 subbranch and prevents incorrect signal flow from Hkr1 to Kss1. PMID:26787842

Buddleja officinalis suppresses high glucose-induced vascular smooth muscle cell proliferation: role of mitogen-activated protein kinases, nuclear factor-kappaB and matrix metalloproteinases.

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Lee, Yun Jung; Kim, Jin Sook; Kang, Dae Gill; Lee, Ho Sub

2010-02-01

Diabetes mellitus is a well-established risk factor for vascular diseases caused by atherosclerosis. In the development of diabetic atherogenesis, vascular smooth muscle cell proliferation is recognized as a key event. Thus, we aimed to investigate whether an ethanol extract of Buddleja officinalis (EBO) suppresses high glucose-induced proliferation in primary cultured human aortic smooth muscle cells (HASMC). [(3)H]-thymidine incorporation revealed that incubation of HASMC with a high concentration of glucose (25 mmol/L) increased cell proliferation. The expression levels of cell cycle protein were also increased by treatment with high glucose concentration. Pretreatment of HASMC with EBO significantly attenuated the increase of high glucose-induced cell proliferation as well as p38 mitogen-activated protein kinases (MAPK) and JNK phosphorylation. EBO suppressed high glucose-induced matrix metalloproteinase (MMP)-9 activity in a dose-dependent manner. In addition, EBO suppressed nuclear factor-kappaB (NF-kappaB) nuclear translocation and transcriptional activity in high glucose conditions. Taken together, the present data suggest that EBO could suppress high glucose-induced atherosclerotic processes through inhibition of p38, JNK, NF-kappaB and MMP signal pathways in HASMC.

Purification and characterization of a mucin specific mycelial lectin from Aspergillus gorakhpurensis: application for mitogenic and antimicrobial activity.

Directory of Open Access Journals (Sweden)

Ram Sarup Singh

Full Text Available Lectins are carbohydrate binding proteins or glycoproteins that bind reversibly to specific carbohydrates present on the apposing cells, which are responsible for their ability to agglutinate red blood cells, lymphocytes, fibroblasts, etc. Interest in lectins has been intensified due to their carbohydrate specificity as they can be valuable reagents for the investigation of cell surface sugars, purification and characterization of glycoproteins. The present study reports the purification, characterization and evaluation of mitogenic and antimicrobial potential of a mycelial lectin from Aspergillus gorakhpurensis.Affinity chromatography on mucin-sepharose column was carried out for purification of Aspergillus gorakhpurensis lectin. The lectin was characterized for physico-chemical parameters. Mitogenic potential of the lectin was evaluated against splenocytes of Swiss albino mice by MTT assay. Antimicrobial activity of the purified lectin has also been evaluated by disc diffusion assay.Single-step affinity purification resulted in 18.6-fold purification of the mycelial lectin. The molecular mass of the lectin was found to be 70 kDa and it was composed of two subunits of 34.8 kDa as determined by gel filtration chromatography, SDS-PAGE and MALDI-TOF analysis. pH optima of the lectin was found to be 6.5-9.5, while optimum temperature for lectin activity was 20-30 °C. Lectin was stable within a pH range of 7.0-10.5 and showed fair thermostability. EDTA did not affect lectin activity whereas it was found susceptible to the denaturants tested. MTT assay revealed strong mitogenic potential of A. gorakhpurensis lectin at a concentration upto 150 µg/mL. Antimicrobial activity assay showed its potent antibacterial activity against Bacillus cereus, Staphylococcous aureus and Escherichia coli and marginal antifungal activity against Saccharomyces cerevisiae.This is the first report on the mitogenic and antimicrobial potential of Aspergillus gorakhpurensis

Purification and characterization of a mucin specific mycelial lectin from Aspergillus gorakhpurensis: application for mitogenic and antimicrobial activity.

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Singh, Ram Sarup; Kaur, Hemant Preet; Singh, Jatinder

2014-01-01

Lectins are carbohydrate binding proteins or glycoproteins that bind reversibly to specific carbohydrates present on the apposing cells, which are responsible for their ability to agglutinate red blood cells, lymphocytes, fibroblasts, etc. Interest in lectins has been intensified due to their carbohydrate specificity as they can be valuable reagents for the investigation of cell surface sugars, purification and characterization of glycoproteins. The present study reports the purification, characterization and evaluation of mitogenic and antimicrobial potential of a mycelial lectin from Aspergillus gorakhpurensis. Affinity chromatography on mucin-sepharose column was carried out for purification of Aspergillus gorakhpurensis lectin. The lectin was characterized for physico-chemical parameters. Mitogenic potential of the lectin was evaluated against splenocytes of Swiss albino mice by MTT assay. Antimicrobial activity of the purified lectin has also been evaluated by disc diffusion assay. Single-step affinity purification resulted in 18.6-fold purification of the mycelial lectin. The molecular mass of the lectin was found to be 70 kDa and it was composed of two subunits of 34.8 kDa as determined by gel filtration chromatography, SDS-PAGE and MALDI-TOF analysis. pH optima of the lectin was found to be 6.5-9.5, while optimum temperature for lectin activity was 20-30 °C. Lectin was stable within a pH range of 7.0-10.5 and showed fair thermostability. EDTA did not affect lectin activity whereas it was found susceptible to the denaturants tested. MTT assay revealed strong mitogenic potential of A. gorakhpurensis lectin at a concentration upto 150 µg/mL. Antimicrobial activity assay showed its potent antibacterial activity against Bacillus cereus, Staphylococcous aureus and Escherichia coli and marginal antifungal activity against Saccharomyces cerevisiae. This is the first report on the mitogenic and antimicrobial potential of Aspergillus gorakhpurensis lectin. The

Mitogen-activated protein kinase 1 from disk abalone (Haliotis discus discus): Roles in early development and immunity-related transcriptional responses.

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Perera, N C N; Godahewa, G I; Lee, Jehee

2016-12-01

Mitogen-activated protein kinase (MAPK) is involved in the regulation of cellular events by mediating signal transduction pathways. MAPK1 is a member of the extracellular-signal regulated kinases (ERKs), playing roles in cell proliferation, differentiation, and development. This is mainly in response to growth factors, mitogens, and many environmental stresses. In the current study, we have characterized the structural features of a homolog of MAPK1 from disk abalone (AbMAPK1). Further, we have unraveled its expressional kinetics against different experimental pathogenic infections or related chemical stimulants. AbMAPK1 harbors a 5' untranslated region (UTR) of 23 bps, a coding sequence of 1104 bps, and a 3' UTR of 448 bp. The putative peptide comprises a predicted molecular mass of 42.2 kDa, with a theoretical pI of 6.28. Based on the in silico analysis, AbMAPK1 possesses two N-glycosylation sites, one S_TK catalytic domain, and a conserved His-Arg-Asp domain (HRD). In addition, a conservative glycine rich ATP-phosphate-binding loop and a threonine-x-tyrosine motif (TEY) important for the autophosphorylation were also identified in the protein. Homology assessment of AbMAPK1 showed several conserved regions, and ark clam (Aplysia californica) showed the highest sequence identity (87.9%). The phylogenetic analysis supported close evolutionary kinship with molluscan orthologs. Constitutive expression of AbMAPK1 was observed in six different tissues of disk abalone, with the highest expression in the digestive tract, followed by the gills and hemocytes. Highest AbMAPK1 mRNA expression level was detected at the trochophore developmental stage, suggesting its role in abalone cell differentiation and proliferation. Significant modulation of AbMAPK1 expression under pathogenic stress suggested its putative involvement in the immune defense mechanism. Copyright © 2016 Elsevier Ltd. All rights reserved.

Syndecan-1/CD147 association is essential for cyclophilin B-induced activation of p44/42 mitogen-activated protein kinases and promotion of cell adhesion and chemotaxis.

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Pakula, Rachel; Melchior, Aurélie; Denys, Agnès; Vanpouille, Christophe; Mazurier, Joël; Allain, Fabrice

2007-05-01

Many of the biological functions attributed to cell surface proteoglycans are dependent on the interaction with extracellular mediators through their heparan sulphate (HS) moieties and the participation of their core proteins in signaling events. A class of recently identified inflammatory mediators is secreted cyclophilins, which are mostly known as cyclosporin A-binding proteins. We previously demonstrated that cyclophilin B (CyPB) triggers chemotaxis and integrin-mediated adhesion of T lymphocytes mainly of the CD4+/CD45RO+ phenotype. These activities are related to interactions with two types of binding sites, CD147 and cell surface HS. Here, we demonstrate that CyPB-mediated adhesion of CD4+/CD45RO+ T cells is related to p44/42 mitogen-activated protein kinase (MAPK) activation by a mechanism involving CD147 and HS proteoglycans (HSPG). Although HSPG core proteins are represented by syndecan-1, -2, -4, CD44v3 and betaglycan in CD4+/CD45RO+ T cells, we found that only syndecan-1 is physically associated with CD147. The intensity of the heterocomplex increased in response to CyPB, suggesting a transient enhancement and/or stabilization in the association of CD147 to syndecan-1. Pretreatment with anti-syndecan-1 antibodies or knockdown of syndecan-1 expression by RNA interference dramatically reduced CyPB-induced p44/p42 MAPK activation and consequent migration and adhesion, supporting the model in which syndecan-1 serves as a binding subunit to form the fully active receptor of CyPB. Altogether, our findings provide a novel example of a soluble mediator in which a member of the syndecan family plays a critical role in efficient interaction with signaling receptors and initiation of cellular responses.

Heat- and radiation effects on the hemaglutinating- and mitogenic activity of phytohemaglutinins

International Nuclear Information System (INIS)

Mancini Filho, J.; Vizeu, D.M.; Lajolo, F.M.

1975-01-01

The effect of ionizing radiation on hemaglutinating and mitogenic activity of phytohemaglutinins (PHA) in solution is studied. 10 Krad (electron beam) are neede for the destruction of 50% of the aglutinating capacity. The mitogenic effect is more resistent to irradiation (70 Krad for 50% inactivation) may be because both effects are due to different molecules. Changes were also followed by electrophoresis in polyacrylamida. The resistence to irradiation showed to be exponential function of the concentration of PHA in solution. (author) [pt

The pearl millet mitogen-activated protein kinase PgMPK4 is involved in responses to downy mildew infection and in jasmonic- and salicylic acid-mediated defense.

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Melvin, Prasad; Prabhu, S Ashok; Veena, Mariswamy; Shailasree, Sekhar; Petersen, Morten; Mundy, John; Shetty, Shekar H; Kini, K Ramachandra

2015-02-01

Plant mitogen-activated protein kinases (MPKs) transduce signals required for the induction of immunity triggered by host recognition of pathogen-associated molecular patterns. We isolated a full-length cDNA of a group B MPK (PgMPK4) from pearl millet. Autophosphorylation assay of recombinant PgMPK4 produced in Escherichia coli confirmed it as a kinase. Differential accumulation of PgMPK4 mRNA and kinase activity was observed between pearl millet cultivars 852B and IP18292 in response to inoculation with the downy mildew oomycete pathogen Sclerospora graminicola. This increased accumulation of PgMPK4 mRNA, kinase activity as well as nuclear-localization of PgMPK protein(s) was only detected in the S. graminicola resistant cultivar IP18292 with a ~tenfold peak at 9 h post inoculation. In the susceptible cultivar 852B, PgMPK4 mRNA and immuno-detectable nuclear PgMPK could be induced by application of the chemical elicitor β-amino butyric acid, the non-pathogenic bacteria Pseudomonas fluorescens, or by the phytohormones jasmonic acid (JA) or salicylic acid (SA). Furthermore, kinase inhibitor treatments indicated that PgMPK4 is involved in the JA- and SA-mediated expression of three defense genes, lipoxygenase, catalase 3 and polygalacturonase-inhibitor protein. These findings indicate that PgMPK/s contribute to pearl millet defense against the downy mildew pathogen by activating the expression of defense proteins.

p38 mitogen-activated protein kinase up-regulates LPS-induced NF-κB activation in the development of lung injury and RAW 264.7 macrophages

International Nuclear Information System (INIS)

Kim, Hee J.; Lee, Hui S.; Chong, Young H.; Kang, Jihee Lee

2006-01-01

Clarification of the key regulatory steps that lead to nuclear factor-kappa B (NF-κB) under cellular and pathological conditions is very important. The action of p38 mitogen-activated protein kinase (MAPK) on the upstream of NF-κB activation remains controversial. To examine this issue using an in vivo lung injury model, SB203580, a p38 MAPK inhibitor was given intraorally 1 h prior to lipopolysaccharide (LPS) treatment (intratracheally). The mice were sacrificed 4 h after LPS treatment. SB203580 substantially suppressed LPS-induced rises in p38 MAPK phosphorylation, neutrophil recruitment, total protein content in bronchoalveolar lavage fluid, and apoptosis of bronchoalveolar cells. Furthermore, SB203580 blocked LPS-induced NF-κB activation in lung tissue through down-regulation of serine phosphorylation, degradation of IκB-α, and consequent translocation of the p65 subunit of NF-κB to the nucleus. It is likely that, in cultured RAW 264.7 macrophages, SB203580 also blocked LPS-induced NF-κB activation in a dose-dependent manner. SB203580 inhibited LPS-induced serine phosphorylation, degradation of IκB-α, and tyrosine phosphorylation of p65 NF-κB. These data indicate that p38 MAPK acts upstream of LPS-induced NF-κB activation by modulating the phosphorylation of IκB-α and p65 NF-κB during acute lung injury. Because LPS-stimulated macrophages may contribute to inflammatory lung injury, the inhibition of the p38 MAPK-mediated intracellular signaling pathway leading to NF-κB activation represents a target for the attenuation of lung inflammation and parenchymal damage

Mitogen-activated protein kinase signaling in the heart: angels versus demons in a heart-breaking tale.

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Rose, Beth A; Force, Thomas; Wang, Yibin

2010-10-01

Among the myriad of intracellular signaling networks that govern the cardiac development and pathogenesis, mitogen-activated protein kinases (MAPKs) are prominent players that have been the focus of extensive investigations in the past decades. The four best characterized MAPK subfamilies, ERK1/2, JNK, p38, and ERK5, are the targets of pharmacological and genetic manipulations to uncover their roles in cardiac development, function, and diseases. However, information reported in the literature from these efforts has not yet resulted in a clear view about the roles of specific MAPK pathways in heart. Rather, controversies from contradictive results have led to a perception that MAPKs are ambiguous characters in heart with both protective and detrimental effects. The primary object of this review is to provide a comprehensive overview of the current progress, in an effort to highlight the areas where consensus is established verses the ones where controversy remains. MAPKs in cardiac development, cardiac hypertrophy, ischemia/reperfusion injury, and pathological remodeling are the main focuses of this review as these represent the most critical issues for evaluating MAPKs as viable targets of therapeutic development. The studies presented in this review will help to reveal the major challenges in the field and the limitations of current approaches and point to a critical need in future studies to gain better understanding of the fundamental mechanisms of MAPK function and regulation in the heart.

Phosphorylation of mitogen-activated protein kinase (MAPK) is required for cytokinesis and progression of cell cycle in tobacco BY-2 cells.

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Ma, Zhaowu; Yu, Guanghui

2010-02-15

The role of mitogen-activated protein kinase (MAPK) in plant cytokinesis remains largely uncharacterized. To elucidate its role, tobacco Bright Yellow-2 (BY-2) cells have been synchronized using a two-step procedure, and the different phases of the cell cycle identified by Histone 4 gene expression and the mitotic index. MAPK expression was analyzed by semi-quantitative (SQ) RT-PCR and protein gel blot analysis for phosphorylated MAPK during cell cycle progression. The SQ RT-PCR analysis indicated that MAPK expression is lower in mitosis than in interphase (G1, G2 and S). However, the amount of phosphorylated MAPK remained stable throughout the cell cycle, indicating that MAPK activity is predominantly regulated at the post-translational level and that phosphorylation of MAPK plays an important role in mitosis. Application of the specific MAPK phosphorylation inhibitor U0126 revealed that while U0126 treatment decreases the phosphorylation of MAPK and the progression from telophase to early cytokinesis is significantly inhibited. The formation of the phragmoplast is also negatively affected at this stage. These results demonstrate that MAPK phosphorylation is involved in the formation of the cell plate within the phragmoplast during cytokinesis and that MAPK predominantly functions during the cytokinesis stage of the cell cycle in tobacco BY-2 cells. Copyright 2009 Elsevier GmbH. All rights reserved.

Reduced response of splenocytes after mitogen-stimulation in the prion protein (PrP) gene-deficient mouse: PrPLP/Doppel production and cerebral degeneration

International Nuclear Information System (INIS)

Kim, Chi-Kyeong; Hirose, Yuko; Sakudo, Akikazu; Takeyama, Natsumi; Kang, Chung-Boo; Taniuchi, Yojiro; Matsumoto, Yoshitsugu; Itohara, Shigeyoshi; Sakaguchi, Suehiro; Onodera, Takashi

2007-01-01

Splenocytes of wild-type (Prnp +/+ ) and prion protein gene-deficient (Prnp -/- ) mice were treated with various activation stimuli such as T cell mitogen concanavalin A (ConA), phorbol 12-myristate 13-acetate (PMA) + ionomycin (Io), or B cell mitogen lipopolysaccharide (LPS). Cellular prion protein (PrP C ) expression was enhanced following ConA stimulation, but not PMA + Io or LPS in Prnp +/+ splenocytes. Rikn Prnp -/- splenocytes elicited lower cell proliferations than Prnp +/+ or Zrch I Prnp -/- splenocytes after LPS stimulation and showed sporadic nerve cells in the cerebral cortex and deeper structure. Around the degenerated nerve cells, mild vacuolation in the neuropil was observed. This neural alteration correlated well to the suppressed response of B cells in the spleen. The finding that discrete lesions within the central nervous systems induced marked modulation of immune function probably indicates the existence of a delicately balanced neural-endocrine network by PrP C and PrPLP/Doppel

RNA interference-based (RNAi) suppression of AtMPK6, an Arabidopsis mitogen-activated protein kinase, results in hypersensitivity to ozone and misregulation of AtMPK3

International Nuclear Information System (INIS)

Miles, Godfrey P.; Samuel, Marcus A.; Zhang Yuelin; Ellis, Brian E.

2005-01-01

The recent increase in tropospheric ozone (O 3 ) concentrations promotes additional oxidative stress through the production of reactive oxygen species (ROS) in plant tissues, resulting in the activation of genes whose products enable the stressed cells to retain their integrity and function. This response is made possible by an integration of highly regulated signaling networks that mediate the perception of, and response to, this oxidative assault. In Arabidopsis thaliana, ROS-induced signaling has been shown to flow through a protein phosphorylation cascade involving the mitogen-activated protein kinases (MAPKs) AtMPK3 (MPK3) and AtMPK6 (MPK6). We found that RNAi-mediated silencing of MPK6 renders the plant more sensitive to ozone, as determined by visible leaf damage. The MPK6-RNAi genotype also displayed a more intense and prolonged activation of MPK3 compared to that of WT plants. An MPK3 loss-of-function genotype is similarly very sensitive to ozone, and displays an abnormally prolonged MPK6 activation profile, suggesting reciprocity in regulation between these two MAPKs. - MPK6 is pivotal in the overall response to oxidative stress and regulation of MPK3 in Arabidopsis thaliana

Bacteroides fragilis Enterotoxin Induces Formation of Autophagosomes in Endothelial Cells but Interferes with Fusion with Lysosomes for Complete Autophagic Flux through a Mitogen-Activated Protein Kinase-, AP-1-, and C/EBP Homologous Protein-Dependent Pathway.

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Ko, Su Hyuk; Jeon, Jong Ik; Myung, Hyun Soo; Kim, Young-Jeon; Kim, Jung Mogg

2017-10-01

Bacteroides fragilis enterotoxin (BFT), a virulence factor of enterotoxigenic B. fragilis (ETBF), plays an essential role in mucosal inflammation. Although autophagy contributes to the pathogenesis of diverse infectious diseases, little is known about autophagy in ETBF infection. This study was conducted to investigate the role of BFT in the autophagic process in endothelial cells (ECs). Stimulation of human umbilical vein ECs (HUVECs) with BFT increased light chain 3 protein II (LC3-II) conversion from LC3-I and protein expression of p62, Atg5, and Atg12. In addition, BFT-exposed ECs showed increased indices of autophagosomal fusion with lysosomes such as LC3-lysosome-associated protein 2 (LAMP2) colocalization and the percentage of red vesicles monitored by the expression of dual-tagged LC3B. BFT also upregulated expression of C/EBP homologous protein (CHOP), and inhibition of CHOP significantly increased indices of autophagosomal fusion with lysosomes. BFT activated an AP-1 transcription factor, in which suppression of AP-1 activity significantly downregulated CHOP and augmented autophagosomal fusion with lysosomes. Furthermore, suppression of Jun N-terminal protein kinase (JNK) mitogen-activated protein kinase (MAPK) significantly inhibited the AP-1 and CHOP signals, leading to an increase in autophagosomal fusion with lysosomes in BFT-stimulated ECs. These results suggest that BFT induced accumulation of autophagosomes in ECs, but activation of a signaling pathway involving JNK, AP-1, and CHOP may interfere with complete autophagy. Copyright © 2017 American Society for Microbiology.

Cancer Cell-derived Exosomes Induce Mitogen-activated Protein Kinase-dependent Monocyte Survival by Transport of Functional Receptor Tyrosine Kinases*

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Song, Xiao; Ding, Yanping; Liu, Gang; Yang, Xiao; Zhao, Ruifang; Zhang, Yinlong; Zhao, Xiao; Anderson, Gregory J.; Nie, Guangjun

2016-01-01

Tumor-associated macrophages (TAM) play pivotal roles in cancer initiation and progression. Monocytes, the precursors of TAMs, normally undergo spontaneous apoptosis within 2 days, but can subsist in the inflammatory tumor microenvironment for continuous survival and generation of sufficient TAMs. The mechanisms underlying tumor-driving monocyte survival remain obscure. Here we report that cancer cell-derived exosomes were crucial mediators for monocyte survival in the inflammatory niche. Analysis of the survival-promoting molecules in monocytes revealed that cancer cell-derived exosomes activated Ras and extracellular signal-regulated kinases in the mitogen-activated protein kinase (MAPK) pathway, resulting in the prevention of caspase cleavage. Phosphorylated receptor tyrosine kinases (RTKs), such as phosphorylated epidermal growth factor receptor (EGFR) and human epidermal growth factor receptor 2 (HER-2), were abundantly expressed in cancer cell-derived exosomes. Knock-out of EGFR or/and HER-2, or alternatively, inhibitors against their phosphorylation significantly disturbed the exosome-mediated activation of the MAPK pathway, inhibition of caspase cleavage, and increase in survival rate in monocytes. Moreover, the deprived survival-stimulating activity of exosomes due to null expression of EGFR and HER-2 could be restored by activation of another RTK, insulin receptor. Overall, our study uncovered a mechanism of tumor-associated monocyte survival and demonstrated that cancer cell-derived exosomes can stimulate the MAPK pathway in monocytes through transport of functional RTKs, leading to inactivation of apoptosis-related caspases. This work provides insights into the long sought question on monocyte survival prior to formation of plentiful TAMs in the tumor microenvironment. PMID:26895960

Ethanol extract of the seed of Zizyphus jujuba var. spinosa potentiates hippocampal synaptic transmission through mitogen-activated protein kinase, adenylyl cyclase, and protein kinase A pathways.

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Jo, So Yeon; Jung, In Ho; Yi, Jee Hyun; Choi, Tae Joon; Lee, Seungheon; Jung, Ji Wook; Yun, Jeanho; Lee, Young Choon; Ryu, Jong Hoon; Kim, Dong Hyun

2017-03-22

As the seed of Zizyphus jujuba var. spinosa (Bunge) Hu ex H.F. Chow (Rhamnaceae) has been used to sleep disturbances in traditional Chinese and Korean medicine, many previous studies have focused on its sedative effect. Recently, we reported the neuroprotective effect of the effect of Z. jujuba var. spinosa. However, its effects on synaptic function have not yet been studied. In this project, we examined the action of ethanol extract of the seed of Z. jujuba var. spinosa (DHP1401) on synaptic transmission in the hippocampus. To investigate the effects of DHP1401, field recordings were conducted using hippocampal slices (400µm). Object recognition test was introduced to examine whether DHP1401 affect normal recognition memory. DHP1401 (50μg/ml) induced a significant increase in synaptic activity in Shaffer collateral pathway in a concentration-dependent manner. This increase of synaptic responses was blocked by NBQX, a broad spectrum α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor antagonist, but not IEM-1460, a Ca 2+ -permeable AMPAR blocker. Moreover, U0126, a mitogen-activated protein kinase inhibitor, SQ22536, an adenylyl cyclase inhibitor, and PKI, a protein kinase A inhibitor, blocked DHP1401-induced increase in synaptic transmission. Finally, DHP1401 facilitated object recognition memory. These results suggest that DHP1401 increase synaptic transmission through increase of synaptic AMPAR transmission via MAPK, AC and PAK. Copyright © 2017 Elsevier Ireland Ltd. All rights reserved.

Aqueous fraction from Cuscuta japonica seed suppresses melanin synthesis through inhibition of the p38 mitogen-activated protein kinase signaling pathway in B16F10 cells.

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Jang, Ji Yeon; Kim, Ha Neui; Kim, Yu Ri; Choi, Yung Hyun; Kim, Byung Woo; Shin, Hwa Kyoung; Choi, Byung Tae

2012-05-07

Semen cuscutae has been used traditionally to treat pimples and alleviate freckles and melasma in Korea. The present study aimed to investigate the inhibitory effect of Cuscuta japonica Choisy seeds on alpha-melanocyte-stimulating hormone (α-MSH)-induced melanogenesis. The aqueous fraction from Semen cuscutae (AFSC) was used to determine anti-melanogenic effects by examination of cellular melanin contents, tyrosinase activity assay, cAMP assay and Western blot analysis for melanin synthesis-related signaling proteins in B16F10 mouse melanoma cells. AFSC markedly inhibited α-MSH-induced melanin synthesis and tyrosinase activity, and also decreased α-MSH-induced expression of microphthalmia-associated transcription factor (MITF) and tyrosinase-related proteins (TRPs). Moreover, AFSC significantly decreased the level of phosphorylated p38 mitogen-activated protein kinase (MAPK) signaling through the down-regulation of α-MSH-induced cAMP. Furthermore, we confirmed that the specific inhibitor of p38 MAPK (SB203580)-mediated suppressed melanin synthesis and tyrosinase activity was further attenuated by AFSC. AFSC also further decreased SB203580-mediated suppression of MITF and TRP expression. These results indicate that AFSC inhibits p38 MAPK phosphorylation with suppressed cAMP levels and subsequently down-regulate MITF and TRP expression, which results in a marked reduction of melanin synthesis and tyrosinase activity in α-MSH-stimulated B16F10 cells. Copyright © 2012 Elsevier Ireland Ltd. All rights reserved.

Regulation of brain capillary endothelial cells by P2Y receptors coupled to Ca2+, phospholipase C and mitogen-activated protein kinase.

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Albert, J L; Boyle, J P; Roberts, J A; Challiss, R A; Gubby, S E; Boarder, M R

1997-11-01

1. The blood-brain barrier is formed by capillary endothelial cells and is regulated by cell-surface receptors, such as the G protein-coupled P2Y receptors for nucleotides. Here we investigated some of the characteristics of control of brain endothelial cells by these receptors, characterizing the phospholipase C and Ca2+ response and investigating the possible involvement of mitogen-activated protein kinases (MAPK). 2. Using an unpassaged primary culture of rat brain capillary endothelial cells we showed that ATP, UTP and 2-methylthio ATP (2MeSATP) give similar and substantial increases in cytosolic Ca2+, with a rapid rise to peak followed by a slower decline towards basal or to a sustained plateau. Removal of extracellular Ca2+ had little effect on the peak Ca2+-response, but resulted in a more rapid decline to basal. There was no response to alpha,beta-MethylATP (alpha,beta MeATP) in these unpassaged cells, but a response to this P2X agonist was seen after a single passage. 3. ATP (log EC50 -5.1+/-0.2) also caused an increase in the total [3H]-inositol (poly)phosphates ([3H]-InsPx) in the presence of lithium with a rank order of agonist potency of ATP=UTP=UDP>ADP, with 2MeSATP and alpha,beta MeATP giving no detectable response. 4. Stimulating the cells with ATP or UTP gave a rapid rise in the level of inositol 1,4,5-trisphosphate (Ins(1,4,5)P3), with a peak at 10 s followed by a decline to a sustained plateau phase. 2MeSATP gave no detectable increase in the level of Ins(1,4,5)P3. 5. None of the nucleotides tested affected basal cyclic AMP, while ATP and ATPgammaS, but not 2MeSATP, stimulated cyclic AMP levels in the presence of 5 microM forskolin. 6. Both UTP and ATP stimulated tyrosine phosphorylation of p42 and p44 mitogen-activated protein kinase (MAPK), while 2MeSATP gave a smaller increase in this index of MAPK activation. By use of a peptide kinase assay, UTP gave a substantial increase in MAPK activity with a concentration-dependency consistent with

Mitogenic activity of new lectins from seeds of wild Artocarpus species from Vietnam.

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Blasco, E; Ngoc, L D; Aucouturier, P; Preud'Homme, J L; Barra, A

1996-05-01

Proliferative response of human peripheral blood mononuclear cells (PBMC) stimulated by new lectins purified from seeds of differents Artocarpus species from Vietnam (A. asperulus, A. heterophyllus, A. masticata, A. melinoxylus, A. parva and A. petelotii) was studied and compared to those of the lectin jacalin purified from jackfruit (A. heterophyllus) seeds collected in the island La Réunion. All lectins stimulated human PBMC to proliferate, with a variable efficiency of the mitogenic activity. Phenotypic analysis of cells recovered after 7 day-cultures showed that these lectins mostly stimulated CD4+ T lymphocytes. These results suggest that these lectins from different Artocarpus species are similar in terms of their mitogenic activity although their structural features are not identical.

Cancer Cell-derived Exosomes Induce Mitogen-activated Protein Kinase-dependent Monocyte Survival by Transport of Functional Receptor Tyrosine Kinases.

Science.gov (United States)

Song, Xiao; Ding, Yanping; Liu, Gang; Yang, Xiao; Zhao, Ruifang; Zhang, Yinlong; Zhao, Xiao; Anderson, Gregory J; Nie, Guangjun

2016-04-15

Tumor-associated macrophages (TAM) play pivotal roles in cancer initiation and progression. Monocytes, the precursors of TAMs, normally undergo spontaneous apoptosis within 2 days, but can subsist in the inflammatory tumor microenvironment for continuous survival and generation of sufficient TAMs. The mechanisms underlying tumor-driving monocyte survival remain obscure. Here we report that cancer cell-derived exosomes were crucial mediators for monocyte survival in the inflammatory niche. Analysis of the survival-promoting molecules in monocytes revealed that cancer cell-derived exosomes activated Ras and extracellular signal-regulated kinases in the mitogen-activated protein kinase (MAPK) pathway, resulting in the prevention of caspase cleavage. Phosphorylated receptor tyrosine kinases (RTKs), such as phosphorylated epidermal growth factor receptor (EGFR) and human epidermal growth factor receptor 2 (HER-2), were abundantly expressed in cancer cell-derived exosomes. Knock-out of EGFR or/and HER-2, or alternatively, inhibitors against their phosphorylation significantly disturbed the exosome-mediated activation of the MAPK pathway, inhibition of caspase cleavage, and increase in survival rate in monocytes. Moreover, the deprived survival-stimulating activity of exosomes due to null expression of EGFR and HER-2 could be restored by activation of another RTK, insulin receptor. Overall, our study uncovered a mechanism of tumor-associated monocyte survival and demonstrated that cancer cell-derived exosomes can stimulate the MAPK pathway in monocytes through transport of functional RTKs, leading to inactivation of apoptosis-related caspases. This work provides insights into the long sought question on monocyte survival prior to formation of plentiful TAMs in the tumor microenvironment. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

Phosphorylation and activation of p42 and p44 mitogen-activated protein kinase are required for the P2 purinoceptor stimulation of endothelial prostacyclin production.

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Patel, V; Brown, C; Goodwin, A; Wilkie, N; Boarder, M R

1996-11-15

Extracellular ATP and ADP, released from platelets and other sites stimulate the endothelial production of prostacyclin (PGI2) by acting on G-protein-coupled P2Y2 and P2Y2 purinoceptors, contributing to the maintenance of a non-thrombogenic surface. The mechanism, widely described as being dependent on elevated cytosolic [Ca2+], also requires protein tyrosine phosphorylation. Here we show that activation of both these P2 receptor types leads to the tyrosine phosphorylation and activation of both the p42 and p44 forms of mitogen-activated protein kinase (MAPK). 2-Methylthio-ATP and UTP, selectively activating P2Y1 and P2Y2 purinoceptors respectively, and ATP, a non-selective agonist at these two receptors, stimulate the tyrosine phosphorylation of both p42mapk and p44mapk, as revealed by Western blots with an antiserum specific for the tyrosine-phosphorylated forms of the enzymes. By using separation on Resource Q columns, peptide kinase activity associated with the phosphorylated MAPK enzymes distributes into two peaks, one mainly p42mapk and one mainly p44mapk, both of which are stimulated by ATP with respect to kinase activity and phospho-MAPK immunoreactivity. Stimulation of P2Y1 or P2Y2 purinoceptors leads to a severalfold increase in PGI2 efflux; this was blocked in a dose-dependent manner by the selective MAPK kinase inhibitor PD98059. This drug also blocked the agonist-stimulated increase in phospho-MAPK immunoreactivity for both p42mapk and p44mapk but left the phospholipase C response to P2 agonists essentially unchanged. Olomoucine has been reported to inhibit p44mapk activity. Here we show that in the same concentration range olomoucine inhibits activity in both peaks from the Resource Q column and also the agonist stimulation of 6-keto-PGF1, but has no effect on agonist-stimulated phospho-MAPK immunoreactivity. These results provide direct evidence for the involvement of p42 and p44 MAPK in the PGI2 response of intact endothelial cells: we have shown

Toward a comprehensive phylogenetic reconstruction of the evolutionary history of mitogen-activated protein kinases in the plant kingdom.

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Janitza, Philipp; Ullrich, Kristian Karsten; Quint, Marcel

2012-01-01

The mitogen-activated protein kinase (MAPK) pathway is a three-tier signaling cascade that transmits cellular information from the plasma membrane to the cytoplasm where it triggers downstream responses. The MAPKs represent the last step in this cascade and are activated when both tyrosine and threonine residues in a conserved TxY motif are phosphorylated by MAPK kinases, which in turn are themselves activated by phosphorylation by MAPK kinase kinases. To understand the molecular evolution of MAPKs in the plant kingdom, we systematically conducted a Hidden-Markov-Model based screen to identify MAPKs in 13 completely sequenced plant genomes. In this analysis, we included green algae, bryophytes, lycophytes, and several mono- and eudicotyledonous species covering >800 million years of evolution. The phylogenetic relationships of the 204 identified MAPKs based on Bayesian inference facilitated the retraction of the sequence of emergence of the four major clades that are characterized by the presence of a TDY or TEY-A/TEY-B/TEY-C type kinase activation loop. We present evidence that after the split of TDY- and TEY-type MAPKs, initially the TEY-C clade emerged. This was followed by the TEY-B clade in early land plants until the TEY-A clade finally emerged in flowering plants. In addition to these well characterized clades, we identified another highly conserved clade of 45 MAPK-likes, members of which were previously described as Mak-homologous kinases. In agreement with their essential functions, molecular population genetic analysis of MAPK genes in Arabidopsis thaliana accessions reveal that purifying selection drove the evolution of the MAPK family, implying strong functional constraints on MAPK genes. Closely related MAPKs most likely subfunctionalized, a process in which differential transcriptional regulation of duplicates may be involved.

Towards a comprehensive phylogenetic reconstruction of the evolutionary history of mitogen-activated protein kinases in the plant kingdom

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Philipp eJanitza

2012-12-01

Full Text Available The mitogen-activated protein kinase (MAPK pathway is a three-tier signaling cascade that transmits cellular information from the plasma membrane to the cytoplasm where it triggers downstream responses. The MAPKs represent the last step in this cascade and are activated when both tyrosine and threonine residues in a conserved TxY motif are phosphorylated by MAPK kinases, which in turn are themselves activated by phosphorylation by MAPK kinase kinases. To understand the molecular evolution of MAPKs in the plant kingdom, we systematically conducted a Hidden-Markov-Model based screen to identify MAPKs in 13 completely sequenced plant genomes. In this analysis, we included green algae, bryophytes, lycophytes, and several mono- and dicotyledonous species covering >800 million years of evolution. The phylogenetic relationships of the 204 identified MAPKs based on Bayesian inference facilitated the retraction of the sequence of emergence of the four major clades that are characterized by the presence of a TDY or TEY-A/TEY-B/TEY-C type kinase activation loop. We present evidence that after the split of TDY- and TEY-type MAPKs, initially the TEY-C clade emerged. This was followed by the TEY-B clade in early land plants until the TEY-A clade finally emerged in flowering plants. In addition to these well characterized clades, we identified another highly conserved clade of 45 MAPK-likes, members of which were previously described as MHKs. In agreement with their essential functions, molecular population genetic analysis of MAPK genes in Arabidopsis thaliana accessions reveal that purifying selection drove the evolution of the MAPK family, implying strong functional constraints on MAPK genes. Closely related MAPKs most likely subfunctionalized, a process in which differential transcriptional regulation of duplicates may be involved.

Pentachlorophenol-Induced Cytotoxic, Mitogenic, and Endocrine-Disrupting Activities in Channel Catfish, Ictalurus punctatus

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Paul B. Tchounwou

2004-09-01

Full Text Available Pentachlorophenol (PCP is an organochlorine compound that has been widely used as a biocide in several industrial, agricultural, and domestic applications. Although it has been shown to induce systemic toxicity and carcinogenesis in several experimental studies, the literature is scarce regarding its toxic mechanisms of action at the cellular and molecular levels. Recent investigations in our laboratory have shown that PCP induces cytotoxicity and transcriptionally activates stress genes in human liver carcinoma (HepG2 cells [1]. In this research, we hypothesize that environmental exposure to PCP may trigger cytotoxic, mitogenic, and endocrine-disrupting activities in aquatic organisms including fish. To test this hypothesis, we carried out in vitro cultures of male channel catfish hepatocytes, and performed the fluorescein diacetate assay (FDA to assess for cell viability, and the Western Blot analysis to assess for vitellogenin expression following exposure to PCP. Data obtained from FDA experiments indicated a strong dose-response relationship with respect to PCP cytotoxicity. Upon 48 hrs of exposure, the chemical dose required to cause 50% reduction in cell viability (LD50 was computed to be 1,987.0 + 9.6 μg PCP/mL. The NOAEL and LOAEL were 62.5 + 10.3 μg PCP/mL and 125.0+15.2 μg PCP/mL, respectively. At lower levels of exposure, PCP was found to be mitogenic, showing a strong dose- and time-dependent response with regard to cell proliferation. Western Blot analysis demonstrated the potential of PCP to cause endocrine-disrupting activity, as evidenced by the up regulation of the 125-kDa vitellogenin protein the hepatocytes of male channel catfish.

The Histone Deacetylase Inhibitors MS-275 and SAHA Suppress the p38 Mitogen-Activated Protein Kinase Signaling Pathway and Chemotaxis in Rheumatoid Arthritic Synovial Fibroblastic E11 Cells

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Hai-Shu Lin

2013-11-01

Full Text Available MS-275 (entinostat and SAHA (vorinostat, two histone deacetylase (HDAC inhibitors currently in oncological trials, have displayed potent anti-rheumatic activities in rodent models of rheumatoid arthritis (RA. To further elucidate their anti-inflammatory mechanisms, the impact of MS-275 and SAHA on the p38 mitogen-activated protein kinase (MAPK signaling pathway and chemotaxis was assessed in human rheumatoid arthritic synovial fibroblastic E11 cells. MS-275 and SAHA significantly suppressed the expression of p38α  MAPK, but induced the expression of MAPK phosphatase-1 (MKP-1, an endogenous suppressor of p38α  in E11 cells. At the same time, the association between p38α and MKP-1 was up-regulated and consequently, the activation (phosphorylation of p38α  was inhibited. Moreover, MS-275 and SAHA suppressed granulocyte chemotactic protein-2 (GCP-2, monocyte chemotactic protein-2 (MCP-2 and macrophage migration inhibitory factor (MIF in E11 cells in a concentration-dependent manner. Subsequently, E11-driven migration of THP-1 and U937 monocytes was inhibited. In summary, suppression of the p38 MAPK signaling pathway and chemotaxis appear to be important anti-rheumatic mechanisms of action of these HDAC inhibitors.

Carprofen induction of p75NTR-dependent apoptosis via the p38 mitogen-activated protein kinase pathway in prostate cancer cells.

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Khwaja, Fatima S; Quann, Emily J; Pattabiraman, Nagarajan; Wynne, Shehla; Djakiew, Daniel

2008-11-01

The p75 neurotrophin receptor (p75(NTR)) functions as a tumor suppressor in prostate epithelial cells, where its expression declines with progression to malignant cancer. Previously, we showed that treatment with R-flurbiprofen or ibuprofen induced p75(NTR) expression in several prostate cancer cell lines leading to p75(NTR)-mediated decreased survival. Using the 2-phenyl propionic acid moiety of these profens as a pharmacophore, we screened an in silico database of 30 million compounds and identified carprofen as having an order of magnitude greater activity for induction of p75(NTR) levels and inhibition of cell survival. Prostate (PC-3 and DU-145) and bladder (T24) cancer cells were more sensitive to carprofen induction of p75(NTR)-associated loss of survival than breast (MCF-7) and fibroblast (3T3) cells. Transfection of prostate cell lines with a dominant-negative form of p75(NTR) before carprofen treatment partially rescued cell survival, showing a cause-and-effect relationship between carprofen induction of p75(NTR) levels and inhibition of survival. Carprofen induced apoptotic nuclear fragmentation in prostate but not in MCF-7 and 3T3 cells. Furthermore, small interfering RNA knockdown of the p38 mitogen-activated protein kinase (MAPK) protein prevented induction of p75(NTR) by carprofen in both prostate cell lines. Carprofen treatment induced phosphorylation of p38 MAPK as early as within 1 min. Expression of a dominant-negative form of MK2, the kinase downstream of p38 MAPK frequently associated with signaling cascades leading to apoptosis, prevented carprofen induction of the p75(NTR) protein. Collectively, we identify carprofen as a highly potent profen capable of inducing p75(NTR)-dependent apoptosis via the p38 MAPK pathway in prostate cancer cells.

The Air Liquid-interface, a Skin Microenvironment, Promotes Growth of Melanoma Cells, but not Their Apoptosis and Invasion, through Activation of Mitogen-activated Protein Kinase

International Nuclear Information System (INIS)

Hong Yee, Chong; Aoki, Shigehisa; Uchihashi, Kazuyoshi; Matsunobu, Aki; Yamasaki, Fumio; Misago, Noriyuki; Piao, Meihua; Tetsuji, Uemura; Yonemitsu, Nobuhisa; Sugihara, Hajime; Toda, Shuji

2010-01-01

The air-liquid interface (ALI) is a common microenvironment of the skin, but it is unknown whether the ALI affects melanoma cell behaviors. Using a collagen gel invasion assay, immunohistochemistry, and Western blots, here we show that melanoma cell proliferation in cultures with an ALI is higher than melanoma cell proliferation in submerged cultures. Bromodeoxyuridine (BrdU) uptake, an indicator of cell proliferation, of melanoma cells at the ALI was about 3 times that of submerged cells, while ALI and submerged melanoma cells had similar levels of single-stranded DNA (a marker of apoptosis). The ALI enhanced the expression of Raf-1, MEK-1 and pERK-1/2 components of the mitogen-activated protein kinase (MAPK) cascade, in cells more than the submerged condition did. The increases in BrdU uptake and pERK-1/2 expression promoted by ALI was abolished by the MEK inhibitor, PD-98059. ALI-treated and submerged melanoma cells did not infiltrate into the collagen gel, and they showed no significant difference in the expression of the invasion- and motility-related molecules, matrix metalloproteinase-1 and -9, laminin 5, and filamin A. Our data indicate that the ALI, a skin microenvironment, accelerates the growth, but not the apoptosis or invasion, of melanoma cells through MAPK activation

Novel mitogen-activated protein kinase MpkC of Aspergillus fumigatus is required for utilization of polyalcohol sugars.

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Reyes, Guadalupe; Romans, Angela; Nguyen, C Kim; May, Gregory S

2006-11-01

The genome of Aspergillus fumigatus has four genes that encode mitogen-activated protein kinases (MAPKs), sakA/hogA, mpkA, mpkB, and mpkC. The functions of the MpkB and MpkC MAPKs are unknown for A. fumigatus and the closely related and genetically amenable species Aspergillus nidulans. mpkC deletion mutants of A. fumigatus were made and their phenotypes characterized. The mpkC deletion mutants were viable and had normal conidial germination and hyphal growth on minimal or complete media. This is in contrast to deletion mutants with deletions in the closely related MAPK gene sakA/hogA that we previously reported had a nitrogen source-dependent germination phenotype. Similarly, the growth of the mpkC deletion mutants was wild type on high-osmolarity medium. Consistent with these two MAP kinase genes regulating different cellular responses, we determined that the mpkC deletion mutants were unable to grow on minimal medium with sorbitol or mannitol as the sole carbon source. This result implicates MpkC signaling in carbon source utilization. Changes in mRNA levels for sakA and mpkC were measured in response to hypertonic stress, oxidative stress, and a shift from glucose to sorbitol to determine if there was overlap in the SakA and MpkC signaling pathways. These studies demonstrated that SakA- and MpkC-dependent patterns of change in mRNA levels are distinct and have minimal overlap in response to these environmental stresses.

Influence of protein depletion and repletion on lymphocyte activation in adult mice

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Elliott, T.S.; Olson, L.M.; Visek, W.J.

1986-01-01

Male 8 month old Balb/c mice were fed 0.5% (Depletion, n = 126) or 6% (Control, n = 16) protein diets for 5 wks. All control and 16 depleted mice were killed (time 0), and the remaining 110 mice were randomly assigned to 1 of 3 diets (3, 6, or 12% protein) and fed for 4, 8, or 32 additional days. Diets were formulated following AIN-76A guidelines with skim milk powder used as the source of protein. Serum albumin and 3 H-thymidine incorporation into splenocytes stimulated by the T-cell mitogens concanavalin A (Con A), or phytohemagglutinin (PHA), and the B-cell mitogen lipopolysaccharide (LPS) were determined. At time 0, mice fed 0.5% protein had lost 32% of their initial body weight and weighed significantly less than controls (19.5 +/- 0.1 g vs 29.9 +/- 0.7 g, p < 0.0001). Protein level did not affect food intake or spleen weight/body weight. Depletion significantly decreased serum albumin concentrations (4.6 +/- 0.1 g/dl vs 6.4 +/- 0.6 g/dl) and responses to PHA and LPS. During repletion mice fed 3% protein weighted significantly less than mice fed 6 or 12% ptn (p < 0.001) despite a significantly higher food intake. The activation of splenocytes by all mitogens increased with time of repletion but was independent of dietary protein concentration

Stress-induced activation of protein kinase CK2 by direct interaction with p38 mitogen-activated protein kinase

DEFF Research Database (Denmark)

Sayed, M; Kim, S O; Salh, B S

2000-01-01

Protein kinase CK2 has been implicated in the regulation of a wide range of proteins that are important in cell proliferation and differentiation. Here we demonstrate that the stress signaling agents anisomycin, arsenite, and tumor necrosis factor-alpha stimulate the specific enzyme activity of CK2...... in the human cervical carcinoma HeLa cells by up to 8-fold, and this could be blocked by the p38 MAP kinase inhibitor SB203580. We show that p38alpha MAP kinase, in a phosphorylation-dependent manner, can directly interact with the alpha and beta subunits of CK2 to activate the holoenzyme through what appears...

Proteomics reveal energy metabolism and mitogen-activated protein kinase signal transduction perturbation in human Borna disease virus Hu-H1-infected oligodendroglial cells.

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Liu, X; Yang, Y; Zhao, M; Bode, L; Zhang, L; Pan, J; Lv, L; Zhan, Y; Liu, S; Zhang, L; Wang, X; Huang, R; Zhou, J; Xie, P

2014-05-30

Borna disease virus (BDV) is a neurotropic, non-cytolytic RNA virus which replicates in the cell nucleus targeting mainly hippocampal neurons, but also astroglial and oligodendroglial cells in the brain. BDV is associated with a large spectrum of neuropsychiatric pathologies in animals. Its relationship to human neuropsychiatric illness still remains controversial. We could recently demonstrate that human BDV strain Hu-H1 promoted apoptosis and inhibited cell proliferation in a human oligodendroglial cell line (OL cells) whereas laboratory BDV strain V acted contrariwise. Here, differential protein expression between BDV Hu-H1-infected OL cells and non-infected OL cells was assessed through a proteomics approach, using two-dimensional electrophoresis followed by matrix-assisted laser desorption ionization-time of flight tandem mass spectrometry. A total of 63 differential host proteins were identified in BDV Hu-H1-infected OL cells compared to non-infected OL cells. We found that most changes referred to alterations related to the pentose phosphate pathway, glyoxylate and dicarboxylate metabolism, the tricarboxylic acid (TCA) cycle, and glycolysis /gluconeogenesis. By manual querying, two differential proteins were found to be associated with mitogen-activated protein kinase (MAPK) signal transduction. Five key signaling proteins of this pathway (i.e., p-Raf, p-MEK, p-ERK1/2, p-RSK, and p-MSK) were selected for Western blotting validation. p-ERK1/2 and p-RSK were found to be significantly up-regulated, and p-MSK was found to be significantly down-regulated in BDV Hu-H1-infected OL cells compared to non-infected OL cell. Although BDV Hu-H1 constitutively activated the ERK-RSK pathway, host cell proliferation and nuclear translocation of activated pERK in BDV Hu-H1-infected OL cells were impaired. These findings indicate that BDV Hu-H1 infection of human oligodendroglial cells significantly perturbs host energy metabolism, activates the downstream ERK-RSK complex of

Barley disease susceptibility factor RACB acts in epidermal cell polarity and positioning of the nucleus.

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Scheler, Björn; Schnepf, Vera; Galgenmüller, Carolina; Ranf, Stefanie; Hückelhoven, Ralph

2016-05-01

RHO GTPases are regulators of cell polarity and immunity in eukaryotes. In plants, RHO-like RAC/ROP GTPases are regulators of cell shaping, hormone responses, and responses to microbial pathogens. The barley (Hordeum vulgare L.) RAC/ROP protein RACB is required for full susceptibility to penetration by Blumeria graminis f.sp. hordei (Bgh), the barley powdery mildew fungus. Disease susceptibility factors often control host immune responses. Here we show that RACB does not interfere with early microbe-associated molecular pattern-triggered immune responses such as the oxidative burst or activation of mitogen-activated protein kinases. RACB also supports rather than restricts expression of defence-related genes in barley. Instead, silencing of RACB expression by RNAi leads to defects in cell polarity. In particular, initiation and maintenance of root hair growth and development of stomatal subsidiary cells by asymmetric cell division is affected by silencing expression of RACB. Nucleus migration is a common factor of developmental cell polarity and cell-autonomous interaction with Bgh RACB is required for positioning of the nucleus near the site of attack from Bgh We therefore suggest that Bgh profits from RACB's function in cell polarity rather than from immunity-regulating functions of RACB. © The Author 2016. Published by Oxford University Press on behalf of the Society for Experimental Biology.

Monosodium iodoacetate-induced joint pain is associated with increased phosphorylation of mitogen activated protein kinases in the rat spinal cord

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Jarvis Michael F

2011-05-01

Full Text Available Abstract Background Intra-articular injection of monosodium iodoacetate (MIA in the knee joint of rats disrupts chondrocyte metabolism resulting in cartilage degeneration and subsequent nociceptive behavior that has been described as a model of osteoarthritis (OA pain. Central sensitization through activation of mitogen activated protein kinases (MAPKs is recognized as a pathogenic mechanism in chronic pain. In the present studies, induction of central sensitization as indicated by spinal dorsal horn MAPK activation, specifically ERK and p38 phosphorylation, was assessed in the MIA-OA model. Results Behaviorally, MIA-injected rats displayed reduced hind limb grip force 1, 2, and 3 weeks post-MIA treatment. In the same animals, activation of phospho ERK1/2 was gradually increased, reaching a significant level at post injection week 3. Conversely, phosphorylation of p38 MAPK was enhanced maximally at post injection week 1 and decreased, but remained elevated, thereafter. Double labeling from 3-wk MIA rats demonstrated spinal pERK1/2 expression in neurons, but not glia. In contrast, p-p38 was expressed by microglia and a subpopulation of neurons, but not astrocytes. Additionally, there was increased ipsilateral expression of microglia, but not astrocytes, in 3-wk MIA-OA rats. Consistent with increased MAPK immunoreactivity in the contralateral dorsal horn, mechanical allodynia to the contralateral hind-limb was observed 3-wk following MIA. Finally, intrathecal injection of the MEK1 inhibitor PD98059 blocked both reduced hind-limb grip force and pERK1/2 induction in MIA-OA rats. Conclusion Results of these studies support the role of MAPK activation in the progression and maintenance of central sensitization in the MIA-OA experimental pain model.

The investigation of Mitogen-Activated Protein kinase Phosphatase-1 as a potential pharmacological target in non-small cell lung carcinomas, assisted by non-invasive molecular imaging

International Nuclear Information System (INIS)

Tai, Cheng-Jeng; Lee, Horng-Mo; Deng, Win-Ping; Wu, Alexander TH; Chiou, Jeng-Feng; Jan, Hsun-Jin; Wei, Hon-Jian; Hsu, Chung-Huei; Lin, Che-Tong; Chiu, Wen-Ta; Wu, Cheng-Wen

2010-01-01

Invasiveness and metastasis are the most common characteristics of non small cell lung cancer (NSCLC) and causes of tumour-related morbidity and mortality. Mitogen-activated protein kinases (MAPKs) signalling pathways have been shown to play critical roles in tumorigenesis. However, the precise pathological role(s) of mitogen-activated protein kinase phosphatase-1 (MKP-1) in different cancers has been controversial such that the up-regulation of MKP-1 in different cancers does not always correlate to a better prognosis. In this study, we showed that the induction of MKP-1 lead to a significant retardation of proliferation and metastasis in NSCLC cells. We also established that rosiglitazone (a PPARγ agonist) elevated MKP-1 expression level in NSCLC cells and inhibited tumour metastasis. Both wildtype and dominant negative forms of MKP-1 were constitutively expressed in NSCLC cell line H441GL. The migration and invasion abilities of these cells were examined in vitro. MKP-1 modulating agents such as rosiglitazone and triptolide were used to demonstrate MKP-1's role in tumorigenesis. Bioluminescent imaging was utilized to study tumorigenesis of MKP-1 over-expressing H441GL cells and anti-metastatic effect of rosiglitazone. Over-expression of MKP-1 reduced NSCLC cell proliferation rate as well as cell invasive and migratory abilities, evident by the reduced expression levels of MMP-2 and CXCR4. Mice inoculated with MKP-1 over-expressing H441 cells did not develop NSCLC while their control wildtype H441 inoculated littermates developed NSCLC and bone metastasis. Pharmacologically, rosiglitazone, a peroxisome proliferator activated receptor-γ (PPARγ) agonist appeared to induce MKP-1 expression while reduce MMP-2 and CXCR4 expression. H441GL-inoculated mice receiving daily oral rosiglitazone treatment demonstrated a significant inhibition of bone metastasis when compared to mice receiving sham treatment. We found that rosiglitazone treatment impeded the ability

Lysophosphatidylcholine Promotes Phagosome Maturation and Regulates Inflammatory Mediator Production Through the Protein Kinase A–Phosphatidylinositol 3 Kinase–p38 Mitogen-Activated Protein Kinase Signaling Pathway During Mycobacterium tuberculosis Infection in Mouse Macrophages

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Hyo-Ji Lee

2018-04-01

Full Text Available Tuberculosis is caused by the infectious agent Mycobacterium tuberculosis (Mtb. Mtb has various survival strategies, including blockade of phagosome maturation and inhibition of antigen presentation. Lysophosphatidylcholine (LPC is a major phospholipid component of oxidized low-density lipoprotein and is involved in various cellular responses, such as activation of second messengers and bactericidal activity in neutrophils. In this study, macrophages were infected with a low infectious dose of Mtb and treated with LPC to investigate the bactericidal activity of LPC against Mtb. In macrophages infected with Mtb strain, H37Ra or H37Rv, LPC suppressed bacterial growth; however, this effect was suppressed in bone marrow-derived macrophages (BMDMs isolated from G2A (a G protein-coupled receptor involved in some LPC actions knockout mice. LPC also promoted phagosome maturation via phosphatidylinositol 3 kinase (PI3K–p38 mitogen-activated protein kinase (MAPK-mediated reactive oxygen species production and intracellular Ca2+ release during Mtb infection. In addition, LPC induced increased levels of intracellular cyclic adenosine monophosphate (cAMP and phosphorylated glycogen synthase kinase 3 beta (GSK3β in Mtb-infected macrophages. Protein kinase A (PKA-induced phosphorylation of GSK3β suppressed activation of NF-κB in LPC-treated macrophages during Mtb infection, leading to decreased secretion of pro-inflammatory cytokines and increased secretion of anti-inflammatory cytokines. These results suggest that LPC can effectively control Mtb growth by promoting phagosome maturation via cAMP-induced activation of the PKA–PI3K–p38 MAPK pathway. Moreover, LPC can regulate excessive production of pro-inflammatory cytokines associated with bacterial infection of macrophages.

KFC, a Ste20-like kinase with mitogenic potential and capability to activate the SAPK/JNK pathway.

Science.gov (United States)

Yustein, J T; Li, D; Robinson, D; Kung, H J

2000-02-03

The Sterile-20 (Ste20) family of serine-threonine kinases has been implicated in the activation of the stress-activated protein kinase pathways. However, the physiological role has remained ambiguous for most of the investigated mammalian Ste20's. Here we report the cloning of a novel Ste20-like kinase, from chicken embryo fibroblast (CEF) cells, which we have named KFC, for Kinase From Chicken. The 898 amino acid full-length KFC protein contains an amino-terminal kinase domain, an adjacent downstream serine-rich region, and a C-terminal tail containing a coiled-coil domain. Here we show that the coiled-coil domain of KFC negatively regulates the intrinsic kinase activity. We have also identified a splice variant of KFC in which there is a 207 nucleotide in-frame deletion. This deletion of 69 amino acids encompasses the serine-rich region. These two isoforms, called KFCL, for full-length, and KFCS for spliced (or short) form, not only differ in structure, but also in biological properties. Stable CEF cells overexpressing KFCL, but not KFCS, have a significant increase in growth rate when compared to parental cells. This mitogenic effect is the first such reported for this family of kinases. Finally, we found that KFC, when activated by truncation of the regulatory C-terminus, has a specific activation of the stress-activated protein kinase (SAPK/JNK) pathway.

p38 mitogen-activated protein kinase (p38MAPK) upregulates catalase levels in response to low dose H2O2 treatment through enhancement of mRNA stability.

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Sen, Prosenjit; Chakraborty, Prabir Kumar; Raha, Sanghamitra

2005-08-15

V79 fibroblasts were repetitively stressed through multiple exposures to a low dose (30 microM) H2O2 in culture for 4 weeks. Catalase activity, protein levels and mRNA levels increased markedly (5-6-fold) during this time and these augmentations were inhibited by the simultaneous presence of SB203580, an inhibitor of p38 mitogen-activated protein kinase (p38MAPK). p38MAPK became dually phosphorylated and ATF-2, a p38MAPK substrate also became increasingly phosphorylated over the repetitive stress period. Short interfering RNA that induced effective silencing of p38MAPK, was used to silence p38MAPK in V79 fibroblasts. Silencing of p38MAPK drastically hindered the elevation in catalase (protein and mRNA) levels observed after a single low dose (50 microM) of H2O. The rise in catalase mRNA levels induced by low concentration (single and multiple dose) H2O2 treatment was established to be unconnected with transcriptional upregulation but was brought forth primarily by an enhancement in catalase mRNA stability through the action of p38MAPK. Therefore, our data strongly indicate that activation of p38MAPK is a key controlling step in the upregulation of catalase levels by low dose H2O2 treatment.

Novel Mitogen-Activated Protein Kinase MpkC of Aspergillus fumigatus Is Required for Utilization of Polyalcohol Sugars▿

Science.gov (United States)

Reyes, Guadalupe; Romans, Angela; Nguyen, C. Kim; May, Gregory S.

2006-01-01

The genome of Aspergillus fumigatus has four genes that encode mitogen-activated protein kinases (MAPKs), sakA/hogA, mpkA, mpkB, and mpkC. The functions of the MpkB and MpkC MAPKs are unknown for A. fumigatus and the closely related and genetically amenable species Aspergillus nidulans. mpkC deletion mutants of A. fumigatus were made and their phenotypes characterized. The mpkC deletion mutants were viable and had normal conidial germination and hyphal growth on minimal or complete media. This is in contrast to deletion mutants with deletions in the closely related MAPK gene sakA/hogA that we previously reported had a nitrogen source-dependent germination phenotype. Similarly, the growth of the mpkC deletion mutants was wild type on high-osmolarity medium. Consistent with these two MAP kinase genes regulating different cellular responses, we determined that the mpkC deletion mutants were unable to grow on minimal medium with sorbitol or mannitol as the sole carbon source. This result implicates MpkC signaling in carbon source utilization. Changes in mRNA levels for sakA and mpkC were measured in response to hypertonic stress, oxidative stress, and a shift from glucose to sorbitol to determine if there was overlap in the SakA and MpkC signaling pathways. These studies demonstrated that SakA- and MpkC-dependent patterns of change in mRNA levels are distinct and have minimal overlap in response to these environmental stresses. PMID:16998074

Crosstalk between Smad and Mitogen-Activated Protein Kinases for the Regulation of Apoptosis in Cyclosporine A- Induced Renal Tubular Injury

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Hideyuki Iwayama

2011-10-01

Full Text Available Background/Aims: It remains elusive whether there is a crosstalk between Smad and mitogen-activated protein kinases (MAPKs and whether it regulates cyclosporine A (CyA-induced apoptosis in renal proximal tubular cells (RPTCs. Methods: The effect of CyA on nuclear translocation of Smad2/3 and MAPKs (measured by Western blotting or immunofluorescence and apoptosis (determined by Hoechst 33258 staining was examined in HK-2 cells. Results: CyA induced apoptosis at 24 h and nuclear translocation of phosphorylated (p-Smad2/3 at 3 h, which was continued till 24 h. CyA enhanced the expression of p-ERK at 1 h, which was continued till 24 h, and of p-p38MAPK at 1–6 h, which returned to control level at 12 h. CyA did not affect JNK. An inhibitor of ERK, PD98059, prevented CyA-induced nuclear translocation of Smad2/3 and apoptosis. An inhibitor of p38MAPK, SB202190, deteriorated CyA-induced nuclear translocation of p-Smad2/3. Epidermal growth factor (EGF activated ERK and p38MAPK but not JNK. EGF-induced activation of MAPKs ameliorated CyA-induced nuclear translocation of p-Smad2/3 and apoptosis. Inhibition of p38MAPK but not of ERK abolished the protective effect of EGF on CyA-induced nuclear translocation of p-Smad2/3 and apoptosis. Conclusion: Crosstalk between R-Smad and p38MAPK/ERK, but not JNK differentially regulates apoptosis in CyA-induced RPTC injury.

17beta-estradiol promotes the odonto/osteogenic differentiation of stem cells from apical papilla via mitogen-activated protein kinase pathway.

Science.gov (United States)

Li, Yao; Yan, Ming; Wang, Zilu; Zheng, Yangyu; Li, Junjun; Ma, Shu; Liu, Genxia; Yu, Jinhua

2014-11-17

Estrogen plays an important role in the osteogenic differentiation of mesenchymal stem cells, while stem cells from apical papilla (SCAP) can contribute to the formation of dentin/bone-like tissues. To date, the effects of estrogen on the differentiation of SCAP remain unclear. SCAP was isolated and treated with 10⁻⁷ M 17beta-estradiol (E2). The odonto/osteogenic potency and the involvement of mitogen-activated protein kinase (MAPK) signaling pathway were subsequently investigated by using methyl-thiazolyl-tetrazolium (MTT) assay, and other methods. MTT and flow cytometry results demonstrated that E2 treatment had no effect on the proliferation of SCAP in vitro, while alkaline phosphatase (ALP) assay and alizarin red staining showed that E2 can significantly promote ALP activity and mineralization ability in SCAP. Real-time reverse transcription polymerase chain reaction (RT-PCR) and western blot assay revealed that the odonto/osteogenic markers (ALP, DMP1/DMP1, DSPP/DSP, RUNX2/RUNX2, OSX/OSX and OCN/OCN) were significantly upregulated in E2-treated SCAP. In addition, the expression of phosphor-p38 and phosphor-JNK in these stem cells was enhanced by E2 treatment, as was the expression of the nuclear downstream transcription factors including phosphor-Sp1, phosphor-Elk-1, phosphor-c-Jun and phosphor-c-Fos, indicating the activation of MAPK signaling pathway during the odonto/osteogenic differentiation of E2-treated SCAP. Conversely, the differentiation of E2-treated SCAP was inhibited in the presence of MAPK specific inhibitors. The ondonto/osteogenic differentiation of SCAP is enhanced by 10⁻⁷ M 17beta-estradiol via the activation of MAPK signaling pathway.

Lack of direct mitogenic activity of dichloroacetate and trichloroacetate in cultured rat hepatocytes

International Nuclear Information System (INIS)

Walgren, Jennie L.; Kurtz, David T.; McMillan, JoEllyn M.

2005-01-01

Dichloroacetate (DCA) and trichloroacetate (TCA) are hepatocarcinogenic metabolites of the common groundwater contaminant, 1,1,2-trichloroethylene. DCA and TCA have been shown to induce hepatocyte proliferation in vivo, but it is not known if this response is the result of direct mitogenic activity or whether cell replication occurs indirectly in response to tissue injury or inflammation. In this study we used primary cultures of rat hepatocytes, a species susceptible to DCA- but not TCA-induced hepatocarcinogenesis, to determine whether DCA and TCA are direct hepatocyte mitogens. Rat hepatocytes, cultured in growth factor-free medium, were treated with 0.01-1.0 mM DCA or TCA for 10-40 h; cell replication was then assessed by measuring incorporation of 3 H-thymidine into DNA and by cell counts. DCA or TCA treatment did not alter 3 H-thymidine incorporation in the cultured hepatocytes. Although an increase in cell number was not observed, DCA treatment significantly abrogated the normal background cell loss, suggesting an ability to inhibit apoptotic cell death in primary hepatocyte cultures. Furthermore, treatment with DCA synergistically enhanced the mitogenic response to epidermal growth factor. The data indicate that DCA and TCA are not direct mitogens in hepatocyte cultures, which is of interest in view of their ability to stimulate hepatocyte replication in vivo. Nevertheless, the synergistic enhancement of epidermal growth factor-induced hepatocyte replication by DCA is of particular interest and warrants further study

Induced overexpression of protein kinase D1 stimulates mitogenic signaling in human pancreatic carcinoma PANC-1 cells.

Science.gov (United States)

Kisfalvi, Krisztina; Hurd, Cliff; Guha, Sushovan; Rozengurt, Enrique

2010-05-01

Neurotensin (NT) stimulates protein kinase D1 (PKD1), extracellular signal regulated kinase (ERK), c-Jun N-terminal Kinase (JNK), and DNA synthesis in the human pancreatic adenocarcinoma cell line PANC-1. To determine the effect of PKD1 overexpression on these biological responses, we generated inducible stable PANC-1 clones that express wild-type (WT) or kinase-dead (K618N) forms of PKD1 in response to the ecdysone analog ponasterone-A (PonA). NT potently stimulated c-Jun Ser(63) phosphorylation in both wild type and clonal derivatives of PANC-1 cells. PonA-induced expression of WT, but not K618N PKD1, rapidly blocked NT-mediated c-Jun Ser(63) phosphorylation either at the level of or upstream of MKK4, a dual-specificity kinase that leads to JNK activation. This is the first demonstration that PKD1 suppresses NT-induced JNK/cJun activation in PANC-1 cells. In contrast, PKD1 overexpression markedly increased the duration of NT-induced ERK activation in these cells. The reciprocal influence of PKD1 signaling on pro-mitogenicERK and pro-apopotic JNK/c-Jun pathways prompted us to examine whether PKD1 overexpression promotes DNA synthesis and proliferation of PANC-1 cells. Our results show that PKD1 overexpression increased DNA synthesis and cell numbers of PANC-1 cells cultured in regular dishes or in polyhydroxyethylmethacrylate [Poly-(HEMA)]-coated dishes to eliminate cell adhesion (anchorage-independent growth). Furthermore, PKD1 overexpression markedly enhanced DNA synthesis induced by NT (1-10 nM). These results indicate that PKD1 mediates mitogenic signaling in PANC-1 and suggests that this enzyme could be a novel target for the development of therapeutic drugs that restrict the proliferation of these cells.

Differential expression of insulin like growth factor I and other fibroblast mitogens in porcine colostrum and milk

International Nuclear Information System (INIS)

Tan, T.J.; Simmen, R.C.M.; Simmen, F.A.

1987-01-01

Sow mammary secretions contain at least 3 distinct growth factor activities, distinguished by their size and relative abundance in colostrum or later milk. Gel filtration of colostrum in Sephadex G-200 columns, followed by acid-ethanol extraction and radioimmunoassay (RIA) for insulin like growth factor I (IGF-I) revealed high levels of this factor in the 150K and 50K MW regions, characteristic of IGF-I: binding protein complexes. Acid treatment of these fractions yielded free IGF-I peptide (7.5K). Parallel mitogen assays with a fibroblast cell line (AKR-2B) demonstrated a predominant peak of high MW activity (sow colostral growth factor-I, SCGF-I) eluting near the column void volume (MW > 150K). Treatment of SCGF-I with 1M acetic acid resulted in a size reduction of the mitogenic activity (MW < 10K), suggesting association of SCGF-I with a binding protein. The SCGF-I peptide was noncompetitive in IGF-I RIA, was distinct in MW from free IGF-I, and was not mitogenic for chick embryo fibroblasts. Sow milk contains less IGF-I and SCGF-I but does display a predominant peak of small MW (∼ 3K) AKR-2B activity. The changes in expression of these growth factors during lactation may reflect differing roles in lactogenesis and/or neonatal growth and development

ASH1L Suppresses Matrix Metalloproteinase through Mitogen-activated Protein Kinase Signaling Pathway in Pulpitis.

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Bei, Yin; Tianqian, Hui; Fanyuan, Yu; Haiyun, Luo; Xueyang, Liao; Jing, Yang; Chenglin, Wang; Ling, Ye

2017-02-01

Pulpitis is an inflammation of dental pulp produced by a response to external stimuli. The response entails substantial cellular and molecular activities. Both genetic and epigenetic regulators contribute to the occurrence of pulpitis. However, the epigenetic mechanisms are still poorly understood. In this research, we studied the role of the absent, small, or homeotic-like (ASH1L) gene in the process of pulpitis. Human dental pulp cells (HDPCs) were stimulated with proinflammatory cytokine tumor necrosis factor alpha (TNF-α). Gene expression profiling was performed to assess the occurrence of epigenetic regulators. Pulp tissue from rat experimental pulpitis was subjected to immunofluorescence to detect the occurrence of ASH1L and trimethylation of lysine 4 histone 3 (H3K4me3). The presence of ASH1L in HDPCs that had been generated by TNF-α stimulation was analyzed by Western blot procedures and cellular immunofluorescence. Once detected, ASH1L was silenced through the use of specific small interfering RNA. The effects of ASH1L on the occurrence and operation of matrix metalloproteinases (MMPs) were then tested by analysis of quantitative polymerase chain reactions, Western blotting, and zymography. Chromatin immunoprecipitation was performed to detect whether ASH1L and H3K4me3 were present in the promoter regions of MMPs. We then used Western blot procedures to examine the nuclear factor kappa B and the mitogen-activated protein kinase (MAPK) responses to the silencing of ASH1L. We also examined the specific pathway involved in ASH1L regulation of the MMPs. After stimulating HDPCs with TNF-α, ASH1L emerged as 1 of the most strongly induced epigenetic mediators. We found that TNF-α treatment induced the expression of ASH1L through the nuclear factor kappa B and MAPK signal pathways. ASH1L was found in both the nucleus and the cytoplasm. TNF-α treatment was particularly active in inducing the accumulation of ASH1L in cellular cytoplasm. As is also consistent

Nicotine stimulates urokinase-type plasminogen activator receptor expression and cell invasiveness through mitogen-activated protein kinase and reactive oxygen species signaling in ECV304 endothelial cells

Energy Technology Data Exchange (ETDEWEB)

Khoi, Pham Ngoc; Park, Jung Sun; Kim, Nam Ho; Jung, Young Do, E-mail: ydjung@chonnam.ac.kr

2012-03-01

Urokinase-type plasminogen activator receptor (uPAR) expression is elevated during inflammation, tissue remodeling and in many human cancers. This study investigated the effect of nicotine, a major alkaloid in tobacco, on uPAR expression and cell invasiveness in ECV304 endothelial cells. Nicotine stimulated uPAR expression in a dose-dependent manner and activated extracellular signal-regulated kinases-1/2 (Erk-1/2), c-Jun amino-terminal kinase (JNK) and p38 mitogen activated protein kinase (MAPK). Specific inhibitors of MEK-1 (PD98059) and JNK (SP600125) inhibited the nicotine-induced uPAR expression, while the p38 MAPK inhibitor SB203580 did not. Expression vectors encoding dominant negative MEK-1 (pMCL-K97M) and JNK (TAM67) also prevented nicotine-induced uPAR promoter activity. The intracellular hydrogen peroxide (H{sub 2}O{sub 2}) content was increased by nicotine treatment. The antioxidant N-acetylcysteine prevented nicotine-activated production of reactive oxygen species (ROS) and uPAR expression. Furthermore, exogenous H{sub 2}O{sub 2} increased uPAR mRNA expression. Deleted and site-directed mutagenesis demonstrated the involvement of the binding sites of transcription factor nuclear factor-kappaB (NF-κB) and activator protein (AP)-1 in the nicotine-induced uPAR expression. Studies with expression vectors encoding mutated NF-κB signaling molecules and AP-1 decoy confirmed that NF-κB and AP-1 were essential for the nicotine-stimulated uPAR expression. MAPK (Erk-1/2 and JNK) and ROS functioned as upstream signaling molecules in the activation of AP-1 and NF-κB, respectively. In addition, ECV304 endothelial cells treated with nicotine displayed markedly enhanced invasiveness, which was partially abrogated by uPAR neutralizing antibodies. The data indicate that nicotine induces uPAR expression via the MAPK/AP-1 and ROS/NF-κB signaling pathways and, in turn, stimulates invasiveness in human ECV304 endothelial cells. -- Highlights: ► Endothelial cells

Nicotine stimulates urokinase-type plasminogen activator receptor expression and cell invasiveness through mitogen-activated protein kinase and reactive oxygen species signaling in ECV304 endothelial cells

International Nuclear Information System (INIS)

Khoi, Pham Ngoc; Park, Jung Sun; Kim, Nam Ho; Jung, Young Do

2012-01-01

Urokinase-type plasminogen activator receptor (uPAR) expression is elevated during inflammation, tissue remodeling and in many human cancers. This study investigated the effect of nicotine, a major alkaloid in tobacco, on uPAR expression and cell invasiveness in ECV304 endothelial cells. Nicotine stimulated uPAR expression in a dose-dependent manner and activated extracellular signal-regulated kinases-1/2 (Erk-1/2), c-Jun amino-terminal kinase (JNK) and p38 mitogen activated protein kinase (MAPK). Specific inhibitors of MEK-1 (PD98059) and JNK (SP600125) inhibited the nicotine-induced uPAR expression, while the p38 MAPK inhibitor SB203580 did not. Expression vectors encoding dominant negative MEK-1 (pMCL-K97M) and JNK (TAM67) also prevented nicotine-induced uPAR promoter activity. The intracellular hydrogen peroxide (H 2 O 2 ) content was increased by nicotine treatment. The antioxidant N-acetylcysteine prevented nicotine-activated production of reactive oxygen species (ROS) and uPAR expression. Furthermore, exogenous H 2 O 2 increased uPAR mRNA expression. Deleted and site-directed mutagenesis demonstrated the involvement of the binding sites of transcription factor nuclear factor-kappaB (NF-κB) and activator protein (AP)-1 in the nicotine-induced uPAR expression. Studies with expression vectors encoding mutated NF-κB signaling molecules and AP-1 decoy confirmed that NF-κB and AP-1 were essential for the nicotine-stimulated uPAR expression. MAPK (Erk-1/2 and JNK) and ROS functioned as upstream signaling molecules in the activation of AP-1 and NF-κB, respectively. In addition, ECV304 endothelial cells treated with nicotine displayed markedly enhanced invasiveness, which was partially abrogated by uPAR neutralizing antibodies. The data indicate that nicotine induces uPAR expression via the MAPK/AP-1 and ROS/NF-κB signaling pathways and, in turn, stimulates invasiveness in human ECV304 endothelial cells. -- Highlights: ► Endothelial cells treated with nicotine

Expression Profiling of Mitogen-Activated Protein Kinase Genes Reveals Their Evolutionary and Functional Diversity in Different Rubber Tree (Hevea brasiliensis Cultivars

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Xiang Jin

2017-10-01

Full Text Available Rubber tree (Hevea brasiliensis is the only commercially cultivated plant for producing natural rubber, one of the most essential industrial raw materials. Knowledge of the evolutionary and functional characteristics of kinases in H. brasiliensis is limited because of the long growth period and lack of well annotated genome information. Here, we reported mitogen-activated protein kinases in H. brasiliensis (HbMPKs by manually checking and correcting the rubber tree genome. Of the 20 identified HbMPKs, four members were validated by proteomic data. Protein motif and phylogenetic analyses classified these members into four known groups comprising Thr-Glu-Tyr (TEY and Thr-Asp-Tyr (TDY domains, respectively. Evolutionary and syntenic analyses suggested four duplication events: HbMPK3/HbMPK6, HbMPK8/HbMPK9/HbMPK15, HbMPK10/HbMPK12 and HbMPK11/HbMPK16/HbMPK19. Expression profiling of the identified HbMPKs in roots, stems, leaves and latex obtained from three cultivars with different latex yield ability revealed tissue- and variety-expression specificity of HbMPK paralogues. Gene expression patterns under osmotic, oxidative, salt and cold stresses, combined with cis-element distribution analyses, indicated different regulation patterns of HbMPK paralogues. Further, Ka/Ks and Tajima analyses suggested an accelerated evolutionary rate in paralogues HbMPK10/12. These results revealed HbMPKs have diverse functions in natural rubber biosynthesis, and highlighted the potential possibility of using MPKs to improve stress tolerance in future rubber tree breeding.

Salidroside pretreatment attenuates apoptosis and autophagy during hepatic ischemia–reperfusion injury by inhibiting the mitogen-activated protein kinase pathway in mice

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Feng J

2017-07-01

Full Text Available Jiao Feng,1,* Qinghui Zhang,2,* Wenhui Mo,3,* Liwei Wu,1 Sainan Li,1 Jingjing Li,1 Tong Liu,1 Shizan Xu,4 Xiaoming Fan,5 Chuanyong Guo1 1Department of Gastroenterology, Shanghai Tenth People’s Hospital, Tongji University School of Medicine, Shanghai, 2Department of Clinical Laboratory, Kunshan First People’s Hospital Affiliated to Jiangsu University, Kunshan, JiangSu, 3Department of Gastroenterology, Minhang Hospital, Fudan University, Shanghai, 4Department of Gastroenterology, Shanghai Tenth People’s Hospital, School of Clinical Medicine of Nanjing Medical University, Shanghai, 5Department of Gastroenterology, Jinshan Hospital of Fudan University, Jinshan, Shanghai, China *These authors contributed equally to this work Abstract: Ischemia–reperfusion injury (IRI contributes to liver damage in many clinical situations, such as liver resection and liver transplantation. In the present study, we investigated the effects of the antioxidant, anti-inflammatory, and anticancer agent salidroside (Sal on hepatic IRI in mice. The mice were randomly divided into six groups: normal control, Sham, Sal (20 mg/kg, IRI, IRI + Sal (10 mg/kg, and IRI + Sal (20 mg/kg. We measured liver enzymes, proinflammatory cytokines, TNF-α and interleukin-6, and apoptosis- and autophagy-related marker proteins at 2, 8, and 24 hours after reperfusion. Components of mitogen-activated protein kinase (MAPK signaling, including P-38, jun N-terminal kinase (JNK, and extracellular signal-regulated kinase (ERK, were also measured using an MAPK activator anisomycin to deduce their roles in hepatic IRI. Our results show that Sal safely protects hepatocytes from IRI by reducing levels of liver enzymes in the serum. These findings were confirmed by histopathology. We concluded that Sal protects hepatocytes from IRI partly by inhibiting the activation of MAPK signaling, including the phosphorylation of P38, JNK, and ERK. This ameliorates inflammatory reactions, apoptosis, and

Protection of Human Podocytes from Shiga Toxin 2-Induced Phosphorylation of Mitogen-Activated Protein Kinases and Apoptosis by Human Serum Amyloid P Component

Science.gov (United States)

Dettmar, Anne K.; Binder, Elisabeth; Greiner, Friederike R.; Liebau, Max C.; Kurschat, Christine E.; Jungraithmayr, Therese C.; Saleem, Moin A.; Schmitt, Claus-Peter; Feifel, Elisabeth; Orth-Höller, Dorothea; Kemper, Markus J.; Pepys, Mark; Würzner, Reinhard

2014-01-01

Hemolytic uremic syndrome (HUS) is mainly induced by Shiga toxin 2 (Stx2)-producing Escherichia coli. Proteinuria can occur in the early phase of the disease, and its persistence determines the renal prognosis. Stx2 may injure podocytes and induce proteinuria. Human serum amyloid P component (SAP), a member of the pentraxin family, has been shown to protect against Stx2-induced lethality in mice in vivo, presumably by specific binding to the toxin. We therefore tested the hypothesis that SAP can protect against Stx2-induced injury of human podocytes. To elucidate the mechanisms underlying podocyte injury in HUS-associated proteinuria, we assessed Stx2-induced activation of mitogen-activated protein kinases (MAPKs) and apoptosis in immortalized human podocytes and evaluated the impact of SAP on Stx2-induced damage. Human podocytes express Stx2-binding globotriaosylceramide 3. Stx2 applied to cultured podocytes was internalized and then activated p38α MAPK and c-Jun N-terminal kinase (JNK), important signaling steps in cell differentiation and apoptosis. Stx2 also activated caspase 3, resulting in an increased level of apoptosis. Coincubation of podocytes with SAP and Stx2 mitigated the effects of Stx2 and induced upregulation of antiapoptotic Bcl2. These data suggest that podocytes are a target of Stx2 and that SAP protects podocytes against Stx2-induced injury. SAP may therefore be a useful therapeutic option. PMID:24566618

Extract of Polygala tenuifolia Alleviates Stress-Exacerbated Atopy-Like Skin Dermatitis through the Modulation of Protein Kinase A and p38 Mitogen-Activated Protein Kinase Signaling Pathway

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Bongjun Sur

2017-01-01

Full Text Available Atopic dermatitis (AD and stress create a vicious cycle: stress exacerbates atopic symptoms, and atopic disease elicits stress and anxiety. Targeting multiple pathways including stress and allergic inflammation is, therefore, important for treating AD. In this study, we investigated the remedial value of Polygala tenuifolia Willd. (PTW for treating immobilization (IMO stress-exacerbated atopy-like skin dermatitis and its underlying mechanism. Trimellitic anhydride (TMA was applied to dorsal skin for sensitization and subsequently both ears for eliciting T-cell-dependent contact hypersensitivity in mice, which underwent 2 h-IMO stress and PTW administration for the latter 6 and 9 days in the ear exposure period of TMA, respectively. To elicit in vitro degranulation of human mast cell line-1 (HMC-1, 10 µM substance P (SP and 200 nM corticotrophin-releasing factor (CRF were sequentially added with 48 h-interval. PTW extract (500 µg/mL was added 30 min before CRF treatment. IMO stress exacerbated TMA-induced scratching behavior by 252%, and increased their blood corticosterone levels by two-fold. Treatment with 250 mg/kg PTW significantly restored IMO stress-exacerbated scratching behavior and other indicators such as skin inflammation and water content, lymph node weights, and serum histamine and immunoglobulin E (lgE levels. Furthermore, it also reversed TMA-stimulated expression of tumor necrosis factor (TNF-α and interleukin (IL-4 mRNAs in ear tissues. PTW significantly inhibited SP/CRF-stimulated degranulation of HMC-1 cells, subsequent tryptase secretion, and protein kinase A (PKA activity. PTW also selectively inhibited p38 mitogen-activated protein kinase (MAPK phosphorylation in SP/CRF-treated HMC-1 cells. PTW significantly inhibited HMC-1 cell degranulation and alleviated IMO stress-exacerbated atopic dermatitis symptoms by modulating the PKA/p38 MAPK signaling pathway.

Mitogen activated protein kinase 6 and MAP kinase phosphatase 1 are involved in the response of Arabidopsis roots to L-glutamate.

Science.gov (United States)

López-Bucio, Jesús Salvador; Raya-González, Javier; Ravelo-Ortega, Gustavo; Ruiz-Herrera, León Francisco; Ramos-Vega, Maricela; León, Patricia; López-Bucio, José; Guevara-García, Ángel Arturo

2018-03-01

The function and components of L-glutamate signaling pathways in plants have just begun to be elucidated. Here, using a combination of genetic and biochemical strategies, we demonstrated that a MAPK module is involved in the control of root developmental responses to this amino acid. Root system architecture plays an essential role in plant adaptation to biotic and abiotic factors via adjusting signal transduction and gene expression. L-Glutamate (L-Glu), an amino acid with neurotransmitter functions in animals, inhibits root growth, but the underlying genetic mechanisms are poorly understood. Through a combination of genetic analysis, in-gel kinase assays, detailed cell elongation and division measurements and confocal analysis of expression of auxin, quiescent center and stem cell niche related genes, the critical roles of L-Glu in primary root growth acting through the mitogen-activated protein kinase 6 (MPK6) and the dual specificity serine-threonine-tyrosine phosphatase MKP1 could be revealed. In-gel phosphorylation assays revealed a rapid and dose-dependent induction of MPK6 and MPK3 activities in wild-type Arabidopsis seedlings in response to L-Glu. Mutations in MPK6 or MKP1 reduced or increased root cell division and elongation in response to L-Glu, possibly modulating auxin transport and/or response, but in a PLETHORA1 and 2 independent manner. Our data highlight MPK6 and MKP1 as components of an L-Glu pathway linking the auxin response, and cell division for primary root growth.

Three Fusarium oxysporum mitogen-activated protein kinases (MAPKs) have distinct and complementary roles in stress adaptation and cross-kingdom pathogenicity.

Science.gov (United States)

Segorbe, David; Di Pietro, Antonio; Pérez-Nadales, Elena; Turrà, David

2017-09-01

Mitogen-activated protein kinase (MAPK) cascades mediate cellular responses to environmental signals. Previous studies in the fungal pathogen Fusarium oxysporum have revealed a crucial role of Fmk1, the MAPK orthologous to Saccharomyces cerevisiae Fus3/Kss1, in vegetative hyphal fusion and plant infection. Here, we genetically dissected the individual and combined contributions of the three MAPKs Fmk1, Mpk1 and Hog1 in the regulation of development, stress response and virulence of F. oxysporum on plant and animal hosts. Mutants lacking Fmk1 or Mpk1 were affected in reactive oxygen species (ROS) homeostasis and impaired in hyphal fusion and aggregation. Loss of Mpk1 also led to increased sensitivity to cell wall and heat stress, which was exacerbated by simultaneous inactivation of Fmk1, suggesting that both MAPKs contribute to cellular adaptation to high temperature, a prerequisite for mammalian pathogens. Deletion of Hog1 caused increased sensitivity to hyperosmotic stress and resulted in partial rescue of the restricted colony growth phenotype of the mpk1Δ mutant. Infection assays on tomato plants and the invertebrate animal host Galleria mellonella revealed distinct and additive contributions of the different MAPKs to virulence. Our results indicate that positive and negative cross-talk between the three MAPK pathways regulates stress adaptation, development and virulence in the cross-kingdom pathogen F. oxysporum. © 2016 BSPP AND JOHN WILEY & SONS LTD.

Involvement of a novel p38 mitogen-activated protein kinase in larval metamorphosis of the polychaete Hydroides elegans (Haswell)

KAUST Repository

Wang, Hao

2010-04-19

Hydroides elegans is a common marine fouling organism in most tropical and subtropical waters. The life cycle of H. elegans includes a planktonic larval stage in which swimming larvae normally take 5 days to attain competency to settle. Larval metamorphosis marks the beginning of its benthic life; however, the endogenous molecular mechanisms that regulate metamorphosis remain largely unknown. In this study, a PCR-based suppressive subtractive hybridization (SSH) library was constructed to screen the genes expressed in competent larvae but not in precompetent larvae. Among the transcripts isolated from the library, 21 significantly matched sequences in the GenBank. Many of these isolated transcripts have putative roles in the reactive oxygen species (ROS) signal transduction pathway or in response to ROS stress. A putative novel p38 mitogen-activated protein kinase (MAPK), which was also isolated with SSH screen, was then cloned and characterized. The MAPK inhibitors assay showed that both p38 MAPK inhibitors SB202190 and SB203580 effectively inhibited the biofilm-induced metamorphosis of H. elegans. A cell stressors assay showed that H2O2 effectively induced larval metamorphosis of H. elegans, but the inductivity of H2O2 was also inhibited by both SB inhibitors. The catalase assay showed that the catalase could effetely inhibit H. elegans larvae from responding to inductive biofilm. These results showed that the p38 MAPK-dependent pathway plays critical role in controlling larval metamorphosis of the marine polychaete H. elegans, and the reactive oxygen radicals produced by biofilm could be the cue inducing larval metamorphosis. © 2010 Wiley-Liss, Inc.

Colletotrichum higginsianum Mitogen-Activated Protein Kinase ChMK1: Role in Growth, Cell Wall Integrity, Colony Melanization and Pathogenicity

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Wei Wei

2016-08-01

Full Text Available Colletotrichum higginsianum is an economically important pathogen that causes anthracnose disease in a wide range of cruciferous crops. To facilitate the efficient control of anthracnose disease, it will be important to understand the mechanism by which the cruciferous crops and C. higginsianum interact. A key step in understanding this interaction is characterizing the mitogen-activated protein kinases (MAPK signaling pathway of C. higginsianum. MAPK plays important roles in diverse physiological processes of multiple pathogens. In this study, a Fus3/Kss1-related MAPK gene, ChMK1, from C. higginsianum was analyzed. The results showed that the Fus3/Kss1-related MAPK ChMK1 plays a significant role in cell wall integrity. Targeted deletion of ChMK1 resulted in a hypersensitivity to cell wall inhibitors, reduced conidiation and albinistic colonies. Further, the deletion mutant was also unable to form melanized appressorium, a specialized infection structure that is necessary for successful infection. Therefore, the deletion mutant loses pathogenicity on A. thaliana leaves, demonstrating that ChMK1 plays an essential role in the early infection step. In addition, the ChMK1 deletion mutant showed an attenuated growth rate that is different from that of its homologue in C. lagenarium, indicating the diverse roles that Fus3/Kss1-related MAPKs plays in phytopathogenic fungi. Furthermore, the expression level of three melanin synthesis associated genes were clearly decreased in the albinistic ChMK1 mutant compared to that of the wild type strain, suggesting that ChMK1 is also required for colony melanization in C. higginsianum.

EphA2 modulates radiosensitive of hepatocellular carcinoma cells via p38/mitogen-activated protein kinase-mediated signal pathways

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Qiao Jin

2015-10-01

Full Text Available This experiment was conducted to investigate the role of EPH receptor A2 (EphA2 in the modulation of radiosensitivity of hepatic cellular cancer (HCC cells and to determine whether p38/mitogen-activated protein kinase (p38MAPK signaling mediated EphA2 function in this respect. The protein expressions of EphA2 and phosphorylated p38MAPK were tested in HCC and normal hepatic tissues. In HCC 97H cells, EphA2 was overexpressed and knocked out by transfection with EphA2 expression vector and EphA2-ShRNA, respectively, prior to cell exposure to low-dose irradiation. Significantly upregulated EphA2 and phosphorylated p38MAPK were observed in HCC tissues, compared with those in normal hepatic tissues. Low-dose irradiation (1 Gy only caused minor damage to HCC 97H cells, as assessed by alterations in cell viability, apoptosis rate, and cell healing capacity (p = 0.072, p = 0.078, and p = 0.069 respectively. However, EphA2 knock-out in HCC 97H cells induced significant reduction in cell viability and cell healing capacity after these cells were subjected to low-dose irradiation. Apoptosis rate underwent dramatic increase (p < 0.01. By contrast, EphA2 overexpression in HCC 97H cells reversed these effects and enhanced cell colony formation rate, thus displaying remarkable attenuation of radiosensitivity of HCC 97H cells. Further, SB203580, a specific inhibitor of p38MAPK, was added to HCC 97H cells over-expressing EphA2. The effect of EphA2 overexpression on the radiosensitivity of HCC 97H cells was abrogated. Thus, the present study indicates that EphA2 have the ability to negatively regulate the radiosensitivity of HCC 97H cells, which mainly depends on 38MAPK-mediated signal pathways.

Mitogen-activated protein kinases mediate Mycobacterium ...

Indian Academy of Sciences (India)

2012-01-19

Jan 19, 2012 ... effector molecules and also in the control of intracellular bacterial replication ..... H37Ra in THP-1 cells. The fall and rise in the activation of .... use this distinct role of p38 MAPK to balance the expression of CD44 during ...

Effects of protein kinase C activators on phorbol ester-sensitive and -resistant EL4 thymoma cells.

Science.gov (United States)

Sansbury, H M; Wisehart-Johnson, A E; Qi, C; Fulwood, S; Meier, K E

1997-09-01

Phorbol ester-sensitive EL4 murine thymoma cells respond to phorbol 12-myristate 13-acetate with activation of ERK mitogen-activated protein kinases, synthesis of interleukin-2, and death, whereas phorbol ester-resistant variants of this cell line do not exhibit these responses. Additional aspects of the resistant phenotype were examined, using a newly-established resistant cell line. Phorbol ester induced morphological changes, ERK activation, calcium-dependent activation of the c-Jun N-terminal kinase (JNK), interleukin-2 synthesis, and growth inhibition in sensitive but not resistant cells. A series of protein kinase C activators caused membrane translocation of protein kinase C's (PKCs) alpha, eta, and theta in both cell lines. While PKC eta was expressed at higher levels in sensitive than in resistant cells, overexpression of PKC eta did not restore phorbol ester-induced ERK activation to resistant cells. In sensitive cells, PKC activators had similar effects on cell viability and ERK activation, but differed in their abilities to induce JNK activation and interleukin-2 synthesis. PD 098059, an inhibitor of the mitogen activated protein (MAP)/ERK kinase kinase MEK, partially inhibited ERK activation and completely blocked phorbol ester-induced cell death in sensitive cells. Thus MEK and/or ERK activation, but not JNK activation or interleukin-2 synthesis, appears to be required for phorbol ester-induced toxicity. Alterations in phorbol ester response pathways, rather than altered expression of PKC isoforms, appear to confer phorbol ester resistance to EL4 cells.

Induced Mitogenic Activity in AML-12 Mouse Hepatocytes Exposed to Low-dose Ultra-Wideband Electromagnetic Radiation

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P. B. Tchounwou

2005-04-01

Full Text Available Ultra–wideband (UWB technology has increased with the use of various civilian and military applications. In the present study, we hypothesized that low-dose UWB electromagnetic radiation (UWBR could elicit a mitogenic effect in AML-12 mouse hepatocytes, in vitro. To test this hypothesis, we exposed AML-12 mouse hepatocytes, to UWBR in a specially constructed gigahertz transverse electromagnetic mode (GTEM cell. Cells were exposed to UWBR for 2 h at a temperature of 23°C, a pulse width of 10 ns, a repetition rate of 1 kHz, and field strength of 5-20 kV/m. UWB pulses were triggered by an external pulse generator for UWBR exposure but were not triggered for the sham exposure. We performed an MTT Assay to assess cell viability for UWBR-treated and sham-exposed hepatocytes. Data from viability studies indicated a time-related increase in hepatocytes at time intervals from 8-24 h post exposure. UWBR exerted a statistically significant (p < 0.05 dose-dependent response in cell viability in both serum-treated and serum free medium (SFM -treated hepatocytes. Western blot analysis of hepatocyte lysates demonstrated that cyclin A protein was induced in hepatocytes, suggesting that increased MTT activity after UWBR exposure was due to cell proliferation. This study indicates that UWBR has a mitogenic effect on AML-12 mouse hepatocytes and implicates a possible role for UWBR in hepatocarcinoma.

Short communication: Camel milk ameliorates inflammatory responses and oxidative stress and downregulates mitogen-activated protein kinase signaling pathways in lipopolysaccharide-induced acute respiratory distress syndrome in rats.

Science.gov (United States)

Zhu, Wei-Wei; Kong, Gui-Qing; Ma, Ming-Ming; Li, Yan; Huang, Xiao; Wang, Li-Peng; Peng, Zhen-Yi; Zhang, Xiao-Hua; Liu, Xiang-Yong; Wang, Xiao-Zhi

2016-01-01

Acute respiratory distress syndrome (ARDS) is a complex syndrome disorder with high mortality rate. Camel milk (CM) contains antiinflammatory and antioxidant properties and protects against numerous diseases. This study aimed to demonstrate the function of CM in lipopolysaccharide (LPS)-induced ARDS in rats. Camel milk reduced the lung wet:dry weight ratio and significantly reduced LPS-induced increases in neutrophil infiltration, interstitial and intra-alveolar edema, thickness of the alveolar wall, and lung injury scores of lung tissues. It also had antiinflammatory and antioxidant effects on LPS-induced ARDS. After LPS stimulation, the levels of proinflammatory cytokines (tumor necrosis factor-α, IL-10, and IL-1β) in serum and oxidative stress markers (malondialdehyde, myeloperoxidase, and total antioxidant capacity) in lung tissue were notably attenuated by CM. Camel milk also downregulated mitogen-activated protein kinase signaling pathways. Given these results, CM is a potential complementary food for ARDS treatment. Copyright © 2016 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.

A Member of the p38 Mitogen-Activated Protein Kinase Family Is Responsible for Transcriptional Induction of Dopa decarboxylase in the Epidermis of Drosophila melanogaster during the Innate Immune Response▿ †

Science.gov (United States)

Davis, Monica M.; Primrose, David A.; Hodgetts, Ross B.

2008-01-01

Drosophila innate immunity is controlled primarily by the activation of IMD (immune deficiency) or Toll signaling leading to the production of antimicrobial peptides (AMPs). IMD signaling also activates the JUN N-terminal kinase (JNK) cascade, which is responsible for immune induction of non-antimicrobial peptide immune gene transcription though the transcription factor AP-1. Transcription of the Dopa decarboxylase (Ddc) gene is induced in response to gram-negative and gram-positive septic injury, but not aseptic wounding. Transcription is induced throughout the epidermis and not specifically at the site of infection. Ddc transcripts are detectible within 2 h and remain high for several hours following infection with either gram-negative or gram-positive bacteria. Using Ddc-green fluorescent protein (GFP) reporter gene constructs, we show that a conserved consensus AP-1 binding site upstream of the Ddc transcription start site is required for induction. However, neither the Toll, IMD, nor JNK pathway is involved. Rather, Ddc transcription depends on a previously uncharacterized member of the p38 mitogen-activated protein kinase family, p38c. We propose that the involvement of DDC in a new pathway involved in Drosophila immunity increases the levels of dopamine, which is metabolized to produce reactive quinones that exert an antimicrobial effect on invading bacteria. PMID:18519585

Regulator of differentiation 1 (ROD1) binds to the amphipathic C-terminal peptide of thrombospondin-4 and is involved in its mitogenic activity.

Science.gov (United States)

Sadvakassova, Gulzhakhan; Dobocan, Monica C; Difalco, Marcos R; Congote, Luis F

2009-09-01

The matrix protein thrombospondin-4 has an acidic amphipathic C-terminal peptide (C21) which stimulates erythroid cell proliferation. Here we show that C21 stimulates red cell formation in anemic mice in vivo. In vitro experiments indicated that the peptide-mediated increase of erythroid colony formation in cultures of human CD34+ hematopoietic progenitor cells was possible only under continuous presence of erythropoietin. In the absence of this cytokine, C21 stimulated exclusively myeloid colony formation. Therefore, the peptide is not a specific erythroid differentiation factor. In fact, it is mitogenic in non-erythroid cells, such as skin fibroblasts and kidney epithelial cells. In erythroleukemic TF-1 cells, it actually decreased the production of the erythroid differentiation marker glycophorin A. C21-affinity chromatography revealed regulator of differentiation 1 (ROD1) as a major C21-binding protein. ROD1 is the hematopoietic cell paralog of polypyrimidine tract binding proteins (PTBs), RNA splice regulators which regulate differentiation by repressing tissue-specific exons. ROD1 binding to C21 was strongly inhibited by synthetic RNAs in the order poly A > poly U > poly G = poly C and was weakly inhibited by a synthetic phosphorylated peptide mimicking the C-terminal domain of RNA polymerase II. Cellular overexpression or knockdown experiments of ROD1 suggest a role for this protein in the mitogenic activity of C21. Since the nuclear proteins ROD1 and PTBs regulate differentiation at a posttranscriptional level and there is a fast nuclear uptake of C21, we put forward the idea that the peptide is internalized, goes to the nucleus and maintains cells in a proliferative state by supporting ROD1-mediated inhibition of differentiation.

AMP-activated protein kinase downregulates Kv7.1 cell surface expression

DEFF Research Database (Denmark)

Andersen, Martin N; Krzystanek, Katarzyna; Jespersen, Thomas

2012-01-01

in response to polarization of the epithelial Madin-Darby canine kidney (MDCK) cell line and that this was mediated by activation of protein kinase C (PKC). In this study, the pathway downstream of PKC, which leads to internalization of Kv7.1 upon cell polarization, is elucidated. We show by confocal...... microscopy that Kv7.1 is endocytosed upon initiation of the polarization process and sent for degradation by the lysosomal pathway. The internalization could be mimicked by pharmacological activation of the AMP-activated protein kinase (AMPK) using three different AMPK activators. We demonstrate...

A mannose-specific tetrameric lectin with mitogenic and antibacterial activities from the ovary of a teleost, the cobia (Rachycentron canadum).

Science.gov (United States)

Ngai, Patrick H K; Ng, T B

2007-02-01

A tetrameric lectin, with hemagglutinating activity toward rabbit erythrocytes and with specificity toward D-mannosamine and D(+)-mannose, was isolated from the ovaries of a teleost, the cobia Rachycentron canadum. The isolation protocol comprised ion exchange chromatography on CM-cellulose and Q-Sepharose, ion exchange chromatography by fast protein liquid chromatography (FPLC) on Mono Q, and finally gel filtration by FPLC on Superose 12. The lectin was adsorbed on all ion exchangers used. It exhibited a molecular mass of 180 kDa in gel filtration on Superose 12 and a single 45-kDa band in sodium dodecyl sulfate-polyacrylamide gel electrophoresis, indicating that it is a tetrameric protein. The hemagglutinating activity of the lectin was stable up to 40 degrees C and between pH 4 and pH 10. All hemagglutinating activity disappeared at 60 degrees C and at pH 1 and pH 13. The hemagglutinating activity was doubled in the presence of 0.1 microM FeCl3. The lectin exerted antibacterial activity against Escherichia coli with 50% inhibition at 250 microg. There was no antifungal activity toward Coprinus comatus, Fusarium oxysporum, Mycosphaerella arachidicola, and Rhizoctonia solani at a dose of 300 microg. The lectin exhibited maximal mitogenic response from mouse splenocytes at a concentration of 14 microM.

A Wnt5 Activity Asymmetry and Intercellular Signaling via PCP Proteins Polarize Node Cells for Left-Right Symmetry Breaking.

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Minegishi, Katsura; Hashimoto, Masakazu; Ajima, Rieko; Takaoka, Katsuyoshi; Shinohara, Kyosuke; Ikawa, Yayoi; Nishimura, Hiromi; McMahon, Andrew P; Willert, Karl; Okada, Yasushi; Sasaki, Hiroshi; Shi, Dongbo; Fujimori, Toshihiko; Ohtsuka, Toshihisa; Igarashi, Yasunobu; Yamaguchi, Terry P; Shimono, Akihiko; Shiratori, Hidetaka; Hamada, Hiroshi

2017-03-13

Polarization of node cells along the anterior-posterior axis of mouse embryos is responsible for left-right symmetry breaking. How node cells become polarized has remained unknown, however. Wnt5a and Wnt5b are expressed posteriorly relative to the node, whereas genes for Sfrp inhibitors of Wnt signaling are expressed anteriorly. Here we show that polarization of node cells is impaired in Wnt5a -/- Wnt5b -/- and Sfrp mutant embryos, and also in the presence of a uniform distribution of Wnt5a or Sfrp1, suggesting that Wnt5 and Sfrp proteins act as instructive signals in this process. The absence of planar cell polarity (PCP) core proteins Prickle1 and Prickle2 in individual cells or local forced expression of Wnt5a perturbed polarization of neighboring wild-type cells. Our results suggest that opposing gradients of Wnt5a and Wnt5b and of their Sfrp inhibitors, together with intercellular signaling via PCP proteins, polarize node cells along the anterior-posterior axis for breaking of left-right symmetry. Copyright © 2017 Elsevier Inc. All rights reserved.

Role of Akt/PKB and PFKFB isoenzymes in the control of glycolysis, cell proliferation and protein synthesis in mitogen-stimulated thymocytes.

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Houddane, Amina; Bultot, Laurent; Novellasdemunt, Laura; Johanns, Manuel; Gueuning, Marie-Agnès; Vertommen, Didier; Coulie, Pierre G; Bartrons, Ramon; Hue, Louis; Rider, Mark H

2017-06-01

Proliferating cells depend on glycolysis mainly to supply precursors for macromolecular synthesis. Fructose 2,6-bisphosphate (Fru-2,6-P 2 ) is the most potent positive allosteric effector of 6-phosphofructo-1-kinase (PFK-1), and hence of glycolysis. Mitogen stimulation of rat thymocytes with concanavalin A (ConA) led to time-dependent increases in lactate accumulation (6-fold), Fru-2,6-P 2 content (4-fold), 6-phosphofructo-2-kinase (PFK-2)/fructose-2,6-bisphosphatase isoenzyme 3 and 4 (PFKFB3 and PFKFB4) protein levels (~2-fold and ~15-fold, respectively) and rates of cell proliferation (~40-fold) and protein synthesis (10-fold) after 68h of incubation compared with resting cells. After 54h of ConA stimulation, PFKFB3 mRNA levels were 45-fold higher than those of PFKFB4 mRNA. Although PFKFB3 could be phosphorylated at Ser461 by protein kinase B (PKB) in vitro leading to PFK-2 activation, PFKFB3 Ser461 phosphorylation was barely detectable in resting cells and only increased slightly in ConA-stimulated cells. On the other hand, PFKFB3 and PFKFB4 mRNA levels were decreased (90% and 70%, respectively) by exposure of ConA-stimulated cells to low doses of PKB inhibitor (MK-2206), suggesting control of expression of the two PFKFB isoenzymes by PKB. Incubation of thymocytes with ConA resulted in increased expression and phosphorylation of the translation factors eukaryotic initiation factor-4E-binding protein-1 (4E-BP1) and ribosomal protein S6 (rpS6). Treatment of ConA-stimulated thymocytes with PFK-2 inhibitor (3PO) or MK-2206 led to significant decreases in Fru-2,6-P 2 content, medium lactate accumulation and rates of cell proliferation and protein synthesis. These data were confirmed by using siRNA knockdown of PFKFB3, PFKFB4 and PKB α/β in the more easily transfectable Jurkat E6-1 cell line. The findings suggest that increased PFKFB3 and PFKFB4 expression, but not increased PFKFB3 Ser461 phosphorylation, plays a role in increasing glycolysis in mitogen

Discovery and Characterization of Non-ATP Site Inhibitors of the Mitogen Activated Protein (MAP) Kinases

Energy Technology Data Exchange (ETDEWEB)

Comess, Kenneth M.; Sun, Chaohong; Abad-Zapatero, Cele; Goedken, Eric R.; Gum, Rebecca J.; Borhani, David W.; Argiriadi, Maria; Groebe, Duncan R.; Jia, Yong; Clampit, Jill E.; Haasch, Deanna L.; Smith, Harriet T.; Wang, Sanyi; Song, Danying; Coen, Michael L.; Cloutier, Timothy E.; Tang, Hua; Cheng, Xueheng; Quinn, Christopher; Liu, Bo; Xin, Zhili; Liu, Gang; Fry, Elizabeth H.; Stoll, Vincent; Ng, Teresa I.; Banach, David; Marcotte, Doug; Burns, David J.; Calderwood, David J.; Hajduk, Philip J. (Abbott)

2012-03-02

Inhibition of protein kinases has validated therapeutic utility for cancer, with at least seven kinase inhibitor drugs on the market. Protein kinase inhibition also has significant potential for a variety of other diseases, including diabetes, pain, cognition, and chronic inflammatory and immunologic diseases. However, as the vast majority of current approaches to kinase inhibition target the highly conserved ATP-binding site, the use of kinase inhibitors in treating nononcology diseases may require great selectivity for the target kinase. As protein kinases are signal transducers that are involved in binding to a variety of other proteins, targeting alternative, less conserved sites on the protein may provide an avenue for greater selectivity. Here we report an affinity-based, high-throughput screening technique that allows nonbiased interrogation of small molecule libraries for binding to all exposed sites on a protein surface. This approach was used to screen both the c-Jun N-terminal protein kinase Jnk-1 (involved in insulin signaling) and p38{alpha} (involved in the formation of TNF{alpha} and other cytokines). In addition to canonical ATP-site ligands, compounds were identified that bind to novel allosteric sites. The nature, biological relevance, and mode of binding of these ligands were extensively characterized using two-dimensional {sup 1}H/{sup 13}C NMR spectroscopy, protein X-ray crystallography, surface plasmon resonance, and direct enzymatic activity and activation cascade assays. Jnk-1 and p38{alpha} both belong to the MAP kinase family, and the allosteric ligands for both targets bind similarly on a ledge of the protein surface exposed by the MAP insertion present in the CMGC family of protein kinases and distant from the active site. Medicinal chemistry studies resulted in an improved Jnk-1 ligand able to increase adiponectin secretion in human adipocytes and increase insulin-induced protein kinase PKB phosphorylation in human hepatocytes, in

Loss of monocyte chemoattractant protein-1 alters macrophage polarization and reduces NFκB activation in the foreign body response.

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Moore, Laura Beth; Sawyer, Andrew J; Charokopos, Antonios; Skokos, Eleni A; Kyriakides, Themis R

2015-01-01

Implantation of biomaterials elicits a foreign body response characterized by fusion of macrophages to form foreign body giant cells and fibrotic encapsulation. Studies of the macrophage polarization involved in this response have suggested that alternative (M2) activation is associated with more favorable outcomes. Here we investigated this process in vivo by implanting mixed cellulose ester filters or polydimethylsiloxane disks in the peritoneal cavity of wild-type (WT) and monocyte chemoattractant protein-1 (MCP-1) knockout mice. We analyzed classical (M1) and alternative (M2) gene expression via quantitative polymerase chain reaction, immunohistochemistry and enzyme-linked immunosorbent assay in both non-adherent cells isolated by lavage and implant-adherent cells. Our results show that macrophages undergo unique activation that displays features of both M1 and M2 polarization including induction of tumor necrosis factor α (TNF), which induces the expression and nuclear translocation of p50 and RelA determined by immunofluorescence and Western blot. Both processes were compromised in fusion-deficient MCP-1 KO macrophages in vitro and in vivo. Furthermore, inclusion of BAY 11-7028, an inhibitor of NFκB activation, reduced nuclear translocation of RelA and fusion in WT macrophages. Our studies suggest that peritoneal implants elicit a unique macrophage polarization phenotype leading to induction of TNF and activation of the NFκB pathway. Published by Elsevier Ltd.

Behavior of tight-junction, adherens-junction and cell polarity proteins during HNF-4α-induced epithelial polarization

International Nuclear Information System (INIS)

Satohisa, Seiro; Chiba, Hideki; Osanai, Makoto; Ohno, Shigeo; Kojima, Takashi; Saito, Tsuyoshi; Sawada, Norimasa

2005-01-01

We previously reported that expression of tight-junction molecules occludin, claudin-6 and claudin-7, as well as establishment of epithelial polarity, was triggered in mouse F9 cells expressing hepatocyte nuclear factor (HNF)-4α [H. Chiba, T. Gotoh, T. Kojima, S. Satohisa, K. Kikuchi, M. Osanai, N. Sawada. Hepatocyte nuclear factor (HNF)-4α triggers formation of functional tight junctions and establishment of polarized epithelial morphology in F9 embryonal carcinoma cells, Exp. Cell Res. 286 (2003) 288-297]. Using these cells, we examined in the present study behavior of tight-junction, adherens-junction and cell polarity proteins and elucidated the molecular mechanism behind HNF-4α-initiated junction formation and epithelial polarization. We herein show that not only ZO-1 and ZO-2, but also ZO-3, junctional adhesion molecule (JAM)-B, JAM-C and cell polarity proteins PAR-3, PAR-6 and atypical protein kinase C (aPKC) accumulate at primordial adherens junctions in undifferentiated F9 cells. In contrast, CRB3, Pals1 and PATJ appeared to exhibit distinct subcellular localization in immature cells. Induced expression of HNF-4α led to translocation of these tight-junction and cell polarity proteins to beltlike tight junctions, where occludin, claudin-6 and claudin-7 were assembled, in differentiated cells. Interestingly, PAR-6, aPKC, CRB3 and Pals1, but not PAR-3 or PATJ, were also concentrated on the apical membranes in differentiated cells. These findings indicate that HNF-4α provokes not only expression of tight-junction adhesion molecules, but also modulation of subcellular distribution of junction and cell polarity proteins, resulting in junction formation and epithelial polarization

Targeting p38 Mitogen-Activated Protein Kinase Signaling Restores Subventricular Zone Neural Stem Cells and Corrects Neuromotor Deficits in Atm Knockout Mouse

Science.gov (United States)

Kim, Jeesun

2012-01-01

Ataxia-telangiectasia (A-T) is a progressive degenerative disorder that results in major neurological disability. In A-T patients, necropsy has revealed atrophy of cerebellar cortical layers along with Purkinje and granular cell loss. We have previously identified an oxidative stress-mediated increase in phospho-p38 mitogen-activated protein kinase (MAPK) and the resultant downregulation of Bmi-1 and upregulation of p21 as key components of the mechanism causing defective proliferation of neural stem cells (NSCs) isolated from the subventricular zone (SVZ) of Atm−/− mice. However, the in vivo aspect of alteration in SVZ tissue and the functional significance of p38MAPK activation in NSCs for neuropathogenesis of ATM deficiency remain unknown. Here we show that the NSC population was abnormally decreased in the SVZ of 3-month-old Atm−/− mice; this decrease was accompanied by p38MAPK activation. However, after a 2-month treatment with the p38MAPK inhibitor SB203580, starting at 1 month old, Atm−/− mice showed restoration of normal levels of Bmi-1 and p21 with the rescue of NSC population in the SVZ. In addition, treated Atm−/− mice exhibited more Purkinje cells in the cerebellum. Most importantly, motor coordination of Atm−/− mice was significantly improved in the treatment group. Our results show for the first time in vivo evidence of depleted NSCs in the SVZ of Atm−/− mice and also demonstrate that pharmacologic inhibition of p38MAPK signaling has the potential to treat neurological defects of A-T. This study provides a promising approach targeting the oxidative stress-dependent p38 signaling pathway not only for A-T but also for other neurodegenerative disorders. PMID:23197859

Inhibition of mitogen-activated protein kinase kinase, DNA methyltransferase, and transforming growth factor-β promotes differentiation of human induced pluripotent stem cells into enterocytes.

Science.gov (United States)

Kodama, Nao; Iwao, Takahiro; Kabeya, Tomoki; Horikawa, Takashi; Niwa, Takuro; Kondo, Yuki; Nakamura, Katsunori; Matsunaga, Tamihide

2016-06-01

We previously reported that small-molecule compounds were effective in generating pharmacokinetically functional enterocytes from human induced pluripotent stem (iPS) cells. In this study, to determine whether the compounds promote the differentiation of human iPS cells into enterocytes, we investigated the effects of a combination of mitogen-activated protein kinase kinase (MEK), DNA methyltransferase (DNMT), and transforming growth factor (TGF)-β inhibitors on intestinal differentiation. Human iPS cells cultured on feeder cells were differentiated into endodermal cells by activin A. These endodermal-like cells were then differentiated into intestinal stem cells by fibroblast growth factor 2. Finally, the cells were differentiated into enterocyte cells by epidermal growth factor and small-molecule compounds. After differentiation, mRNA expression levels and drug-metabolizing enzyme activities were measured. The mRNA expression levels of the enterocyte marker sucrase-isomaltase and the major drug-metabolizing enzyme cytochrome P450 (CYP) 3A4 were increased by a combination of MEK, DNMT, and TGF-β inhibitors. The mRNA expression of CYP3A4 was markedly induced by 1α,25-dihydroxyvitamin D3. Metabolic activities of CYP1A1/2, CYP2B6, CYP2C9, CYP2C19, CYP3A4/5, UDP-glucuronosyltransferase, and sulfotransferase were also observed in the differentiated cells. In conclusion, MEK, DNMT, and TGF-β inhibitors can be used to promote the differentiation of human iPS cells into pharmacokinetically functional enterocytes. Copyright © 2016 The Japanese Society for the Study of Xenobiotics. Published by Elsevier Ltd. All rights reserved.

Xingshentongqiao Decoction Mediates Proliferation, Apoptosis, Orexin-A Receptor and Orexin-B Receptor Messenger Ribonucleic Acid Expression and Represses Mitogen-activated Protein Kinase Signaling

Directory of Open Access Journals (Sweden)

Yuanli Dong

2015-01-01

Full Text Available Background: Hypocretin (HCRT signaling plays an important role in the pathogenesis of narcolepsy and can be significantly influenced by Chinese herbal therapy. Our previous study showed that xingshentongqiao decoction (XSTQ is clinically effective for the treatment of narcolepsy. To determine whether XSTQ improves narcolepsy by modulating HCRT signaling, we investigated its effects on SH-SY5Y cell proliferation, apoptosis, and HCRT receptor 1/2 (orexin receptor 1 [OX1R] and orexin receptor 2 [OX2R] expression. The signaling pathways involved in these processes were also assessed. Methods: The effects of XSTQ on proliferation and apoptosis in SH-SY5Y cells were assessed using cell counting kit-8 and annexin V-fluorescein isothiocyanate assays. OX1R and OX2R expression was assessed by quantitative real-time polymerase chain reaction analysis. Western blotting for mitogen-activated protein kinase (MAPK pathway activation was performed to further assess the signaling mechanism of XSTQ. Results: XSTQ reduced the proliferation and induced apoptosis of SH-SY5Y cells. This effect was accompanied by the upregulation of OX1R and OX2R expression and the reduced phosphorylation of extracellular signal-regulated kinase (Erk 1/2, p38 MAPK and c-Jun N-terminal kinase (JNK. Conclusions: XSTQ inhibits proliferation and induces apoptosis in SH-SY5Y cells. XSTQ also promotes OX1R and OX2R expression. These effects are associated with the repression of the Erk1/2, p38 MAPK, and JNK signaling pathways. These results define a molecular mechanism for XSTQ in regulating HCRT and MAPK activation, which may explain its ability to treat narcolepsy.

Dynamically polarized samples for neutron protein crystallography at the Spallation Neutron Source

International Nuclear Information System (INIS)

Zhao, Jinkui; Pierce, Josh; Robertson, J. L.; Herwig, Kenneth W.; Myles, Dean; Cuneo, Matt; Li, Le; Meilleur, Flora; Standaert, Bob

2016-01-01

To prepare for the next generation neutron scattering instruments for the planned second target station at the Spallation Neutron Source (SNS) and to broaden the scientific impact of neutron protein crystallography at the Oak Ridge National Laboratory, we have recently ramped up our efforts to develop a dynamically polarized target for neutron protein crystallography at the SNS. Proteins contain a large amount of hydrogen which contributes to incoherent diffraction background and limits the sensitivity of neutron protein crystallography. This incoherent background can be suppressed by using polarized neutron diffraction, which in the same time also improves the coherent diffraction signal. Our plan is to develop a custom Dynamic Nuclear Polarization (DNP) setup tailored to neutron protein diffraction instruments. Protein crystals will be polarized at a magnetic field of 5 T and temperatures of below 1 K. After the dynamic polarization process, the sample will be brought to a frozen-spin mode in a 0.5 T holding field and at temperatures below 100 mK. In a parallel effort, we are also investigating various ways of incorporating polarization agents needed for DNP, such as site specific spin labels, into protein crystals. (paper)

Mitogen activated protein kinase phosphatase-1 prevents the development of tactile sensitivity in a rodent model of neuropathic pain

Directory of Open Access Journals (Sweden)

Ndong Christian

2012-04-01

Full Text Available Abstract Background Neuropathic pain due to nerve injury is one of the most difficult types of pain to treat. Following peripheral nerve injury, neuronal and glial plastic changes contribute to central sensitization and perpetuation of mechanical hypersensitivity in rodents. The mitogen activated protein kinase (MAPK family is pivotal in this spinal cord plasticity. MAPK phosphatases (MKPs limit inflammatory processes by dephosphorylating MAPKs. For example, MKP-1 preferentially dephosphorylates p-p38. Since spinal p-p38 is pivotal for the development of chronic hypersensitivity in rodent models of pain, and p-p38 inhibitors have shown clinical potential in acute and chronic pain patients, we hypothesize that induction of spinal MKP-1 will prevent the development of peripheral nerve-injury-induced hypersensitivity and p-p38 overexpression. Results We cloned rat spinal cord MKP-1 and optimize MKP-1 cDNA in vitro using transfections to BV-2 cells. We observed that in vitro overexpression of MKP-1 blocked lipopolysaccharide-induced phosphorylation of p38 (and other MAPKs as well as release of pro-algesic effectors (i.e., cytokines, chemokines, nitric oxide. Using this cDNA MKP-1 and a non-viral, in vivo nanoparticle transfection approach, we found that spinal cord overexpression of MKP-1 prevented development of peripheral nerve-injury-induced tactile hypersensitivity and reduced pro-inflammatory cytokines and chemokines and the phosphorylated form of p38. Conclusions Our results indicate that MKP-1, the natural regulator of p-p38, mediates resolution of the spinal cord pro-inflammatory milieu induced by peripheral nerve injury, resulting in prevention of chronic mechanical hypersensitivity. We propose that MKP-1 is a potential therapeutic target for pain treatment or prevention.

Three mitogen-activated protein kinases required for cell wall integrity contribute greatly to biocontrol potential of a fungal entomopathogen.

Directory of Open Access Journals (Sweden)

Ying Chen

Full Text Available Bck1, Mkk1 and Slt2 are three mitogen-activated protein (MAP kinases constituting cell wall integrity (CWI pathway that may control multi-stress responses via crosstalk with high-osmolarity glycerol (HOG pathway in budding yeast. In this study, Bck1, Mkk1 and Slt2 orthologues in Beauveria bassiana were confirmed as the three-module cascade essential for CWI because cell wall impairment occurred in the hyphae and conidia of Δbck1, Δmkk1 and Δslt2 examined in multiple experiments. Strikingly, all the deletion mutants became more sensitive to hyperosmotic NaCl and sorbitol with the Western blot of Hog1 phosphorylation being weakened in Δbck1 and absent in Δmkk1 and Δslt2. Apart from crossing responses to cell wall perturbation and high osmolarity, three deletion mutants exhibited faster growth and conidiation on nutrition-rich medium, much less virulence to Galleria mellonella larvae, and higher sensitivity to nutritional, fungicidal, thermal and UV-B irradiative stresses, accompanied with less accumulation of intracellular mannitol and trehalose. Moreover, Δmkk1 and Δslt2 were equally more sensitive to all the stresses of different types except wet-heat stress than wild type and more or less different from Δbck1 in sensitivity to most of the stresses despite their null responses to two oxidants. All the changes in three deletion mutants were restored by each targeted gene complementation. Taken together, the CWI-required Bck1, Mkk1 and Slt2 are all positive, but differential, regulators of multi-stress tolerance and virulence perhaps due to interplay with the HOG pathway essential for osmoregulation, thereby contributing greatly to the biocontrol potential of the fungal entomopathogen.

Desmoglein 2 regulates the intestinal epithelial barrier via p38 mitogen-activated protein kinase.

Science.gov (United States)

Ungewiß, Hanna; Vielmuth, Franziska; Suzuki, Shintaro T; Maiser, Andreas; Harz, Hartmann; Leonhardt, Heinrich; Kugelmann, Daniela; Schlegel, Nicolas; Waschke, Jens

2017-07-24

Intestinal epithelial barrier properties are maintained by a junctional complex consisting of tight junctions (TJ), adherens junctions (AJ) and desmosomes. Desmoglein 2 (Dsg2), an adhesion molecule of desmosomes and the only Dsg isoform expressed in enterocytes, is required for epithelial barrier properties and may contribute to barrier defects in Crohn's disease. Here, we identified extradesmosomal Dsg2 on the surface of polarized enterocytes by Triton extraction, confocal microscopy, SIM and STED. Atomic force microscopy (AFM) revealed Dsg2-specific binding events along the cell border on the surface of enterocytes with a mean unbinding force of around 30pN. Binding events were blocked by an inhibitory antibody targeting Dsg2 which under same conditions activated p38MAPK but did not reduce cell cohesion. In enterocytes deficient for Dsg2, p38MAPK activity was reduced and both barrier integrity and reformation were impaired. Dsc2 rescue did not restore p38MAPK activity indicating that Dsg2 is required. Accordingly, direct activation of p38MAPK in Dsg2-deficient cells enhanced barrier reformation demonstrating that Dsg2-mediated activation of p38MAPK is crucial for barrier function. Collectively, our data show that Dsg2, beside its adhesion function, regulates intestinal barrier function via p38MAPK signalling. This is in contrast to keratinocytes and points towards tissue-specific signalling functions of desmosomal cadherins.

ImaEdge - a platform for quantitative analysis of the spatiotemporal dynamics of cortical proteins during cell polarization.

Science.gov (United States)

Zhang, Zhen; Lim, Yen Wei; Zhao, Peng; Kanchanawong, Pakorn; Motegi, Fumio

2017-12-15

Cell polarity involves the compartmentalization of the cell cortex. The establishment of cortical compartments arises from the spatial bias in the activity and concentration of cortical proteins. The mechanistic dissection of cell polarity requires the accurate detection of dynamic changes in cortical proteins, but the fluctuations of cell shape and the inhomogeneous distributions of cortical proteins greatly complicate the quantitative extraction of their global and local changes during cell polarization. To address these problems, we introduce an open-source software package, ImaEdge, which automates the segmentation of the cortex from time-lapse movies, and enables quantitative extraction of cortical protein intensities. We demonstrate that ImaEdge enables efficient and rigorous analysis of the dynamic evolution of cortical PAR proteins during Caenorhabditis elegans embryogenesis. It is also capable of accurate tracking of varying levels of transgene expression and discontinuous signals of the actomyosin cytoskeleton during multiple rounds of cell division. ImaEdge provides a unique resource for quantitative studies of cortical polarization, with the potential for application to many types of polarized cells.This article has an associated First Person interview with the first authors of the paper. © 2017. Published by The Company of Biologists Ltd.

2,2',4,4'-Tetrachlorobiphenyl upregulates cyclooxygenase-2 in HL-60 cells via p38 mitogen-activated protein kinase and NF-κB

International Nuclear Information System (INIS)

Bezdecny, Steven A.; Karmaus, Peer; Roth, Robert A.; Ganey, Patricia E.

2007-01-01

Polychlorinated biphenyls (PCBs) are ubiquitous, persistent environmental contaminants that affect a number of cellular systems, including neutrophils. Among the effects caused by the noncoplanar PCB 2,2',4,4'-tetrachlorobiphenyl (2244-TCB) in granulocytic HL-60 cells are increases in superoxide anion production, activation of phospholipase A 2 with subsequent release of arachidonic acid (AA) and upregulation of the inflammatory gene cyclooxygenase-2 (COX-2). The objective of this study was to determine the signal transduction pathways involved in the upregulation of COX-2 by 2244-TCB. Treatment of HL-60 cells with 2244-TCB led to increased expression of COX-2 mRNA. This increase was prevented by the transcriptional inhibitor actinomycin D in cells pretreated with 2244-TCB for 10 min. The increase in COX-2 mRNA was associated with release of 3 H-AA, phosphorylation of p38 and extracellular signal-regulated kinase (ERK) mitogen-activated protein (MAP) kinases, increased levels of nuclear NF-κB and increased superoxide anion production. Bromoenol lactone, an inhibitor of the calcium-independent phospholipase A 2 , reduced 3 H-AA release but had no effect on COX-2 mRNA, protein or activity. Pretreatment with SB-202190 or SB-203580, inhibitors of the p38 MAP kinase pathway, prevented the 2244-TCB-mediated induction of COX-2 and phosphorylation of p38 and ERK MAP kinases. These inhibitors did not alter 3 H-AA release. Treatment with PD 98059 or U 0126, inhibitors of the MAP/ERK (MEK) pathway, prevented the 2244-TCB-mediated activation of ERK but had no effect on COX-2 induction or p38 phosphorylation. 2244-TCB treatment did not affect c-Jun N-terminal kinase (JNK) phosphorylation. 2244-TCB exposure increased the amount of nuclear NF-κB. This increase was prevented by pretreatment with p38 MAP kinase inhibitors, but not by pretreatment with MEK inhibitors. Pretreatment with inhibitors of NF-κB prevented the 2244-TCB-mediated induction of COX-2 mRNA. 2244-TCB

Sodium appetite elicited by low-sodium diet is dependent on p44/42 mitogen-activated protein kinase (extracellular signal-regulated kinase 1/2) activation in the brain.

Science.gov (United States)

Monteiro, L R N; Marangon, P B; Elias, L L K; Reis, L C; Antunes-Rodrigues, J; Mecawi, A S

2017-09-01

Sodium appetite is regulated by several signalling molecules, among which angiotensin II (Ang II) serves as a key driver of robust salt intake by binding to Ang II type 1 receptors (AT1R) in several regions in the brain. The activation of these receptors recruits the mitogen-activated protein kinase (MAPK) pathway, which has previously been linked to Ang II-induced increases in sodium appetite. Thus, we addressed the involvement of MAPK signalling in the induction of sodium appetite after 4 days of low-sodium diet consumption. An increase in extracellular signal-regulated kinase (ERK) phosphorylation in the laminae terminalis and mediobasal hypothalamus was observed after low-sodium diet consumption. This response was reduced by i.c.v. microinjection of an AT1R antagonist into the laminae terminalis but not the hypothalamus. This result indicates that low-sodium diet consumption activates the MAPK pathway via Ang II/AT1R signalling on the laminae terminalis. On the other hand, activation of the MAPK pathway in the mediobasal hypothalamus after low-sodium diet consumption appears to involve another extracellular mediator. We also evaluated whether a low-sodium diet could increase the sensitivity for Ang II in the brain and activate the MAPK pathway. However, i.c.v. injection of Ang II increased ERK phosphorylation on the laminae terminalis and mediobasal hypothalamus; this increase achieved a response magnitude similar to those observed in both the normal and low-sodium diet groups. These data indicate that low-sodium diet consumption for 4 days is insufficient to change the ERK phosphorylation response to Ang II in the brain. To investigate whether the MAPK pathway is involved in sodium appetite after low-sodium diet consumption, we performed i.c.v. microinjections of a MAPK pathway inhibitor (PD98059). PD98059 inhibited both saline and water intake after low-sodium diet consumption. Thus, the MAPK pathway is involved in promoting the sodium appetite after low

Bioactive glass induced osteogenic differentiation of human adipose stem cells is dependent on cell attachment mechanism and mitogen-activated protein kinases

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M Ojansivu

2018-02-01

Full Text Available Bioactive glasses (BaGs are widely utilised in bone tissue engineering (TE but the molecular response of cells to BaGs is poorly understood. To elucidate the mechanisms of cell attachment to BaGs and BaG-induced early osteogenic differentiation, we cultured human adipose stem cells (hASCs on discs of two silica-based BaGs S53P4 (23.0 Na2O - 20.0 CaO - 4.0 P2O5 - 53.0 SiO2 (wt-% and 1-06 (5.9 Na2O - 12.0 K2O - 5.3 MgO - 22.6 CaO - 4.0 P2O5 - 0.2 B2O3 - 50.0 SiO2 in the absence of osteogenic supplements. Both BaGs induced early osteogenic differentiation by increasing alkaline phosphatase activity (ALP and the expression of osteogenic marker genes RUNX2a and OSTERIX. Based on ALP activity, the slower reacting 1-06 glass was a stronger osteoinducer. Regarding the cell attachment, cells cultured on BaGs had enhanced integrinβ1 and vinculin production, and mature focal adhesions were smaller but more dispersed than on cell culture plastic (polystyrene. Focal adhesion kinase (FAK, extracellular signal-regulated kinase (ERK1/2 and c-Jun N-terminal kinase (JNK-induced c-Jun phosphorylations were upregulated by glass contact. Moreover, the BaG-stimulated osteoinduction was significantly reduced by FAK and mitogen-activated protein kinase (MAPK inhibitors, indicating an important role for FAK and MAPKs in the BaG-induced early osteogenic commitment of hASCs. Upon indirect insert culture, the ions released from the BaG discs could not reproduce the observed cellular changes, which highlighted the role of direct cell-BaG interactions in the osteopotential of BaGs. These findings gave valuable insight into the mechanism of BaG-induced osteogenic differentiation and therefore provided knowledge to aid the future design of new functional biomaterials to meet the increasing demand for clinical bone TE treatments.

Calcium Hydroxide-induced Proliferation, Migration, Osteogenic Differentiation, and Mineralization via the Mitogen-activated Protein Kinase Pathway in Human Dental Pulp Stem Cells.

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Chen, Luoping; Zheng, Lisha; Jiang, Jingyi; Gui, Jinpeng; Zhang, Lingyu; Huang, Yan; Chen, Xiaofang; Ji, Jing; Fan, Yubo

2016-09-01

Calcium hydroxide has been extensively used as the gold standard for direct pulp capping in clinical dentistry. It induces proliferation, migration, and mineralization in dental pulp stem cells (DPSCs), but the underlying mechanisms are still unclear. The aim of this study was to investigate the role of the mitogen-activated protein (MAP) kinase pathway in calcium hydroxide-induced proliferation, migration, osteogenic differentiation, and mineralization in human DPSCs. Human DPSCs between passages 3 and 6 were used. DPSCs were preincubated with inhibitors of MAP kinases and cultured with calcium hydroxide. The phosphorylated MAP kinases were detected by Western blot analysis. Cell viability was analyzed via the methylthiazol tetrazolium assay. Cell migration was estimated using the wound healing assay. Alkaline phosphatase (ALP) expression was analyzed using the ALP staining assay. Mineralization was studied by alizarin red staining analysis. Calcium hydroxide significantly promoted the phosphorylation of the c-Jun N-terminal kinase (JNK), p38, and extracellular signal-regulated kinase. The inhibition of JNK and p38 signaling abolished calcium hydroxide-induced proliferation of DPSCs. The inhibition of JNK, p38, and extracellular signal-regulated kinase signaling suppressed the migration, ALP expression, and mineralization of DPSCs. Our study showed that the MAP kinase pathway was involved in calcium hydroxide-induced proliferation, migration, osteogenic differentiation, and mineralization in human DPSCs. Copyright © 2016 American Association of Endodontists. Published by Elsevier Inc. All rights reserved.

Probing membrane protein structure using water polarization transfer solid-state NMR.

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Williams, Jonathan K; Hong, Mei

2014-10-01

Water plays an essential role in the structure and function of proteins, lipid membranes and other biological macromolecules. Solid-state NMR heteronuclear-detected (1)H polarization transfer from water to biomolecules is a versatile approach for studying water-protein, water-membrane, and water-carbohydrate interactions in biology. We review radiofrequency pulse sequences for measuring water polarization transfer to biomolecules, the mechanisms of polarization transfer, and the application of this method to various biological systems. Three polarization transfer mechanisms, chemical exchange, spin diffusion and NOE, manifest themselves at different temperatures, magic-angle-spinning frequencies, and pulse irradiations. Chemical exchange is ubiquitous in all systems examined so far, and spin diffusion plays the key role in polarization transfer within the macromolecule. Tightly bound water molecules with long residence times are rare in proteins at ambient temperature. The water polarization-transfer technique has been used to study the hydration of microcrystalline proteins, lipid membranes, and plant cell wall polysaccharides, and to derive atomic-resolution details of the kinetics and mechanism of ion conduction in channels and pumps. Using this approach, we have measured the water polarization transfer to the transmembrane domain of the influenza M2 protein to obtain information on the structure of this tetrameric proton channel. At short mixing times, the polarization transfer rates are site-specific and depend on the pH, labile protons, sidechain conformation, as well as the radial position of the residues in this four-helix bundle. Despite the multiple dependences, the initial transfer rates reflect the periodic nature of the residue positions from the water-filled pore, thus this technique provides a way of gleaning secondary structure information, helix tilt angle, and the oligomeric structure of membrane proteins. Copyright © 2014 Elsevier Inc. All

Phosphorylation of Icariin Can Alleviate the Oxidative Stress Caused by the Duck Hepatitis Virus A through Mitogen-Activated Protein Kinases Signaling Pathways

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Wen Xiong

2017-09-01

Full Text Available The duck virus hepatitis (DVH caused by the duck hepatitis virus A (DHAV has produced extensive economic losses to the duck industry. The currently licensed commercial vaccine has shown some defects and does not completely prevent the DVH. Accordingly, a new alternative treatment for this disease is urgently needed. Previous studies have shown that icariin (ICA and its phosphorylated derivative (pICA possessed good anti-DHAV effects through direct and indirect antiviral pathways, such as antioxidative stress. But the antioxidant activity showed some differences between ICA and pICA. The aim of this study is to prove that ICA and pICA attenuate oxidative stress caused by DHAV in vitro and in vivo, and to investigate their mechanism of action to explain their differences in antioxidant activities. In vivo, the dynamic deaths, oxidative evaluation indexes and hepatic pathological change scores were detected. When was added the hinokitiol which showed the pro-oxidative effect as an intervention method, pICA still possessed more treatment effect than ICA. The strong correlation between mortality and oxidative stress proves that ICA and pICA alleviate oxidative stress caused by DHAV. This was also demonstrated by the addition of hydrogen peroxide (H2O2 as an intervention method in vitro. pICA can be more effective than ICA to improve duck embryonic hepatocytes (DEHs viability and reduce the virulence of DHAV. The strong correlation between TCID50 and oxidative stress demonstrates that ICA and pICA can achieve anti-DHAV effects by inhibiting oxidative stress. In addition, the superoxide dismutase (SOD and glutathione peroxidase (GSH-Px of ICA and pICA showed significant difference. pICA could significantly inhibit the phosphorylation of p38, extra cellular signal regulated Kinase (ERK 1/2 and c-Jun N-terminal kinase (JNK, which were related to mitogen-activated protein kinases (MAPKs signaling pathways. Ultimately, compared to ICA, pICA exhibited more

Curcumin Modulates Macrophage Polarization Through the Inhibition of the Toll-Like Receptor 4 Expression and its Signaling Pathways

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Yaoyao Zhou

2015-05-01

Full Text Available Background: Curcumin, the active ingredient in curcuma rhizomes, has a wide range of therapeutic effects. However, its atheroprotective activity in human acute monocytic leukemia THP-1 cells remains unclear. We investigated the activity and molecular mechanism of action of curcumin in polarized macrophages. Methods: Phorbol myristate acetate (PMA-treated THP-1 cells were differentiated to macrophages, which were further polarized to M1 cells by lipopolysaccharide (LPS; 1 µg/ml and interferon (IFN-γ (20 ng/ml and treated with varying curcumin concentrations. [3H]thymidine (3H-TdR incorporation assays were utilized to measure curcumin-induced growth inhibition. The expression of tumor necrosis factor-a (TNF-a, interleukin (IL-6, and IL-12B (p40 were measured by quantitative real-time polymerase chain reaction (PCR and enzyme-linked immunosorbent assay (ELISA. Macrophage polarization and its mechanism were evaluated by flow cytometry and western blot. Additionally, toll-like receptor 4 (TLR4 small interfering RNA and mitogen-activated protein kinase (MAPK inhibitors were used to further confirm the molecular mechanism of curcumin on macrophage polarization. Results: Curcumin dose-dependently inhibited M1 macrophage polarization and the production of TNF-a, IL-6, and IL-12B (p40. It also decreased TLR4 expression, which regulates M1 macrophage polarization. Furthermore, curcumin significantly inhibited the phosphorylation of ERK, JNK, p38, and nuclear factor (NF-γB. In contrast, SiTLR4 in combination with p-JNK, p-ERK, and p-p38 inhibition reduced the effect of curcumin on polarization. Conclusions: Curcumin can modulate macrophage polarization through TLR4-mediated signaling pathway inhibition, indicating that its effect on macrophage polarization is related to its anti-inflammatory and atheroprotective effects. Our data suggest that curcumin could be used as a therapeutic agent in atherosclerosis.

The actin cytoskeleton may control the polar distribution of an auxin transport protein

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Muday, G. K.; Hu, S.; Brady, S. R.; Davies, E. (Principal Investigator)

2000-01-01

The gravitropic bending of plants has long been linked to the changes in the transport of the plant hormone auxin. To understand the mechanism by which gravity alters auxin movement, it is critical to know how polar auxin transport is initially established. In shoots, polar auxin transport is basipetal (i.e., from the shoot apex toward the base). It is driven by the basal localization of the auxin efflux carrier complex. One mechanism for localizing this efflux carrier complex to the basal membrane may be through attachment to the actin cytoskeleton. The efflux carrier protein complex is believed to consist of several polypeptides, including a regulatory subunit that binds auxin transport inhibitors, such as naphthylphthalamic acid (NPA). Several lines of experimentation have been used to determine if the NPA binding protein interacts with actin filaments. The NPA binding protein has been shown to partition with the actin cytoskeleton during detergent extraction. Agents that specifically alter the polymerization state of the actin cytoskeleton change the amount of NPA binding protein and actin recovered in these cytoskeletal pellets. Actin-affinity columns were prepared with polymers of actin purified from zucchini hypocotyl tissue. NPA binding activity was eluted in a single peak from the actin filament column. Cytochalasin D, which fragments the actin cytoskeleton, was shown to reduce polar auxin transport in zucchini hypocotyls. The interaction of the NPA binding protein with the actin cytoskeleton may localize it in one plane of the plasma membrane, and thereby control the polarity of auxin transport.

Dynamic nuclear polarization of membrane proteins: covalently bound spin-labels at protein–protein interfaces

International Nuclear Information System (INIS)

Wylie, Benjamin J.; Dzikovski, Boris G.; Pawsey, Shane; Caporini, Marc; Rosay, Melanie; Freed, Jack H.; McDermott, Ann E.

2015-01-01

We demonstrate that dynamic nuclear polarization of membrane proteins in lipid bilayers may be achieved using a novel polarizing agent: pairs of spin labels covalently bound to a protein of interest interacting at an intermolecular interaction surface. For gramicidin A, nitroxide tags attached to the N-terminal intermolecular interface region become proximal only when bimolecular channels forms in the membrane. We obtained signal enhancements of sixfold for the dimeric protein. The enhancement effect was comparable to that of a doubly tagged sample of gramicidin C, with intramolecular spin pairs. This approach could be a powerful and selective means for signal enhancement in membrane proteins, and for recognizing intermolecular interfaces

PSM/SH2-B distributes selected mitogenic receptor signals to distinct components in the PI3-kinase and MAP kinase signaling pathways.

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Deng, Youping; Xu, Hu; Riedel, Heimo

2007-02-15

The Pro-rich, PH, and SH2 domain containing mitogenic signaling adapter PSM/SH2-B has been implicated as a cellular partner of various mitogenic receptor tyrosine kinases and related signaling mechanisms. Here, we report in a direct comparison of three peptide hormones, that PSM participates in the assembly of distinct mitogenic signaling complexes in response to insulin or IGF-I when compared to PDGF in cultured normal fibroblasts. The complex formed in response to insulin or IGF-I involves the respective peptide hormone receptor and presumably the established components leading to MAP kinase activation. However, our data suggest an alternative link from the PDGF receptor via PSM directly to MEK1/2 and consequently also to p44/42 activation, possibly through a scaffold protein. At least two PSM domains participate, the SH2 domain anticipated to link PSM to the respective receptor and the Pro-rich region in an association with an unidentified downstream component resulting in direct MEK1/2 and p44/42 regulation. The PDGF receptor signaling complex formed in response to PDGF involves PI 3-kinase in addition to the same components and interactions as described for insulin or IGF-I. PSM associates with PI 3-kinase via p85 and in addition the PSM PH domain participates in the regulation of PI 3-kinase activity, presumably through membrane interaction. In contrast, the PSM Pro-rich region appears to participate only in the MAP kinase signal. Both pathways contribute to the mitogenic response as shown by cell proliferation, survival, and focus formation. PSM regulates p38 MAP kinase activity in a pathway unrelated to the mitogenic response.

Asthmatic airway smooth muscle CXCL10 production: mitogen-activated protein kinase JNK involvement

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Alrashdan, Yazan A.; Alkhouri, Hatem; Chen, Emily; Lalor, Daniel J.; Poniris, Maree; Henness, Sheridan; Brightling, Christopher E.; Burgess, Janette K.; Armour, Carol L.; Ammit, Alaina J.

2012-01-01

CXCL10 (IP10) is involved in mast cell migration to airway smooth muscle (ASM) bundles in asthma. We aimed to investigate the role of cytokine-induced MAPK activation in CXCL10 production by ASM cells from people with and without asthma. Confluent growth-arrested ASM cells were treated with inhibitors of the MAPKs ERK, p38, and JNK and transcription factor NF-κB, or vehicle, and stimulated with IL-1β, TNF-α, or IFN-γ, alone or combined (cytomix). CXCL10 mRNA and protein, JNK, NF-κB p65 phosphorylation, and Iκ-Bα protein degradation were assessed using real-time PCR, ELISA, and immunoblotting, respectively. Cytomix, IL-1β, and TNF-α induced CXCL10 mRNA expression more rapidly in asthmatic than nonasthmatic ASM cells. IL-1β and/or TNF-α combined with IFN-γ synergistically increased asthmatic ASM cell CXCL10 release. Inhibitor effects were similar in asthmatic and nonasthmatic cells, but cytomix-induced release was least affected, with only JNK and NF-κB inhibitors halving it. Notably, JNK phosphorylation was markedly less in asthmatic compared with nonasthmatic cells. However, in both, the JNK inhibitor SP600125 reduced JNK phosphorylation and CXCL10 mRNA levels but did not affect CXCL10 mRNA stability or Iκ-Bα degradation. Together, the JNK and NF-κB inhibitors completely inhibited their CXCL10 release. We concluded that, in asthmatic compared with nonasthmatic ASM cells, JNK activation was reduced and CXCL10 gene expression was more rapid following cytomix stimulation. However, in both, JNK activation did not regulate early events leading to NF-κB activation. Thus JNK and NF-κB provide independent therapeutic targets for limiting CXCL10 production and mast cell migration to the ASM in asthma. PMID:22387292

Resveratrol inhibits PDGF receptor mitogenic signaling in mesangial cells: role of PTP1B

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Venkatesan, Balachandar; Ghosh-Choudhury, Nandini; Das, Falguni; Mahimainathan, Lenin; Kamat, Amrita; Kasinath, Balakuntalam S.; Abboud, Hanna E.; Choudhury, Goutam Ghosh

2008-01-01

Mesangioproliferative glomerulonephritis is associated with overactive PDGF receptor signal transduction. We show that the phytoalexin resveratrol dose dependently inhibits PDGF-induced DNA synthesis in mesangial cells with an IC50 of 10 μM without inducing apoptosis. Remarkably, the increased SIRT1 deacetylase activity induced by resveratrol was not necessary for this inhibitory effect. Resveratrol significantly blocked PDGF-stimulated c-Src and Akt kinase activation, resulting in reduced cyclin D1 expression and attenuated pRb phosphorylation and cyclin-dependent kinase-2 (CDK2) activity. Furthermore, resveratrol inhibited PDGFR phosphorylation at the PI 3 kinase and Grb-2 binding sites tyrosine-751 and tyrosine-716, respectively. This deficiency in PDGFR phosphorylation resulted in significant inhibition of PI 3 kinase and Erk1/2 MAPK activity. Interestingly, resveratrol increased the activity of protein tyrosine phosphatase PTP1B, which dephosphorylates PDGF-stimulated phosphorylation at tyrosine-751 and tyrosine-716 on PDGFR with concomitant reduction in Akt and Erk1/2 kinase activity. PTP1B significantly inhibited PDGF-induced DNA synthesis without inducing apoptosis. These results for the first time provide evidence that the stilbene resveratrol targets PTP1B to inhibit PDGFR mitogenic signaling.—Venkatesan, B., Ghosh-Choudhury, N., Das, F., Mahimainathan, L., Kamat, A., Kasinath, B. S., Abboud, H. E., Choudhury, G. G. Resveratrol inhibits PDGF receptor mitogenic signaling in mesangial cells: role of PTP1B. PMID:18567737

Arabidopsis Raf-Like Mitogen-Activated Protein Kinase Kinase Kinase Gene Raf43 Is Required for Tolerance to Multiple Abiotic Stresses.

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Nasar Virk

Full Text Available Mitogen-activated protein kinase (MAPK cascades are critical signaling modules that mediate the transduction of extracellular stimuli into intracellular response. A relatively large number of MAPKKKs have been identified in a variety of plant genomes but only a few of them have been studied for their biological function. In the present study, we identified an Arabidopsis Raf-like MAPKKK gene Raf43 and studied its function in biotic and abiotic stress response using a T-DNA insertion mutant raf43-1 and two Raf43-overexpressing lines Raf43-OE#1 and Raf43-OE#13. Expression of Raf43 was induced by multiple abiotic and biotic stresses including treatments with drought, mannitol and oxidative stress or defense signaling molecule salicylic acid and infection with necrotrophic fungal pathogen Botrytis cinerea. Seed germination and seedling root growth of raf43-1 were significantly inhibited on MS medium containing mannitol, NaCl, H2O2 or methyl viologen (MV while seed germination and seedling root growth of the Raf43-OE#1 and Raf43-OE#13 lines was similar to wild type Col-0 under the above stress conditions. Soil-grown raf43-1 plants exhibited reduced tolerance to MV, drought and salt stress. Abscisic acid inhibited significantly seed germination and seedling root growth of the raf43-1 line but had no effect on the two Raf43-overexpressing lines. Expression of stress-responsive RD17 and DREB2A genes was significantly down-regulated in raf43-1 plants. However, the raf43-1 and Raf43-overexpressing plants showed similar disease phenotype to the wild type plants after infection with B. cinerea or Pseudomonas syringae pv. tomato DC3000. Our results demonstrate that Raf43, encoding for a Raf-like MAPKKK, is required for tolerance to multiple abiotic stresses in Arabidopsis.

Duration of streptozotocin-induced diabetes differentially affects p38-mitogen-activated protein kinase (MAPK phosphorylation in renal and vascular dysfunction

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Gupta Akanksha

2005-03-01

Full Text Available Abstract Background In the present study we tested the hypothesis that progression of streptozotocin (STZ-induced diabetes (14-days to 28-days would produce renal and vascular dysfunction that correlate with altered p38- mitogen-activated protein kinase (p38-MAPK phosphorylation in kidneys and thoracic aorta. Methods Male Sprague Dawley rats (350–400 g were randomized into three groups: sham (N = 6, 14-days diabetic (N = 6 and 28-days diabetic rats (N = 6. Diabetes was induced using a single tail vein injection of STZ (60 mg/kg, I.V. on the first day. Rats were monitored for 28 days and food, water intake and plasma glucose levels were noted. At both 14-days and 28-days post diabetes blood samples were collected and kidney cortex, medulla and aorta were harvested from each rat. Results The diabetic rats lost body weight at both 14-days (-10% and 28-days (-13% more significantly as compared to sham (+10% group. Glucose levels were significantly elevated in the diabetic rats at both 14-days and 28-days post-STZ administration. Renal dysfunction as evidenced by renal hypertrophy, increased plasma creatinine concentration and reduced renal blood flow was observed in 14-days and 28-days diabetes. Vascular dysfunction as evidenced by decreased carotid blood flow was observed in 14-days and 28-days diabetes. We observed an up-regulation of inducible nitric oxide synthase (iNOS, prepro endothelin-1 (preproET-1 and phosphorylated p38-MAPK in thoracic aorta and kidney cortex but not in kidney medulla in 28-days diabetes group. Conclusion The study provides evidence that diabetes produces vascular and renal dysfunction with a profound effect on signaling mechanisms at later stage of diabetes.

MaHog1, a Hog1-type mitogen-activated protein kinase gene, contributes to stress tolerance and virulence of the entomopathogenic fungus Metarhizium acridum.

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Jin, Kai; Ming, Yue; Xia, Yu Xian

2012-12-01

Fungal biocontrol agents have great potential in integrated pest management. However, poor efficacy and sensitivity to various adverse factors have hampered their wide application. In eukaryotic cells, Hog1 kinase plays a critical role in stress responses. In this study, MaHog1 (GenBank accession no. EFY85878), encoding a member of the Hog1/Sty1/p38 mitogen-activated protein kinase family in Metarhizium (Me.) acridum, was identified. Targeted gene disruption was used to analyse the role of MaHog1 in virulence and tolerance of adverse factors. Mutants with MaHog1 depletion showed increased sensitivity to high osmotic stress, high temperature and oxidative stress, and exhibited remarkable resistance to cell wall-disturbing agents. These results suggest that Hog1 kinase has a conserved function in regulating multistress responses among fungi, and that MaHog1 might influence cell wall biogenesis in Me. acridum. Bioassays conducted with topical inoculation and intrahaemocoel injection revealed that MaHog1 is required for both penetration and postpenetration development of Me. acridum. MaHog1 disruption resulted in a significant reduction in virulence, likely due to the combination of a decrease in conidial germination, a reduction in appressorium formation and a decline in growth rate in insect haemolymph, which might be caused by impairing fungal tolerance of various stresses during infection.

Activation of transcriptional activities of AP-1 and SRE by a new zinc-finger protein ZNF641

International Nuclear Information System (INIS)

Qi Xingzhu; Li Yongqing; Xiao Jing; Yuan Wuzhou; Yan Yan; Wang Yuequn; Liang Shuyuan; Zhu Chuanbing; Chen Yingduan; Liu Mingyao; Wu Xiushan

2006-01-01

Mitogen-activated protein kinases (MAPKs) are evolutionarily conserved enzymes in cell signal transduction connecting cell-surface receptors to critical regulatory targets within cells and control cell survival, adaptation, and proliferation. Previous studies revealed that zinc-finger proteins are involved in the regulation of the MAPK signaling pathways. Here, we report the identification and characterization of a novel human zinc-finger protein, ZNF641. The cDNA of ZNF641 is 4.9 kb, encoding 438 amino acids in the nucleus. The protein is highly conserved in evolution across different vertebrate species from mouse to human. Northern blot analysis indicates that ZNF641 is expressed in most of the examined human tissues, with a high level in skeletal muscle. Overexpression of pCMV-Tag2B-ZNF641 in the COS-7 cells activates the transcriptional activities of AP-1 and SRE. Deletion analysis indicates that the linker between KRAB box and C 2 H 2 -type zinc-fingers represents the basal activation domain. These results suggest that ZNF641 may be a positive regulator in MAPK-mediated signaling pathways that lead to the activation of AP-1 and SRE

Andrographolide stimulates p38 mitogen-activated protein kinase-nuclear factor erythroid-2-related factor 2-heme oxygenase 1 signaling in primary cerebral endothelial cells for definite protection against ischemic stroke in rats.

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Yen, Ting-Lin; Chen, Ray-Jade; Jayakumar, Thanasekaran; Lu, Wan-Jung; Hsieh, Cheng-Ying; Hsu, Ming-Jen; Yang, Chih-Hao; Chang, Chao-Chien; Lin, Yen-Kuang; Lin, Kuan-Hung; Sheu, Joen-Rong

2016-04-01

Stroke pathogenesis involves complex oxidative stress-related pathways. The nuclear factor erythroid-2-related factor 2 (Nrf2) and heme oxygenase 1 (HO-1) pathways have been considered molecular targets in pharmacologic intervention for ischemic diseases. Andrographolide, a labdane diterpene, has received increasing attention in recent years because of its various pharmacologic activities. We determined that andrographolide modulates the mitogen-activated protein kinase (MAPK)-Nrf2-HO-1 signaling cascade in primary cerebral endothelial cells (CECs) to provide positive protection against middle cerebral artery occlusion (MCAO)-induced ischemic stroke in rats. In the present study, andrographolide (10 μM) increased HO-1 protein and messenger RNA expressions, Nrf2 phosphorylation, and nuclear translocation in CECs, and these activities were disrupted by a p38 MAPK inhibitor, SB203580, but not by the extracellular signal-regulated kinase inhibitor PD98059 or c-Jun amino-terminal kinase inhibitor SP600125. Similar results were observed in confocal microscopy analysis. Moreover, andrographolide-induced Nrf2 and HO-1 protein expressions were significantly inhibited by Nrf2 small interfering RNA. Moreover, HO-1 knockdown attenuated the protective effect of andrographolide against oxygen-glucose deprivation-induced CEC death. Andrographolide (0.1 mg/kg) significantly suppressed free radical formation, blood-brain barrier disruption, and brain infarction in MCAO-insulted rats, and these effects were reversed by the HO-1 inhibitor zinc protoporphyrin IX. The mechanism is attributable to HO-1 activation, as directly evidenced by andrographolide-induced pronounced HO-1 expression in brain tissues, which was highly localized in the cerebral capillary. In conclusion, andrographolide increased Nrf2-HO-1 expression through p38 MAPK regulation, confirming that it provides protection against MCAO-induced brain injury. These findings provide strong evidence that andrographolide could

Expression, purification and crystallization of Streptococcus dysgalactiae-derived mitogen

International Nuclear Information System (INIS)

Papageorgiou, Anastassios C.; Saarinen, Susanna; Ramirez-Bartutis, Rosa; Kato, Hidehito; Uchiyama, Takehiko; Kirikae, Teruo; Miyoshi-Akiyama, Toru

2006-01-01

S. dysgalactiae-derived mitogen, a superantigen, was crystallized. Crystals diffract to 2.4 Å at a synchrotron-radiation source and belong to space group P3/P3 1 /P3 2 , with unit-cell parameters a = b = 52.7, c = 62.4 Å, γ = 120° and one molecule in the crystallographic asymmetric unit. Superantigens are bacterial or viral toxins with potent immunostimulatory properties. Streptococcus dysgalactiae-derived mitogen, a 25 kDa protein, is a recently discovered superantigen isolated from S. dysgalactiae culture supernatant. Sequence considerations suggest that it belongs to a new superantigen family distinct from other superantigens. The protein was expressed in Escherichia coli cells and purified to homogeneity. Crystals were grown at pH 4.2–4.4 in the presence of 18–20%(w/v) PEG 3350 and 0.4 M lithium nitrate. A complete data set to 2.4 Å resolution was collected from a single crystal at liquid-nitrogen temperatures using synchrotron radiation. The crystals belong to space group P3/P3 1 /P3 2 , with unit-cell parameters a = b = 52.7, c = 62.4 Å, γ = 120° and one molecule in the crystallographic asymmetric unit

Detection of proliferating cell nuclear antigens and interleukin-2 beta receptor molecules on mitogen- and antigen-stimulated lymphocytes.

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Hesketh, J; Dobbelaere, D; Griffin, J F; Buchan, G

1993-01-01

The expression of interleukin-2 receptors (IL-2R) and proliferating cell nuclear antigens (PCNA) were compared for their usefulness as markers of lymphocyte activation. Heterologous polyclonal (anti-bovine IL-2R) and monoclonal (anti-human PCNA) antibodies were used to detect the expression of these molecules on activated deer lymphocytes. Both molecules were co-expressed on blast cells which had been activated with mitogen [concanavalin A (Con A)]. There was detectable up-regulation of IL-2R expression in response to antigen [Mycobacterium bovis-derived purified protein derivative (PPD)] stimulation while PCNA expression mimicked lymphocyte transformation (LT) reactivity. PCNA expression was found to more accurately reflect both antigen- and mitogen-activated lymphocyte activation, as estimated by LT activity. The expression of PCNA was used to identify antigen reactive cells from animals exposed to M. bovis. A very low percentage (1.1 +/- 0.4%) of peripheral blood lymphocytes from non-infected animals could be stimulated to express PCNA by in vitro culture with antigen (PPD). Within the infected group both diseased and healthy, 'in-contact', animals expressed significantly higher levels of PCNA upon antigen stimulation. PMID:8104884

Progesterone increases brain-derived neuroptrophic factor expression and protects against glutamate toxicity in a mitogen-activated protein kinase- and phosphoinositide-3 kinase-dependent manner in cerebral cortical explants.

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Kaur, Paramjit; Jodhka, Parmeet K; Underwood, Wendy A; Bowles, Courtney A; de Fiebre, Nancyellen C; de Fiebre, Christopher M; Singh, Meharvan

2007-08-15

The higher prevalence and risk for Alzheimer's disease in women relative to men has been partially attributed to the precipitous decline in gonadal hormone levels that occurs in women following the menopause. Although considerable attention has been focused on the consequence of estrogen loss, and thus estrogen's neuroprotective potential, it is important to recognize that the menopause results in a precipitous decline in progesterone levels as well. In fact, progesterone is neuroprotective, although the precise mechanisms involved remain unclear. Based on our previous observation that progesterone elicits the phosphorylation of ERK and Akt, key effectors of the neuroprotective mitogen-activated protein kinase (MAPK) and phosphoinositide-3 kinase (PI3-K) pathways, respectively, we determined whether activation of either of these pathways was necessary for progesterone-induced protection. With organotypic explants (slice culture) of the cerebral cortex, we found that progesterone protected against glutamate-induced toxicity. Furthermore, these protective effects were inhibited by either the MEK1/2 inhibitor UO126 or the PI3-K inhibitor LY294002, supporting the requirement for both the MAPK and PI3-K pathways in progesterone-induced protection. In addition, at a concentration and duration of treatment consistent with our neuroprotection data, progesterone also increased the expression of brain-derived neurotrophic factor (BDNF), at the level of both protein and mRNA. This induction of BDNF may be relevant to the protective effects of progesterone, in that inhibition of Trk signaling, with K252a, inhibited the protective effects of progesterone. Collectively, these data suggest that progesterone is protective via multiple and potentially related mechanisms. (c) 2007 Wiley-Liss, Inc. Copyright 2007 Wiley-Liss, Inc.

Polarized trafficking: the palmitoylation cycle distributes cytoplasmic proteins to distinct neuronal compartments.

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Tortosa, Elena; Hoogenraad, Casper C

2018-02-01

In neurons, polarized cargo distribution occurs mainly between the soma and axonal and dendritic compartments, and requires coordinated regulation of cytoskeletal remodeling and membrane trafficking. The Golgi complex plays a critical role during neuronal polarization and secretory trafficking has been shown to differentially transport proteins to both axons and dendrites. Besides the Golgi protein sorting, recent data revealed that palmitoylation cycles are an efficient mechanism to localize cytoplasmic, non-transmembrane proteins to particular neuronal compartments, such as the newly formed axon. Palmitoylation allows substrate proteins to bind to and ride with Golgi-derived secretory vesicles to all neuronal compartments. By allowing cytoplasmic proteins to 'hitchhike' on transport carriers in a non-polarized fashion, compartmentalized depalmitoylation may act as a selective retention mechanism. Copyright © 2018 Elsevier Ltd. All rights reserved.

Neuropeptide Y induces potent migration of human immature dendritic cells and promotes a Th2 polarization.

Science.gov (United States)

Buttari, Brigitta; Profumo, Elisabetta; Domenici, Giacomo; Tagliani, Angela; Ippoliti, Flora; Bonini, Sergio; Businaro, Rita; Elenkov, Ilia; Riganò, Rachele

2014-07-01

Neuropeptide Y (NPY), a major autonomic nervous system and stress mediator, is emerging as an important regulator of inflammation, implicated in autoimmunity, asthma, atherosclerosis, and cancer. Yet the role of NPY in regulating phenotype and functions of dendritic cells (DCs), the professional antigen-presenting cells, remains undefined. Here we investigated whether NPY could induce DCs to migrate, mature, and polarize naive T lymphocytes. We found that NPY induced a dose-dependent migration of human monocyte-derived immature DCs through the engagement of NPY Y1 receptor and the activation of ERK and p38 mitogen-activated protein kinases. NPY promoted DC adhesion to endothelial cells and transendothelial migration. It failed to induce phenotypic DC maturation, whereas it conferred a T helper 2 (Th2) polarizing profile to DCs through the up-regulation of interleukin (IL)-6 and IL-10 production. Thus, during an immune/inflammatory response NPY may exert proinflammatory effects through the recruitment of immature DCs, but it may exert antiinflammatory effects by promoting a Th2 polarization. Locally, at inflammatory sites, cell recruitment could be amplified in conditions of intense acute, chronic, or cold stress. Thus, altered or amplified signaling through the NPY-NPY-Y1 receptor-DC axis may have implications for the development of inflammatory conditions.-Buttari, B., Profumo, E., Domenici, G., Tagliani, A., Ippoliti, F., Bonini, S., Businaro, R., Elenkov, I., Riganò, R. Neuropeptide Y induces potent migration of human immature dendritic cells and promotes a Th2 polarization. © FASEB.

Different sets of ER-resident J-proteins regulate distinct polar nuclear-membrane fusion events in Arabidopsis thaliana.

Science.gov (United States)

Maruyama, Daisuke; Yamamoto, Masaya; Endo, Toshiya; Nishikawa, Shuh-ichi

2014-11-01

Angiosperm female gametophytes contain a central cell with two polar nuclei. In many species, including Arabidopsis thaliana, the polar nuclei fuse during female gametogenesis. We previously showed that BiP, an Hsp70 in the endoplasmic reticulum (ER), was essential for membrane fusion during female gametogenesis. Hsp70 function requires partner proteins for full activity. J-domain containing proteins (J-proteins) are the major Hsp70 functional partners. A. thaliana ER contains three soluble J-proteins, AtERdj3A, AtERdj3B, and AtP58(IPK). Here, we analyzed mutants of these proteins and determined that double-mutant ovules lacking AtP58(IPK) and AtERdj3A or AtERdj3B were defective in polar nuclear fusion. Electron microscopy analysis identified that polar nuclei were in close contact, but no membrane fusion occurred in mutant ovules lacking AtP58(IPK) and AtERdj3A. The polar nuclear outer membrane appeared to be connected via the ER remaining at the inner unfused membrane in mutant ovules lacking AtP58(IPK) and AtERdj3B. These results indicate that ER-resident J-proteins, AtP58(IPK)/AtERdj3A and AtP58(IPK)/AtERdj3B, function at distinct steps of polar nuclear-membrane fusion. Similar to the bip1 bip2 double mutant female gametophytes, the aterdj3a atp58(ipk) double mutant female gametophytes defective in fusion of the outer polar nuclear membrane displayed aberrant endosperm proliferation after fertilization with wild-type pollen. However, endosperm proliferated normally after fertilization of the aterdj3b atp58(ipk) double mutant female gametophytes defective in fusion of the inner membrane. Our results indicate that the polar nuclear fusion defect itself does not cause an endosperm proliferation defect. © The Author 2014. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists. All rights reserved. For permissions, please email: journals.permissions@oup.com.

Opposite effects of Ha-Ras and Ki-Ras on radiation-induced apoptosis via differential activation of PI3K/Akt and Rac/p38 mitogen-activated protein kinase signaling

International Nuclear Information System (INIS)

Choi, J.-A.; Kang, C.-M.; Lee, Y.-S.; Lee, S.-J.; Bae, S.-W.; Cho, C.-K.

2003-01-01

It has been well known that Ras signaling is involved in various cellular processes, including proliferation, differentiation, and apoptosis. However, distinct cellular functions of Ras isozymes are not fully understood. Here we show the opposing roles of Ha-Ras and Ki-Ras genes in the modulation of cell sensitivity to ionizing radiation. Overexpression of active isoform of Ha-Ras (12V-Ha- Ras) in Rat2 cells increases resistance to the ionizing radiation. Constitutive activation of phosphoinositide-3-kinase (PI3K) and Akt is detected specifically in 12V-Ha-Ras-overexpressing cells. The specific PI3K inhibitor LY294002 inhibits PI3K/Akt signaling and potentiates the radiation-induced apoptosis, suggesting that activation of PI3K/Akt signaling pathway is involved in the increased radio-resistance in cells overexpressing 12V-Ha-Ras. Overexpression of activated Ki-Ras (12V-Ki-Ras), on the other hand, markedly increases radiation sensitivity. The p38 mitogen-activated protein (MAP) kinase activity is selectively enhanced by ionizing radiation in cells overexpressing 12V-Ki-Ras. The specific p38 MAP kinase inhibitor, PD169316, or dominant-negative p38 MAP kinase decreases radiation-induced cell death. We further show that the mechanism that underlies potentiation of cell death in cells overexpressing 12V-Ki-Ras involves Bax translocation to the mitochondrial membrane. Elevated Bax translocation following ionizing irradiation in 12V-Ki-Ras-overexpressing cells is completely inhibited by PD169316 or dominant-negative p38 MAP kinase. In addition, introduction of cells with RacN17, a dominant negative mutant of Rac, resulted in a marked inhibition of radiation-induced Bax translocation and apoptotic cell death as well as p38 MAP kinase activation. Taken together, these findings explain the opposite effects of Ha-Ras and Ki-Ras on modulation of radio-sensitivity, and suggest that differential activation of PI3K/Akt and Rac/p38 MAP kinase signaling by Ha-Ras and Ki-Ras may

Activation of ERK mitogen-activated protein kinase in human cells by the mycotoxin patulin

International Nuclear Information System (INIS)

Wu, T.-S.; Yu, F.-Y.; Su, C.-C.; Kan, J.-C.; Chung, C.-P.; Liu, B.-H.

2005-01-01

Patulin (PAT), a mycotoxin produced by certain species of Penicillium and Aspergillus, is often detectable in moldy fruits and their derivative products. PAT led to a concentration-dependent and time-dependent increase in phosphorylation of extracellular signal-regulated protein kinases 1 and 2 (ERK1/2) in human embryonic kidney (HEK293) cells, human peripheral blood mononuclear cells (PBMCs), and Madin-Darby canine kidney (MDCK) cells. Exposure of HEK293 cells to concentrations above 5 μM PAT for 30 min induced ERK1/2 phosphorylation; activation of ERK1/2 was also observed after 24 h incubation with 0.05 μM of PAT. Treatment of human PBMCs for 30 min with 30 μM PAT dramatically increased the phosphorylated ERK1/2 levels. Both MEK1/2 inhibitors, U0126 and PD98059, suppressed ERK1/2 activation in either HEK293 or MDCK cells. In HEK293 cells, U0126-mediated inhibition of PAT-induced ERK1/2 phosphorylation resulted in a significant decrease in levels of DNA damage, expressed as tail moment values, in the single cell gel electrophoresis assay. Conversely, U0126 did not affect cell viability, lactate dehydrogenase release, and the DNA synthesis rate in PAT-treated cultures. Exposure of HEK293 cells for 90 min to 15 μM PAT elevated the levels of early growth response gene-1 (egr-1) mRNA, but not of c-fos, fosB, and junB mRNAs. These results indicate that in human cells, PAT causes a rapid and persistent activation of ERK1/2 and this signaling pathway plays an important role in mediating PAT-induced DNA damage and egr-1 gene expression

The Protein Kinase RSK Family - Roles in Prostate Cancer

National Research Council Canada - National Science Library

Lannigan, Deborah

2006-01-01

The Ser/Thr protein kinase p90-kDa ribosomal S6 kinase (RSK) is an important downstream effector of mitogen-activated protein kinase but its roles in prostate cancer have not been previously examined...

Effects of cyclophosphamide on in vitro human lymphocyte culture and mitogenic stimulation

International Nuclear Information System (INIS)

Sharma, B.S.

1983-01-01

Cyclophosphamide (CY) has been reported to be inactive in vitro under certain conditions. In the present study, CY was tested for its ability to inhibit human lymphocyte proliferation and to modulate lymphocyte response to mitogens in vitro. The inhibition of or the increase in 3 H-thymidine incorporation in mitogen-stimulated and unstimulated lymphocytes by CY was used as a measure of CY activity in vitro. The results demonstrate that lymphocytes from 10 different persons had a mean decrease of 74% in 3 H-thymidine incorporation in the presence of CY (P less than 0.005). The effect was maximal at a concentration of 160 micrograms/ml. A mean inhibition of 35 and 55% was caused by 10 and 40 micrograms/ml concentrations of CY, respectively. CY also was able to reduce the number of viable cells during 5 days in culture and had a profound effect on mitogen stimulation of lymphocytes. In all cases, CY modulated the stimulation of lymphocytes by phytohemagglutinin (PHA), concanavalin A (Con A), and pokeweed mitogen (PWM) either by augmenting or suppressing the responses. At low concentrations (10 micrograms/ml) it augmented mitogenic stimulation by 46 to 281%. At higher concentrations (20 to 160 micrograms/ml), CY had a suppressive effect with a maximum suppression of 99%. The CY-induced immunomodulation is perhaps caused by its action on the regulatory T cells. When tested in vitro, CY had inhibitory activity on T cells

Adenosine monophosphate-activated protein kinase modulates the activated phenotype of hepatic stellate cells.

Science.gov (United States)

Caligiuri, Alessandra; Bertolani, Cristiana; Guerra, Cristina Tosti; Aleffi, Sara; Galastri, Sara; Trappoliere, Marco; Vizzutti, Francesco; Gelmini, Stefania; Laffi, Giacomo; Pinzani, Massimo; Marra, Fabio

2008-02-01

Adiponectin limits the development of liver fibrosis and activates adenosine monophosphate-activated protein kinase (AMPK). AMPK is a sensor of the cellular energy status, but its possible modulation of the fibrogenic properties of hepatic stellate cells (HSCs) has not been established. In this study, we investigated the role of AMPK activation in the biology of activated human HSCs. A time-dependent activation of AMPK was observed in response to a number of stimuli, including globular adiponectin, 5-aminoimidazole-4-carboxamide-1-beta-4-ribofuranoside (AICAR), or metformin. All these compounds significantly inhibited platelet-derived growth factor (PDGF)-stimulated proliferation and migration of human HSCs and reduced the secretion of monocyte chemoattractant protein-1. In addition, AICAR limited the secretion of type I procollagen. Knockdown of AMPK by gene silencing increased the mitogenic effects of PDGF, confirming the negative modulation exerted by this pathway on HSCs. AMPK activation did not reduce PDGF-dependent activation of extracellular signal-regulated kinase (ERK) or Akt at early time points, whereas a marked inhibition was observed 24 hours after addition of PDGF, reflecting a block in cell cycle progression. In contrast, AICAR blocked short-term phosphorylation of ribosomal S6 kinase (p70(S6K)) and 4E binding protein-1 (4EBP1), 2 downstream effectors of the mammalian target of rapamycin (mTOR) pathway, by PDGF. The ability of interleukin-a (IL-1) to activate nuclear factor kappa B (NF-kappaB) was also reduced by AICAR. Activation of AMPK negatively modulates the activated phenotype of HSCs.

Expression, purification and crystallization of Streptococcus dysgalactiae-derived mitogen

Energy Technology Data Exchange (ETDEWEB)

Papageorgiou, Anastassios C., E-mail: tassos.papageorgiou@btk.fi; Saarinen, Susanna; Ramirez-Bartutis, Rosa [Turku Centre for Biotechnology, University of Turku and Åbo Akademi University, Turku 20521 (Finland); Kato, Hidehito; Uchiyama, Takehiko [Department of Microbiology and Immunology, Tokyo Women’s Medical University, 8-1 Kawada-cho, Shinjuku-ku, Tokyo 162-8666 (Japan); Kirikae, Teruo; Miyoshi-Akiyama, Toru [Department of Infectious Diseases, International Medical Center of Japan, 1-21-1 Toyama, Shinjuku-ku, Tokyo 162-8655 (Japan); Turku Centre for Biotechnology, University of Turku and Åbo Akademi University, Turku 20521 (Finland)

2006-03-01

S. dysgalactiae-derived mitogen, a superantigen, was crystallized. Crystals diffract to 2.4 Å at a synchrotron-radiation source and belong to space group P3/P3{sub 1}/P3{sub 2}, with unit-cell parameters a = b = 52.7, c = 62.4 Å, γ = 120° and one molecule in the crystallographic asymmetric unit. Superantigens are bacterial or viral toxins with potent immunostimulatory properties. Streptococcus dysgalactiae-derived mitogen, a 25 kDa protein, is a recently discovered superantigen isolated from S. dysgalactiae culture supernatant. Sequence considerations suggest that it belongs to a new superantigen family distinct from other superantigens. The protein was expressed in Escherichia coli cells and purified to homogeneity. Crystals were grown at pH 4.2–4.4 in the presence of 18–20%(w/v) PEG 3350 and 0.4 M lithium nitrate. A complete data set to 2.4 Å resolution was collected from a single crystal at liquid-nitrogen temperatures using synchrotron radiation. The crystals belong to space group P3/P3{sub 1}/P3{sub 2}, with unit-cell parameters a = b = 52.7, c = 62.4 Å, γ = 120° and one molecule in the crystallographic asymmetric unit.

Common and distinctive localization patterns of Crumbs polarity complex proteins in the mammalian eye.

Science.gov (United States)

Kim, Jin Young; Song, Ji Yun; Karnam, Santi; Park, Jun Young; Lee, Jamie J H; Kim, Seonhee; Cho, Seo-Hee

2015-01-01

Crumbs polarity complex proteins are essential for cellular and tissue polarity, and for adhesion of epithelial cells. In epithelial tissues deletion of any of three core proteins disrupts localization of the other proteins, indicating structural and functional interdependence among core components. Despite previous studies of function and co-localization that illustrated the properties that these proteins share, it is not known whether an individual component of the complex plays a distinct role in a unique cellular and developmental context. In order to investigate this question, we primarily used confocal imaging to determine the expression and subcellular localization of the core Crumbs polarity complex proteins during ocular development. Here we show that in developing ocular tissues core Crumbs polarity complex proteins, Crb, Pals1 and Patj, generally appear in an overlapping pattern with some exceptions. All three core complex proteins localize to the apical junction of the retinal and lens epithelia. Pals1 is also localized in the Golgi of the retinal cells and Patj localizes to the nuclei of the apically located subset of progenitor cells. These findings suggest that core Crumbs polarity complex proteins exert common and independent functions depending on cellular context. Copyright © 2015 Elsevier B.V. All rights reserved.

Periodic mechanical stress activates EGFR-dependent Rac1 mitogenic signals in rat nucleus pulpous cells via ERK1/2

Energy Technology Data Exchange (ETDEWEB)

Gao, Gongming [Department of Orthopedics, The Affiliated Changzhou No. 2 Hospital of Nanjing Medical University, Changzhou 213003 (China); Shen, Nan [Department of Clinical Pharmacy, The Affiliated Jiangyin Hospital of Southeast University Medical School, Jiangyin 214400 (China); Jiang, Xuefeng; Sun, Huiqing [Department of Orthopedics, The Affiliated Jiangyin Hospital of Southeast University Medical School, Jiangyin 214400 (China); Xu, Nanwei; Zhou, Dong [Department of Orthopedics, The Affiliated Changzhou No. 2 Hospital of Nanjing Medical University, Changzhou 213003 (China); Nong, Luming, E-mail: lumingnong@hotmail.com [Department of Orthopedics, The Affiliated Changzhou No. 2 Hospital of Nanjing Medical University, Changzhou 213003 (China); Ren, Kewei, E-mail: keweiren@hotmail.com [Department of Orthopedics, The Affiliated Jiangyin Hospital of Southeast University Medical School, Jiangyin 214400 (China)

2016-01-15

The mitogenic effects of periodic mechanical stress on nucleus pulpous cells have been studied extensively but the mechanisms whereby nucleus pulpous cells sense and respond to mechanical stimulation remain a matter of debate. We explored this question by performing cell culture experiments in our self-developed periodic stress field and perfusion culture system. Under periodic mechanical stress, rat nucleus pulpous cell proliferation was significantly increased (p < 0.05 for each) and was associated with increases in the phosphorylation and activation of EGFR, Rac1, and ERK1/2 (p < 0.05 for each). Pretreatment with the ERK1/2 selective inhibitor PD98059 reduced periodic mechanical stress-induced nucleus pulpous cell proliferation (p < 0.05 for each), while the activation levels of EGFR and Rac1 were not inhibited. Proliferation and phosphorylation of ERK1/2 were inhibited after pretreatment with the Rac1 inhibitor NSC23766 in nucleus pulpous cells in response to periodic mechanical stress (p < 0.05 for each), while the phosphorylation site of EGFR was not affected. Inhibition of EGFR activity with AG1478 abrogated nucleus pulpous cell proliferation (p < 0.05 for each) and attenuated Rac1 and ERK1/2 activation in nucleus pulpous cells subjected to periodic mechanical stress (p < 0.05 for each). These findings suggest that periodic mechanical stress promotes nucleus pulpous cell proliferation in part through the EGFR-Rac1-ERK1/2 signaling pathway, which links these three important signaling molecules into a mitogenic cascade. - Highlights: • The mechanism involved in nucleus pulpous cells to respond to mechanical stimuli. • Periodic mechanical stress can stimulate the phosphorylation of EGFR. • EGFR activates Rac1 and leads to rat nucleus pulpous cell proliferation. • EGFR and Rac1 activate ERK1/2 mitogenic signals in nucleus pulpous cells. • EGFR-Rac1-ERK1/2 is constitutes at least one critical signal transduction pathway.

Periodic mechanical stress activates EGFR-dependent Rac1 mitogenic signals in rat nucleus pulpous cells via ERK1/2

International Nuclear Information System (INIS)

Gao, Gongming; Shen, Nan; Jiang, Xuefeng; Sun, Huiqing; Xu, Nanwei; Zhou, Dong; Nong, Luming; Ren, Kewei

2016-01-01

The mitogenic effects of periodic mechanical stress on nucleus pulpous cells have been studied extensively but the mechanisms whereby nucleus pulpous cells sense and respond to mechanical stimulation remain a matter of debate. We explored this question by performing cell culture experiments in our self-developed periodic stress field and perfusion culture system. Under periodic mechanical stress, rat nucleus pulpous cell proliferation was significantly increased (p < 0.05 for each) and was associated with increases in the phosphorylation and activation of EGFR, Rac1, and ERK1/2 (p < 0.05 for each). Pretreatment with the ERK1/2 selective inhibitor PD98059 reduced periodic mechanical stress-induced nucleus pulpous cell proliferation (p < 0.05 for each), while the activation levels of EGFR and Rac1 were not inhibited. Proliferation and phosphorylation of ERK1/2 were inhibited after pretreatment with the Rac1 inhibitor NSC23766 in nucleus pulpous cells in response to periodic mechanical stress (p < 0.05 for each), while the phosphorylation site of EGFR was not affected. Inhibition of EGFR activity with AG1478 abrogated nucleus pulpous cell proliferation (p < 0.05 for each) and attenuated Rac1 and ERK1/2 activation in nucleus pulpous cells subjected to periodic mechanical stress (p < 0.05 for each). These findings suggest that periodic mechanical stress promotes nucleus pulpous cell proliferation in part through the EGFR-Rac1-ERK1/2 signaling pathway, which links these three important signaling molecules into a mitogenic cascade. - Highlights: • The mechanism involved in nucleus pulpous cells to respond to mechanical stimuli. • Periodic mechanical stress can stimulate the phosphorylation of EGFR. • EGFR activates Rac1 and leads to rat nucleus pulpous cell proliferation. • EGFR and Rac1 activate ERK1/2 mitogenic signals in nucleus pulpous cells. • EGFR-Rac1-ERK1/2 is constitutes at least one critical signal transduction pathway.

Regulation of mitogen-activated protein kinase pathways by the plasma membrane Na+/H+ exchanger, NHE1

DEFF Research Database (Denmark)

Pedersen, Stine Helene Falsig; Darborg, Barbara Vasek; Rentsch, Maria Louise

2006-01-01

activity is regulated by a three-tiered phosphorelay system, which is in turn regulated by a complex network of signaling events and scaffolding proteins. The ubiquitous plasma membrane Na(+)/H(+) exchanger NHE1 is activated by, and implicated in, the physiological/pathophysiological responses to many...... of the same stimuli that modulate MAPK activity. While under some conditions, NHE1 is regulated by MAPKs, a number of studies have, conversely, implicated NHE1 in the regulation of MAPK activity. Here, we discuss the current evidence indicating the involvement of NHE1 in MAPK regulation, the mechanisms...

Tim-4 inhibition of T-cell activation and T helper type 17 differentiation requires both the immunoglobulin V and mucin domains and occurs via the mitogen-activated protein kinase pathway.

LENUS (Irish Health Repository)

Cao, Wei

2011-06-01

Emerging experimental data suggest an important role for the T-cell immunoglobulin mucin 1 (Tim-1):Tim-4 pathway in autoimmune and alloimmune responses in vivo. Using a Tim-4 ectodomain human IgG Fc fusion protein we studied the role of Tim-4 in T-cell activation, signalling and differentiation responses in vitro. We demonstrate that Tim-4Fc can inhibit naive and pre-activated T-cell activation, proliferation and cytokine secretion via a Tim-1-independent pathway. Tim-4 contains immunoglobulin variable (IgV) and mucin domains; to identify which domain accounts for the inhibitory effect novel Tim-4 fusion proteins containing either the IgV or mucin domain were generated. We demonstrate that both IgV and mucin domains are required for the inhibitory effects and that they are mediated at least in part by inhibition of extracellular signal-regulated kinase pathway activity. Given the emerging interest in the role of the Tim family in T helper type 17 (Th17) cells, which play an important role in autoimmune disease and transplantation tolerance, our data show that Tim-4Fc can prevent polarization of CD4(+) T cells to the Th17 phenotype. Collectively, our results highlight an inhibitory role for Tim-4Fc in vitro, which we propose is mediated by a receptor other than Tim-1. In addition, this study provides new insights into the role of Tim-4Fc in regulating Th17 immune responses and may open a new avenue for autoimmune therapy.

Tim-4 inhibition of T-cell activation and T helper type 17 differentiation requires both the immunoglobulin V and mucin domains and occurs via the mitogen-activated protein kinase pathway

Science.gov (United States)

Cao, Wei; Ryan, Michelle; Buckley, Deirdre; O'Connor, Rosemary; Clarkson, Michael R

2011-01-01

Emerging experimental data suggest an important role for the T-cell immunoglobulin mucin 1 (Tim-1):Tim-4 pathway in autoimmune and alloimmune responses in vivo. Using a Tim-4 ectodomain human IgG Fc fusion protein we studied the role of Tim-4 in T-cell activation, signalling and differentiation responses in vitro. We demonstrate that Tim-4Fc can inhibit naive and pre-activated T-cell activation, proliferation and cytokine secretion via a Tim-1-independent pathway. Tim-4 contains immunoglobulin variable (IgV) and mucin domains; to identify which domain accounts for the inhibitory effect novel Tim-4 fusion proteins containing either the IgV or mucin domain were generated. We demonstrate that both IgV and mucin domains are required for the inhibitory effects and that they are mediated at least in part by inhibition of extracellular signal-regulated kinase pathway activity. Given the emerging interest in the role of the Tim family in T helper type 17 (Th17) cells, which play an important role in autoimmune disease and transplantation tolerance, our data show that Tim-4Fc can prevent polarization of CD4+ T cells to the Th17 phenotype. Collectively, our results highlight an inhibitory role for Tim-4Fc in vitro, which we propose is mediated by a receptor other than Tim-1. In addition, this study provides new insights into the role of Tim-4Fc in regulating Th17 immune responses and may open a new avenue for autoimmune therapy. PMID:21463297

Cisplatin Induces Cytotoxicity through the Mitogen-Activated Protein Kinase Pathways ana Activating Transcription Factor 3

Directory of Open Access Journals (Sweden)

Carly St. Germain

2010-07-01

Full Text Available The mechanisms underlying the proapoptotic effect of the chemotherapeutic agent, cisplatin, are largely undefined. Understanding the mechanisms regulating cisplatin cytotoxicity may uncover strategies to enhance the efficacy of this important therapeutic agent. This study evaluates the role of activating transcription factor 3 (ATF3 as a mediator of cisplatin-induced cytotoxicity. Cytotoxic doses of cisplatin and carboplatin treatments consistently induced ATF3 expression in five tumor-derived cell lines. Characterization of this induction revealed a p53, BRCA1, and integrated stress response-independent mechanism, all previously implicated in stress-mediated ATF3 induction. Analysis of mitogenactivated protein kinase (MAPK pathway involvement in ATF3 induction by cisplatin revealed a MAPK-dependent mechanism. Cisplatin treatment combined with specific inhibitors to each MAPK pathway (c-Jun N-terminal kinase, extracellularsignal-regulated kinase, and p38 resulted in decreasedATF3 induction at the protein level. MAPK pathway inhibition led to decreased ATF3 messenger RNA expression and reduced cytotoxic effects of cisplatin as measured by the 3-(4,5-dimethylthiazol-2-ylF2,5-diphenyltetrazolium bromide cell viability assay. In A549 lung carcinoma cells, targeting ATF3 with specific small hairpin RNA also attenuated the cytotoxic effects of cisplatin. Similarly, ATF3-/murine embryonic fibroblasts (MEFs were shown to be less sensitive to cisplatin-induced cytotoxicity compared with ATF3+/+ MEFs. This study identifies cisplatin as a MAPK pathway-dependent inducer of ATF3, whose expression influences cisplatin’s cytotoxic effects.

Upstream and Downstream Co-inhibition of Mitogen-Activated Protein Kinase and PI3K/Akt/mTOR Pathways in Pancreatic Ductal Adenocarcinoma

Directory of Open Access Journals (Sweden)

Matthew H. Wong

2016-07-01

Full Text Available BACKGROUND: Extensive cross talk exists between PI3K/Akt/mTOR and mitogen-activated protein kinase (MAPK pathways, and both are upregulated in pancreatic ductal adenocarcinoma (PDAC. Our previous study suggested that epidermal growth factor receptor inhibitor erlotinib which acts upstream of these pathways acts synergistically with PI3K inhibitors in PDAC. Horizontal combined blockade upstream and downstream of these two pathways is therefore explored. METHODS: Erlotinib paired with PI3K inhibitor (BYL719 was tested against erlotinib plus dual PI3K/mTOR inhibitor BEZ-235, and MEK inhibitor (PD98059 plus BEZ235, on five primary PDAC cell lines and on two pairs of parent and erlotinib-resistant (ER cell lines. A range of in vitro assays including cell proliferation, Western blotting, migration, clonogenic, cell cycle, and apopotic assays was used to test for the efficacy of combined blockade. RESULTS: Dual downstream blockade of the MAPK and PAM pathways was more effective in attenuating downstream molecular signals. Synergy was demonstrated for erlotinib and BEZ235 and for PD-98059 and BEZ-235. This resulted in a trend of increased growth cell cycle arrest, apoptosis, cell proliferation, and colony and migration suppression. This combination showed more efficacy in cell lines with acquired resistance to erlotinib. CONCLUSIONS: The additional mTOR blockade provided by BEZ235 in combined blockade resulted in increased anticancer effect. The hypersensitivity of ER cell lines to additional mTOR blockade suggested PAM pathway oncogenic dependence via mTOR. Dual downstream combined blockade of MAPK and PAM pathways with MEK and PI3K/mTOR inhibitor appeared most effective and represents an attractive therapeutic strategy against pancreatic cancer and its associated drug resistance.

Neuroprotective effects of ebselen in traumatic brain injury model: involvement of nitric oxide and p38 mitogen-activated protein kinase signalling pathway.

Science.gov (United States)

Wei, Liang; Zhang, Yanfei; Yang, Cheng; Wang, Qi; Zhuang, Zhongwei; Sun, Zhiyang

2014-02-01

Previous investigations have found that ebselen is able to treat neurodegenerative diseases caused by radical and acute total cerebral ischaemia. The aim of the present study was to investigate the neuroprotective effects of ebselen in a traumatic brain injury (TBI) model. Ninety Sprague-Dawley rats were randomly divided into five groups (n = 18 in each): (i) sham operation; (ii) an injury model group; (iii) low-dose (3 mg/kg) ebselen-treated group; (iv) a moderate-dose (10 mg/kg) ebselen-treated group; and (v) a high-dose (30 mg/kg) ebselen-treated group. The TBI model was created according using a modified weight-drop model. Neurological severity score (NSS), brain water content and histopathological deficits were assessed as parameters of injury severity. Expression of nitric oxide (NO), inducible NO synthase (iNOS) mRNA, Toll-like receptor (TLR) and phosphorylated (p-) p38 mitogen-activated protein kinase (MAPK) were examined by chemical colorimetry, quantitative polymerase chain reaction and western blotting 24 h after intragastric ebselen administration. Rats in the TBI model group exhibited markedly more severe neurological injury (higher NSS, more brain water content and more histopathological deficits) than those in the sham-operated group. Ebselen treatment significantly ameliorated the neurological injury of TBI rats in a dose-dependent manner. Moreover, ebselen significantly reduced the NO and iNOS mRNA levels and inhibited TLR4 and p-p38 MAPK expression, indicating the involvement of NO and p38 MAPK signalling pathways in the neuroprotection afforded by ebselen. In conclusion, ebselen ameliorated neurological injury, possibly by reducing NO levels and modulating the TLR4-mediated p38 MAPK signalling pathway. Therefore, ebselen may have potential to treat secondary injuries of TBI. © 2013 Wiley Publishing Asia Pty Ltd.

Networking for proteins : A yeast two-hybrid and RNAi profiling approach to uncover C. elegans cell polarity regulators

NARCIS (Netherlands)

Koorman, T.|info:eu-repo/dai/nl/337456038

2016-01-01

Cell polarity is a near universal trait of life and guides many aspects of animal development. Although a number of key polarity proteins have been identified, many interactions with proteins acting downstream likely remain to be elucidated. Mutations in polarity proteins or deregulation of polarity

Novel Indole-based Tambjamine-Analogues Induce Apoptotic Lung Cancer Cell Death through p38 Mitogen-Activated Protein Kinase Activation.

Science.gov (United States)

Manuel-Manresa, Pilar; Korrodi-Gregório, Luís; Hernando, Elsa; Villanueva, Alberto; Martínez-García, David; Rodilla, Ananda M; Ramos, Ricard; Fardilha, Margarida; Moya, Juan; Quesada, Roberto; Soto-Cerrato, Vanessa; Pérez-Tomás, Ricardo

2017-07-01

Lung cancer has become the leading killer cancer worldwide, due to late diagnosis and lack of efficient anticancer drugs. We have recently described novel natural-derived tambjamine analogues that are potent anion transporters capable of disrupting cellular ion balance, inducing acidification of the cytosol and hyperpolarization of cellular plasma membranes. Although these tambjamine analogues were able to compromise cell survival, their molecular mechanism of action remains largely unknown. Herein we characterize the molecular cell responses induced by highly active indole-based tambjamine analogues treatment in lung cancer cells. Expression changes produced after compounds treatment comprised genes related to apoptosis, cell cycle, growth factors and its receptors, protein kinases and topoisomerases, among others. Dysregulation of BCL2 and BIRC5 /survivin genes suggested the apoptotic pathway as the induced molecular cell death mechanism. In fact, activation of several proapoptotic markers (caspase-9, caspase-3, and PARP) and reversion of the cytotoxic effect upon treatment with an apoptosis inhibitor (Z-VAD-FMK) were observed. Moreover, members of the Bcl-2 protein family suffered changes after tambjamine analogues treatment, with a concomitant protein decrease towards the prosurvival members. Besides this, it was observed cellular accumulation of ROS upon compound treatment and an activation of the stress-kinase p38 MAPK route that, when inhibited, reverted the cytotoxic effect of the tambjamine analogues. Finally, a significant therapeutic effect of these compounds was observed in subcutaneous and orthotopic lung cancer mice models. Taken together, these results shed light on the mechanism of action of novel cytotoxic anionophores and demonstrate the therapeutic effects against lung cancer. Mol Cancer Ther; 16(7); 1224-35. ©2017 AACR . ©2017 American Association for Cancer Research.

The relationship between lymphocytes activated by pokeweed mitogen and by lipopolysaccharides and their radiosensitivity

International Nuclear Information System (INIS)

Su Liaoyuan; Liu Fenju; Liu Keliang; Xu Changshao; Xu Yingdong; Geng Yongzhi

1992-07-01

Human whole blood was incubated in vitro. Lymphocytes were activated by poke-weed mitogen (PWM) and by Lipopolysaccharides (LPS). The relationship between the two kinds of lymphocytes was investigated using radioactive compound incorporation. The study showed that PWM-activated lymphocytes were able to promote the stimulating effect of LPS on B lymphocytes. The stimulating effect of PWM-activated lymphocytes was obviously decreased after they were irradiated with 10 Gy gamma rays. When PWM-activated lymphocytes and LPS-activated lymphocytes were incubated together after one of the cell populations had been exposed 10 Gy 60 Co gamma rays, the incorporation of [ 3 H] TdR was much decreased and the synergistic function disappeared, especially when the PWM-activated lymphocytes were irradiated. In cells from patients treated with 60 Co gamma rays for carcinoma of nasopharynx, the incorporation in LPS-activated lymphocytes approached normal levels while that in PWM-activated lymphocytes was reduced significantly and the stimulating effect of PWM-activated lymphocytes on LPS-activated lymphocytes was also markedly reduced. These demonstrate that PWM-activated lymphocytes have a similar function to T-helper cells and seem to be more radiosensitive than LPS-activated lymphocytes

Lipoxin A4 regulates expression of the estrogen receptor and inhibits 17β-estradiol induced p38 mitogen-activated protein kinase phosphorylation in human endometriotic stromal cells.

Science.gov (United States)

Chen, Shuo; Wu, Rong-Feng; Su, Lin; Zhou, Wei-Dong; Zhu, Mao-Bi; Chen, Qiong-Hua

2014-07-01

To study the role of lipoxin A4 (LXA4) in endometriosis. Molecular analysis in human samples and primary human endometriotic stromal cells (ESCs). University hospital. Forty-nine premenopausal women (30 patients with endometriosis and 19 controls). Normal and ectopic endometrial biopsies obtained during surgery performed during the proliferative phase of the menstrual cycle; ESCs used for in vitro studies. Levels of LXA4 measured by enzyme-linked immunosorbent assay (ELISA); mRNA levels of the estrogen receptor (ER), progestogen receptor (PR), tumor necrosis factor α (TNF-α), and interleukin 6 (IL-6) quantified by quantitative reverse-transcription polymerase chain reaction (qRT-PCR); and p38 mitogen-activated protein kinase (p38 MAPK) phosphorylation evaluated by Western blotting. The LXA4 expression level decreased in ectopic tissue as well as ERα and PR, although the expression of ERβ increased in ectopic endometrium compared with the controls. Investigations with correlation analysis revealed the expression of LXA4 was positively correlated with ERα and negatively correlated with ERβ in vivo. Moreover, administering LXA4 could augment ERβ expression in ESCs and inhibit the 17β-estradiol-induced phosphorylation of p38 MAPK very likely through ERβ. Our findings indicate that LXA4 regulates ERβ expression and inhibits 17β-estradiol-induced phosphorylation of p38 MAPK, very likely through ERβ in ESCs. Copyright © 2014. Published by Elsevier Inc.

On the importance of polar interactions for complexes containing intrinsically disordered proteins.

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Eric T C Wong

Full Text Available There is a growing recognition for the importance of proteins with large intrinsically disordered (ID segments in cell signaling and regulation. ID segments in these proteins often harbor regions that mediate molecular recognition. Coupled folding and binding of the recognition regions has been proposed to confer high specificity to interactions involving ID segments. However, researchers recently questioned the origin of the interaction specificity of ID proteins because of the overrepresentation of hydrophobic residues in their interaction interfaces. Here, we focused on the role of polar and charged residues in interactions mediated by ID segments. Making use of the extended nature of most ID segments when in complex with globular proteins, we first identified large numbers of complexes between globular proteins and ID segments by using radius-of-gyration-based selection criteria. Consistent with previous studies, we found the interfaces of these complexes to be enriched in hydrophobic residues, and that these residues contribute significantly to the stability of the interaction interface. However, our analyses also show that polar interactions play a larger role in these complexes than in structured protein complexes. Computational alanine scanning and salt-bridge analysis indicate that interfaces in ID complexes are highly complementary with respect to electrostatics, more so than interfaces of globular proteins. Follow-up calculations of the electrostatic contributions to the free energy of binding uncovered significantly stronger Coulombic interactions in complexes harbouring ID segments than in structured protein complexes. However, they are counter-balanced by even higher polar-desolvation penalties. We propose that polar interactions are a key contributing factor to the observed high specificity of ID segment-mediated interactions.

The N-terminal of a heparin-binding sperm membrane mitogen possess lectin-like sequence

International Nuclear Information System (INIS)

Mor, Visesato; Chatterjee, Tapati

2007-01-01

Glycosaminoglycans like heparin and heparin sulfate in follicular fluid induce changes in the intracellular environment during the spermatozoal functional maturation. We previously reported the isolation, purification and partial characterization of a heparin binding sperm membrane protein (HBSM). In the present study, the amino acids analysis provided evidence of a single sequence, which suggest the homogeneity of the purified HBSM. Fourteen amino acids- 1 A D T I V A V E L D T Y P N 14 -correspond to the amino terminal sequence of Concanavalin A (Con A) and contain 45.2% carbohydrate by weight. HBSM possess mitogenic property on lymphocytes with comparable magnitude to the well-known mitogen; Con A, inducing 83% radiolabel thymidine incorporation in growing lymphocytes. Unlike Con A, there was no agglutination of cell by HBSM upto 5 ng/ml concentration. Interestingly, we found that heparin and chondroitin sulfate-conjugated HBSM inhibit the proliferative activity. Similar effect was also found with an in-house isolate sulfated glycans; G-I (28% sulfate). In contrast, there was no inhibition by the desulfated form; G-ID. Altogether, our data suggest that the mechanism of cell proliferative pathway may be different for HBSM and Con A

Transcriptional integration of mitogenic and mechanical signals by Myc and YAP.

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Croci, Ottavio; De Fazio, Serena; Biagioni, Francesca; Donato, Elisa; Caganova, Marieta; Curti, Laura; Doni, Mirko; Sberna, Silvia; Aldeghi, Deborah; Biancotto, Chiara; Verrecchia, Alessandro; Olivero, Daniela; Amati, Bruno; Campaner, Stefano

2017-10-15

Mammalian cells must integrate environmental cues to determine coherent physiological responses. The transcription factors Myc and YAP-TEAD act downstream from mitogenic signals, with the latter responding also to mechanical cues. Here, we show that these factors coordinately regulate genes required for cell proliferation. Activation of Myc led to extensive association with its genomic targets, most of which were prebound by TEAD. At these loci, recruitment of YAP was Myc-dependent and led to full transcriptional activation. This cooperation was critical for cell cycle entry, organ growth, and tumorigenesis. Thus, Myc and YAP-TEAD integrate mitogenic and mechanical cues at the transcriptional level to provide multifactorial control of cell proliferation. © 2017 Croci et al.; Published by Cold Spring Harbor Laboratory Press.

A role for Pyk2 and Src in linking G-protein-coupled receptors with MAP kinase activation.

Science.gov (United States)

Dikic, I; Tokiwa, G; Lev, S; Courtneidge, S A; Schlessinger, J

1996-10-10

The mechanisms by which mitogenic G-protein-coupled receptors activate the MAP kinase signalling pathway are poorly understood. Candidate protein tyrosine kinases that link G-protein-coupled receptors with MAP kinase include Src family kinases, the epidermal growth factor receptor, Lyn and Syk. Here we show that lysophosphatidic acid (LPA) and bradykinin induce tyrosine phosphorylation of Pyk2 and complex formation between Pyk2 and activated Src. Moreover, tyrosine phosphorylation of Pyk2 leads to binding of the SH2 domain of Src to tyrosine 402 of Pyk2 and activation of Src. Transient overexpression of a dominant interfering mutant of Pyk2 or the protein tyrosine kinase Csk reduces LPA- or bradykinin-induced activation of MAP kinase. LPA- or bradykinin-induced MAP kinase activation was also inhibited by overexpression of dominant interfering mutants of Grb2 and Sos. We propose that Pyk2 acts with Src to link Gi- and Gq-coupled receptors with Grb2 and Sos to activate the MAP kinase signalling pathway in PC12 cells.

Shc adaptor proteins are key transducers of mitogenic signaling mediated by the G protein-coupled thrombin receptor

DEFF Research Database (Denmark)

Chen, Y; Grall, D; Salcini, A E

1996-01-01

The serine protease thrombin activates G protein signaling systems that lead to Ras activation and, in certain cells, proliferation. Whereas the steps leading to Ras activation by G protein-coupled receptors are not well defined, the mechanisms of Ras activation by receptor tyrosine kinases have......-insensitive proteins appear to mediate this effect, since (i) pertussis toxin pre-treatment of cells does not blunt the action of thrombin and (ii) Shc phosphorylation on tyrosine can be stimulated by the muscarinic m1 receptor. Shc phosphorylation does not appear to involve protein kinase C, since the addition of 4...

Ghrelin protects against depleted uranium-induced apoptosis of MC3T3-E1 cells through oxidative stress-mediated p38-mitogen-activated protein kinase pathway

Energy Technology Data Exchange (ETDEWEB)

Hao, Yuhui; Liu, Cong; Huang, Jiawei; Gu, Ying; Li, Hong; Yang, Zhangyou; Liu, Jing [State Key Laboratory of Trauma, Burns and Combined Injury, Institute of Combined Injury, Chongqing Engineering Research Center for Nanomedicine, College of Preventive Medicine, Third Military Medical University, No. 30 Gaotanyan Street, Shapingba District, Chongqing 400038 (China); Wang, Weidong, E-mail: wwdwyl@sina.com [Department of Radiation Oncology, Shanghai Jiao Tong University Affiliated Sixth People Hospital, Shanghai 200233 (China); Li, Rong, E-mail: yuhui_hao@126.com [State Key Laboratory of Trauma, Burns and Combined Injury, Institute of Combined Injury, Chongqing Engineering Research Center for Nanomedicine, College of Preventive Medicine, Third Military Medical University, No. 30 Gaotanyan Street, Shapingba District, Chongqing 400038 (China)

2016-01-01

Depleted uranium (DU) mainly accumulates in the bone over the long term. Osteoblast cells are responsible for the formation of bone, and they are sensitive to DU damage. However, studies investigating methods of reducing DU damage in osteoblasts are rarely reported. Ghrelin is a stomach hormone that stimulates growth hormones released from the hypothalamic–pituitary axis, and it is believed to play an important physiological role in bone metabolism. This study evaluates the impact of ghrelin on DU-induced apoptosis of the osteoblast MC3T3-E1 and investigates its underlying mechanisms. The results show that ghrelin relieved the intracellular oxidative stress induced by DU, eliminated reactive oxygen species (ROS) and reduced lipid peroxidation by increasing intracellular GSH levels; in addition, ghrelin effectively suppressed apoptosis, enhanced mitochondrial membrane potential, and inhibited cytochrome c release and caspase-3 activation after DU exposure. Moreover, ghrelin significantly reduced the expression of DU-induced phosphorylated p38-mitogen-activated protein kinase (MAPK). A specific inhibitor (SB203580) or specific siRNA of p38-MAPK could significantly suppress DU-induced apoptosis and related signals, whereas ROS production was not affected. In addition, ghrelin receptor inhibition could reduce the anti-apoptosis effect of ghrelin on DU and reverse the effect of ghrelin on intracellular ROS and p38-MAPK after DU exposure. These results suggest that ghrelin can suppress DU-induced apoptosis of MC3T3-E1 cells, reduce DU-induced oxidative stress by interacting with its receptor, and inhibit downstream p38-MAPK activation, thereby suppressing the mitochondrial-dependent apoptosis pathway. - Highlights: • Ghrelin suppressed DU-induced apoptosis of MC3T3-E1 cells. • Ghrelin inhibited DU-induced oxidative stress and further p38-MAPK activation. • Ghrelin further suppressed mitochondrial-dependent apoptosis pathway. • The anti-oxidation effect of

Yeast G-proteins mediate directional sensing and polarization behaviors in response to changes in pheromone gradient direction

Science.gov (United States)

Moore, Travis I.; Tanaka, Hiromasa; Kim, Hyung Joon; Jeon, Noo Li; Yi, Tau-Mu

2013-01-01

Yeast cells polarize by projecting up mating pheromone gradients, a classic cell polarity behavior. However, these chemical gradients may shift direction. We examine how yeast cells sense and respond to a 180o switch in the direction of microfluidically generated pheromone gradients. We identify two behaviors: at low concentrations of α-factor, the initial projection grows by bending, whereas at high concentrations, cells form a second projection toward the new source. Mutations that increase heterotrimeric G-protein activity expand the bending-growth morphology to high concentrations; mutations that increase Cdc42 activity result in second projections at low concentrations. Gradient-sensing projection bending requires interaction between Gβγ and Cdc24, whereas gradient-nonsensing projection extension is stimulated by Bem1 and hyperactivated Cdc42. Of interest, a mutation in Gα affects both bending and extension. Finally, we find a genetic perturbation that exhibits both behaviors. Overexpression of the formin Bni1, a component of the polarisome, makes both bending-growth projections and second projections at low and high α-factor concentrations, suggesting a role for Bni1 downstream of the heterotrimeric G-protein and Cdc42 during gradient sensing and response. Thus we demonstrate that G-proteins modulate in a ligand-dependent manner two fundamental cell-polarity behaviors in response to gradient directional change. PMID:23242998

Coherent active polarization control without loss

Science.gov (United States)

Ye, Yuqian; Hay, Darrick; Shi, Zhimin

2017-11-01

We propose a lossless active polarization control mechanism utilizing an anisotropic dielectric medium with two coherent inputs. Using scattering matrix analysis, we derive analytically the required optical properties of the anisotropic medium that can behave as a switchable polarizing beam splitter. We also show that such a designed anisotropic medium can produce linearly polarized light at any azimuthal direction through coherent control of two inputs with a specific polarization state. Furthermore, we present a straightforward design-on-demand procedure of a subwavelength-thick metastructure that can possess the desired optical anisotropy at a flexible working wavelength. Our lossless coherent polarization control technique may lead to fast, broadband and integrated polarization control elements for applications in imaging, spectroscopy, and telecommunication.

Camel Milk Attenuates Rheumatoid Arthritis Via Inhibition of Mitogen Activated Protein Kinase Pathway

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Hany H. Arab

2017-09-01

Full Text Available Background/Aims: Camel milk (CM has shown beneficial anti-inflammatory actions in several experimental and clinical settings. So far, its effect on rheumatoid arthritis (RA has not been previously explored. Thus, the current work aimed to evaluate the effects of CM in Adjuvant-induced arthritis and air pouch edema models in rats, which mimic human RA. Methods: CM was administered at 10 ml/kg orally for 3 weeks starting on the day of Freund’s adjuvant paw inoculation. The levels of TNF-α and IL-10 were measured by ELISA while the protein expression of NF-κBp65, COX-2 and iNOS was detected by immunohistochemistry. The expression of MAPK target proteins was assessed by Western blotting. Results: CM attenuated paw edema, arthritic index and gait score along with dorsal pouch inflammatory cell migration. CM lowered the TNF-α and augmented the anti-inflammatory IL-10 levels in sera and exudates of arthritic rats. It also attenuated the expression of activated NF-κBp65, COX-2 and iNOS in the lining of the dorsal pouch. Notably, CM inhibited the MAPK pathway signal transduction via lowering the phosphorylation of p38 MAPK, ERK1/2 and JNK1/2 in rat hind paws. Additionally, CM administration lowered the lipid peroxide and nitric oxide levels and boosted glutathione and total anti-oxidant capacity in sera and exudates of animals. Conclusion: The observed CM downregulation of the arthritic process may support the interest of CM consumption as an adjunct approach for the management of RA.

Magnolol Alleviates Inflammatory Responses and Lipid Accumulation by AMP-Activated Protein Kinase-Dependent Peroxisome Proliferator-Activated Receptor α Activation

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Ye Tian

2018-02-01

Full Text Available Magnolol (MG is a kind of lignin isolated from Magnolia officinalis, which serves several different biological functions, such as antifungal, anticancer, antioxidant, and hepatoprotective functions. This study aimed to evaluate the protective effect of MG against oleic acid (OA-induced hepatic steatosis and inflammatory damage in HepG2 cells and in a tyloxapol (Ty-induced hyperlipidemia mouse model. Our findings indicated that MG can effectively inhibit OA-stimulated tumor necrosis factor α (TNF-α secretion, reactive oxygen species generation, and triglyceride (TG accumulation. Further study manifested that MG significantly suppressed OA-activated mitogen-activated protein kinase (MAPK and nuclear factor-kappa B (NF-κB signaling pathways and that these inflammatory responses can be negated by pretreatment with inhibitors of extracellular regulated protein kinase and c-Jun N-terminal kinase (U0126 and SP600125, respectively. In addition, MG dramatically upregulated peroxisome proliferator-activated receptor α (PPARα translocation and reduced sterol regulatory element-binding protein 1c (SREBP-1c protein synthesis and excretion, both of which are dependent upon the phosphorylation of adenosine monophosphate (AMP-activated protein kinase (AMPK, acetyl-CoA carboxylase, and AKT kinase (AKT. However, MG suspended the activation of PPARα expression and was thus blocked by pretreatment with LY294002 and compound c (specific inhibitors of AKT and AMPK. Furthermore, MG clearly alleviated serum TG and total cholesterol release; upregulated AKT, AMPK, and PPARα expression; suppressed SREBP-1c generation; and alleviated hepatic steatosis and dyslipidemia in Ty-induced hyperlipidemia mice. Taken together, these results suggest that MG exerts protective effects against steatosis, hyperlipidemia, and the underlying mechanism, which may be closely associated with AKT/AMPK/PPARα activation and MAPK/NF-κB/SREBP-1c inhibition.

Coherent active polarization control without loss

Directory of Open Access Journals (Sweden)

Yuqian Ye

2017-11-01

Full Text Available We propose a lossless active polarization control mechanism utilizing an anisotropic dielectric medium with two coherent inputs. Using scattering matrix analysis, we derive analytically the required optical properties of the anisotropic medium that can behave as a switchable polarizing beam splitter. We also show that such a designed anisotropic medium can produce linearly polarized light at any azimuthal direction through coherent control of two inputs with a specific polarization state. Furthermore, we present a straightforward design-on-demand procedure of a subwavelength-thick metastructure that can possess the desired optical anisotropy at a flexible working wavelength. Our lossless coherent polarization control technique may lead to fast, broadband and integrated polarization control elements for applications in imaging, spectroscopy, and telecommunication.

Association analysis between mitogen-activated protein/extracellular signal-regulated kinase (MEK) gene polymorphisms and depressive disorder in the Han Chinese population.

Science.gov (United States)

Hu, Yingyan; Hong, Wu; Smith, Alicia; Yu, Shunying; Li, Zezhi; Wang, Dongxiang; Yuan, Chengmei; Cao, Lan; Wu, Zhiguo; Huang, Jia; Fralick, Drew; Phillips, Michael Robert; Fang, Yiru

2017-11-01

Recent research findings suggest that BDNF and BDNF signaling pathways participate in the development of major depressive disorder. Mitogen-activated extracellular signal-regulated kinase (MEK) is the most important kinase in the extracellular signal-regulated kinase pathway, and the extracellular signal-regulated kinase pathway is the key signaling pathway of BDNF, so it may play a role in development of depressive disorder. The aim of this study is to investigate the association between polymorphisms of the MAP2K1 (also known as MEK) gene and depressive disorder. Three single nucleotide polymorphisms (SNPs), were significantly associated with depressive disorder: rs1549854 (p = 0.006), rs1432441 (p = 0.025), and rs7182853 (p = 0.039). When subdividing the sample by gender, two of the SNPs remained statistically associated with depressive disorder in females: rs1549854 (p = 0.013) and rs1432441 (p = 0.04). The rs1549854 and rs1432441 polymorphisms of the MAP2K1 gene may be associated with major depressive disorder, especially in females. This study is the first to report that the MAP2K1 gene may be a genetic marker for depressive disorder. Copyright © 2017 Elsevier B.V. All rights reserved.

Cyclophilin B induces integrin-mediated cell adhesion by a mechanism involving CD98-dependent activation of protein kinase C-delta and p44/42 mitogen-activated protein kinases.

Science.gov (United States)

Melchior, Aurélie; Denys, Agnès; Deligny, Audrey; Mazurier, Joël; Allain, Fabrice

2008-02-01

Initially identified as a cyclosporin-A binding protein, cyclophilin B (CyPB) is an inflammatory mediator that induces adhesion of T lymphocytes to fibronectin, by a mechanism dependent on CD147 and alpha 4 beta 1 integrins. Recent findings have suggested that another cell membrane protein, CD98, may cooperate with CD147 to regulate beta1 integrin functions. Based on these functional relationships, we examined the contribution of CD98 in the pro-adhesive activity of CyPB, by utilizing the responsive promonocyte cell line THP-1. We demonstrated that cross-linking CD98 with CD98-AHN-18 antibody mimicked the responses induced by CyPB, i.e. homotypic aggregation, integrin-mediated adhesion to fibronectin and activation of p44/42 MAPK. Consistent with previous data, immunoprecipitation confirmed the existence of a heterocomplex wherein CD147, CD98 and beta1 integrins were associated. We then demonstrated that CyPB-induced cell adhesion and p44/42 MAPK activation were dependent on the participation of phosphoinositide 3-kinase and subsequent activation of protein kinase C-delta. Finally, silencing the expression of CD98 by RNA interference potently reduced CyPB-induced cell responses, thus confirming the role of CD98 in the pro-adhesive activity of CyPB. Altogether, our results support a model whereby CyPB induces integrin-mediated adhesion via interaction with a multimolecular unit formed by the association between CD147, CD98 and beta1 integrins.

Type I and Type III Interferons Display Different Dependency on Mitogen-Activated Protein Kinases to Mount an Antiviral State in the Human Gut.

Science.gov (United States)

Pervolaraki, Kalliopi; Stanifer, Megan L; Münchau, Stephanie; Renn, Lynnsey A; Albrecht, Dorothee; Kurzhals, Stefan; Senís, Elena; Grimm, Dirk; Schröder-Braunstein, Jutta; Rabin, Ronald L; Boulant, Steeve

2017-01-01

Intestinal epithelial cells (IECs) are constantly exposed to commensal flora and pathogen challenges. How IECs regulate their innate immune response to maintain gut homeostasis remains unclear. Interferons (IFNs) are cytokines produced during infections. While type I IFN receptors are ubiquitously expressed, type III IFN receptors are expressed only on epithelial cells. This epithelium specificity strongly suggests exclusive functions at epithelial surfaces, but the relative roles of type I and III IFNs in the establishment of an antiviral innate immune response in human IECs are not clearly defined. Here, we used mini-gut organoids to define the functions of types I and III IFNs to protect the human gut against viral infection. We show that primary non-transformed human IECs, upon viral challenge, upregulate the expression of both type I and type III IFNs at the transcriptional level but only secrete type III IFN in the supernatant. However, human IECs respond to both type I and type III IFNs by producing IFN-stimulated genes that in turn induce an antiviral state. Using genetic ablation of either type I or type III IFN receptors, we show that either IFN can independently restrict virus infection in human IECs. Importantly, we report, for the first time, differences in the mechanisms by which each IFN establishes the antiviral state. Contrary to type I IFN, the antiviral activity induced by type III IFN is strongly dependent on the mitogen-activated protein kinases signaling pathway, suggesting a pathway used by type III IFNs that non-redundantly contributes to the antiviral state. In conclusion, we demonstrate that human intestinal epithelial cells specifically regulate their innate immune response favoring type III IFN-mediated signaling, which allows for efficient protection against pathogens without producing excessive inflammation. Our results strongly suggest that type III IFN constitutes the frontline of antiviral response in the human gut. We propose that

Manifestation of radiaton injury of human lymphocytes using PHA mitogenic stimulation in different culture systems

International Nuclear Information System (INIS)

Horky, J.

1986-01-01

The proliferative response of human lymphocytes to phytohemagglutinin in vitro is affected by X-irradiation. Dose-related changes in mitogenic stimulation of irradiated lymphocytes were compared for two culture systems - the cultivation of separated lymphocytes and the cultivation of whole blood. In the whole blood cultures, the proliferative activity of stimulated lyphocytes was markedly and reproducibly depressed by irradiation. An exponential curve could be fitted to the values of mitogenic response within a dose range from 0 to 2.5 Gy with high correlation. In a modified test where the mitogenic stimulus was given after a 24 h delay, depression in the response was even more pronounced. Radiosensitivity of human lymphocytes as determined by means of mitogenic stimulation in the whole blood cultures appears to be a characteristic individual feature. The mean D 37 value of the radiation-induced depression in mitogenic response in a group of 20 healthy donors was 2.5 Gy in the standard test and 2.0 Gy in the test with a delayed mitogenic stimulus. In contrast, the data obtained from separated lymphocyte cultures were characterized by a high degree of test-to-test variability and by much lower radiosensitivity. The possible mechanisms of these distinctive manifestations of the same primary radiation injury are discussed. (author) 3 tabs., 2 figs., 12 refs

Mitogen-activated protein kinase and abscisic acid signal transduction

NARCIS (Netherlands)

Heimovaara-Dijkstra, S.; Testerink, C.; Wang, M.

1998-01-01

The phytohormone abscisic acid (ABA) is a classical plant hormone, responsible for regulation of abscission, diverse aspects of plant and seed development, stress responses and germination. It was found that ABA signal transduction in plants can involve the activity of type 2C-phosphatases (PP2C),

Exposure to Cerium Oxide Nanoparticles Is Associated With Activation of Mitogen-activated Protein Kinases Signaling and Apoptosis in Rat Lungs

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Kevin M. Rice

2015-05-01

Full Text Available Objectives: With recent advances in nanoparticle manufacturing and applications, potential exposure to nanoparticles in various settings is becoming increasing likely. No investigation has yet been performed to assess whether respiratory tract exposure to cerium oxide (CeO2 nanoparticles is associated with alterations in protein signaling, inflammation, and apoptosis in rat lungs. Methods: Specific-pathogen-free male Sprague-Dawley rats were instilled with either vehicle (saline or CeO2 nanoparticles at a dosage of 7.0 mg/kg and euthanized 1, 3, 14, 28, 56, or 90 days after exposure. Lung tissues were collected and evaluated for the expression of proteins associated with inflammation and cellular apoptosis. Results: No change in lung weight was detected over the course of the study; however, cerium accumulation in the lungs, gross histological changes, an increased Bax to Bcl-2 ratio, elevated cleaved caspase-3 protein levels, increased phosphorylation of p38 MAPK, and diminished phosphorylation of ERK-1/2-MAPK were detected after CeO2 instillation (p<0.05. Conclusions: Taken together, these data suggest that high-dose respiratory exposure to CeO2 nanoparticles is associated with lung inflammation, the activation of signaling protein kinases, and cellular apoptosis, which may be indicative of a long-term localized inflammatory response.

Conversion of proteins from a non-polarized to an apical secretory pattern in MDCK cells

International Nuclear Information System (INIS)

Vogel, Lotte K.; Larsen, Jakob E.; Hansen, Martin; Truffer, Renato

2005-01-01

Previously it was shown that fusion proteins containing the amino terminus of an apical targeted member of the serpin family fused to the corresponding carboxyl terminus of the non-polarized secreted serpin, antithrombin, are secreted mainly to the apical side of MDCK cells. The present study shows that this is neither due to the transfer of an apical sorting signal from the apically expressed proteins, since a sequence of random amino acids acts the same, nor is it due to the deletion of a conserved signal for correct targeting from the non-polarized secreted protein. Our results suggest that the polarity of secretion is determined by conformational sensitive sorting signals

Spatio-temporal dynamics of an active, polar, viscoelastic ring.

Science.gov (United States)

Marcq, Philippe

2014-04-01

Constitutive equations for a one-dimensional, active, polar, viscoelastic liquid are derived by treating the strain field as a slow hydrodynamic variable. Taking into account the couplings between strain and polarity allowed by symmetry, the hydrodynamics of an active, polar, viscoelastic body include an evolution equation for the polarity field that generalizes the damped Kuramoto-Sivashinsky equation. Beyond thresholds of the active coupling coefficients between the polarity and the stress or the strain rate, bifurcations of the homogeneous state lead first to stationary waves, then to propagating waves of the strain, stress and polarity fields. I argue that these results are relevant to living matter, and may explain rotating actomyosin rings in cells and mechanical waves in epithelial cell monolayers.

Puerarin reduces apoptosis in rat hippocampal neurons culturea in high glucose medium by modulating the p38 mitogen activated protein kinase and c-Jun N-terminal kinase signaling pathways.

Science.gov (United States)

Xu, Xiaohan; Wang, Jingbo; Zhang, Hong; Tian, Guoqing; Liu, Yuqin

2016-02-01

To investigate the neuroprotective etfect of puerarin on rat hippocampal neurons cultured in high glucose medium, and to examine the role of the p38 mitogen activated protein kinase (p38 MAPK) and c-Jun N-terminal kinase (JNK) signaling pathways in this effect. Primary cultures of hippocampal neurons were prepared from newborn Sprague Dawley rats. Neuron-specific enolase immunocytochemistry was used to identify neurons. The neurons were cultured with normal medium (control group) or with high-glucose medium (high-glucose group), and puerarin (puerarin group), a p38 MAPK inhibitor (SB239063; p38 MAPK inhibitor group) or a JNK inhibitor (SP600125; JNK inhibitor group) were added. After 72 h of treatment, terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling assay was performed to detect apoptosis, and western blotting was used to assess protein levels of p-p38, p38, p-JNK and JNK. In the high-glucose group, the neuronal apoptosis rate and the p-p38/p38 and p-JNK/JNK ratios were higher than in the control group. The p38 MAPK and JNK inhibitors prevented this increase in the apoptosis rate. The apoptosis rates in the puerarin group, the p38 MAPK inhibitor group and the JNK inhibitor group were significantly decreased compared with the high-glucose group. Moreover, protein levels of p-p38 and p-JNK were significantly reduced, and the p-p38/p38 and p-JNK/JNK ratios were decreased in the puerarin group compared with the high-glucose group. In addition, compared with the high-glucose group, p-p38 levels and the p-p38/p38 ratio were reduced in the p38 MAPK inhibitor group, and p-JNK levels and the p-JNK/JNK ratio were decreased in the JNK inhibitor group. Puerarin attenuates neuronal apoptosis induced by high glucose by reducing the phosphorylation of p38 and JNK.

Reciprocal and dynamic polarization of planar cell polarity core components and myosin

Science.gov (United States)

Newman-Smith, Erin; Kourakis, Matthew J; Reeves, Wendy; Veeman, Michael; Smith, William C

2015-01-01

The Ciona notochord displays planar cell polarity (PCP), with anterior localization of Prickle (Pk) and Strabismus (Stbm). We report that a myosin is polarized anteriorly in these cells and strongly colocalizes with Stbm. Disruption of the actin/myosin machinery with cytochalasin or blebbistatin disrupts polarization of Pk and Stbm, but not of myosin complexes, suggesting a PCP-independent aspect of myosin localization. Wash out of cytochalasin restored Pk polarization, but not if done in the presence of blebbistatin, suggesting an active role for myosin in core PCP protein localization. On the other hand, in the pk mutant line, aimless, myosin polarization is disrupted in approximately one third of the cells, indicating a reciprocal action of core PCP signaling on myosin localization. Our results indicate a complex relationship between the actomyosin cytoskeleton and core PCP components in which myosin is not simply a downstream target of PCP signaling, but also required for PCP protein localization. DOI: http://dx.doi.org/10.7554/eLife.05361.001 PMID:25866928

Phosphodiesterase inhibitors suppress Lactobacillus casei cell-wall-induced NF-κB and MAPK activations and cell proliferation through protein kinase A--or exchange protein activated by cAMP-dependent signal pathway.

Science.gov (United States)

Saito, Takekatsu; Sugimoto, Naotoshi; Ohta, Kunio; Shimizu, Tohru; Ohtani, Kaori; Nakayama, Yuko; Nakamura, Taichi; Hitomi, Yashiaki; Nakamura, Hiroyuki; Koizumi, Shoichi; Yachie, Akihiro

2012-01-01

Specific strains of Lactobacillus have been found to be beneficial in treating some types of diarrhea and vaginosis. However, a high mortality rate results from underlying immunosuppressive conditions in patients with Lactobacillus casei bacteremia. Cyclic AMP (cAMP) is a small second messenger molecule that mediates signal transduction. The onset and progression of inflammatory responses are sensitive to changes in steady-state cAMP levels. L. casei cell wall extract (LCWE) develops arteritis in mice through Toll-like receptor-2 signaling. The purpose of this study was to investigate whether intracellular cAMP affects LCWE-induced pathological signaling. LCWE was shown to induce phosphorylation of the nuclear factor κB (NF-κB) and mitogen-activated protein kinase (MAPK) signaling pathways and cell proliferation in mice fibroblast cells. Theophylline and phosphodiesterase inhibitor increased intracellular cAMP and inhibited LCWE-induced cell proliferation as well as phosphorylation of NF-κB and MAPK. Protein kinase A inhibitor H89 prevented cAMP-induced MAPK inhibition, but not cAMP-induced NF-κB inhibition. An exchange protein activated by cAMP (Epac) agonist inhibited NF-κB activation but not MAPK activation. These results indicate that an increase in intracellular cAMP prevents LCWE induction of pathological signaling pathways dependent on PKA and Epac signaling.

SUMO-, MAPK- and resistance protein-signaling converge at transcription complexes that regulate plant innate immunity

NARCIS (Netherlands)

Burg, van den H.A.; Takken, F.L.W.

2010-01-01

Upon pathogen perception plant innate immune receptors activate various signaling pathways that trigger host defenses. PAMP-triggered defense signaling requires mitogen-activated protein kinase (MAPK) pathways, which modulate the activity of transcription factors through phosphorylation. Here, we

SUMO-, MAPK-, and resistance protein-signaling converge at transcription complexes that regulate plant innate immunity

NARCIS (Netherlands)

van den Burg, H.A.; Takken, F.L.W.

2010-01-01

Upon pathogen perception plant innate immune receptors activate various signaling pathways that trigger host defenses. PAMP-triggered defense signaling requires mitogen-activated protein kinase (MAPK) pathways, which modulate the activity of transcription factors through phosphorylation. Here, we

Ligand binding affinity at the insulin receptor isoform A (IR-A and subsequent IR-A tyrosine phosphorylation kinetics are important determinants of mitogenic biological outcomes.

Directory of Open Access Journals (Sweden)

Harinda eRajapaksha

2015-07-01

Full Text Available The insulin receptor (IR is a tyrosine kinase receptor that can mediate both metabolic and mitogenic biological actions. The IR isoform-A (IR-A arises from alternative splicing of exon 11 and has different ligand binding and signalling properties compared to the IR isoform-B. The IR-A not only binds insulin but also insulin-like growth factor-II (IGF-II with high affinity. IGF-II acting through the IR-A promotes cancer cell proliferation, survival and migration by activating some unique signalling molecules compared to those activated by insulin. This observation led us to investigate whether the different IR-A signalling outcomes in response to IGF-II and insulin could be attributed to phosphorylation of a different subset of IR-A tyrosine residues or to the phosphorylation kinetics. We correlated IR-A phosphorylation to activation of molecules involved in mitogenic and metabolic signalling (MAPK and Akt and receptor internalisation rates (related to mitogenic signalling. We also extended this study to incorporate two ligands that are known to promote predominantly mitogenic ([His4, Tyr15, Thr49, Ile51] IGF-I, qIGF-I or metabolic (S597 peptide biological actions, to see if common mechanisms can be used to define mitogenic or metabolic signalling through the IR-A. The 3-fold lower mitogenic action of IGF-II compared to insulin was associated with a decreased potency in activation of Y960, Y1146, Y1150, Y1151, Y1316 and Y1322, in MAPK phosphorylation and in IR-A internalization. With the poorly mitogenic S597 peptide it was a decreased rate of tyrosine phosphorylation rather than potency that was associated with a low mitogenic potential. We conclude that both decreased affinity of IR-A binding and the kinetics of IR-A phosphorylation can independently lead to a lower mitogenic activity. None of the studied parameters could account for the lower metabolic activity of qIGF-I.

Parameters of the labeling of mitogen-activated murine lymphocytes by [35S]methionine for two-dimensional gel electrophoresis. I

International Nuclear Information System (INIS)

Kettman, J.R.

1986-01-01

Labeling with [ 35 S]methionine at a high specific activity is essential to the facile preparation of 2-dimensional gel electrophoretograms with the analytical 2-dimensional charge-size separation procedure. Mitogen-activated T and B lymphocytes subjected to low methionine concentrations would not proceed through cell cycle. In the case of activated B lymphocytes, the use of fetal bovine serum, dialyzed to lower endogenous methionine concentrations, prevented B cell growth even in the presence of otherwise satisfactory levels of methionine. High concentrations of [ 35 S]methionine induced B cell death, apparently by radiation damage. Despite these problems, good radioautograms and radiofluorograms of 2D electrophoretograms could be prepared by labeling activated B or T cells in bulk with high specific activity [ 35 S]methionine. (Auth.)

A Mitogen-Activated Protein Kinase Tmk3 Participates in High Osmolarity Resistance, Cell Wall Integrity Maintenance and Cellulase Production Regulation in Trichoderma reesei

Science.gov (United States)

Wang, Mingyu; Zhao, Qiushuang; Yang, Jinghua; Jiang, Baojie; Wang, Fangzhong; Liu, Kuimei; Fang, Xu

2013-01-01

The mitogen-activated protein kinase (MAPK) pathways are important signal transduction pathways conserved in essentially all eukaryotes, but haven't been subjected to functional studies in the most important cellulase-producing filamentous fungus Trichoderma reesei. Previous reports suggested the presence of three MAPKs in T. reesei: Tmk1, Tmk2, and Tmk3. By exploring the phenotypic features of T. reesei Δtmk3, we first showed elevated NaCl sensitivity and repressed transcription of genes involved in glycerol/trehalose biosynthesis under higher osmolarity, suggesting Tmk3 participates in high osmolarity resistance via derepression of genes involved in osmotic stabilizer biosynthesis. We also showed significant downregulation of genes encoding chitin synthases and a β-1,3-glucan synthase, decreased chitin content, ‘budded’ hyphal appearance typical to cell wall defective strains, and increased sensitivity to calcofluor white/Congo red in the tmk3 deficient strain, suggesting Tmk3 is involved in cell wall integrity maintenance in T. reesei. We further observed the decrease of cellulase transcription and production in T. reesei Δtmk3 during submerged cultivation, as well as the presence of MAPK phosphorylation sites on known transcription factors involved in cellulase regulation, suggesting Tmk3 is also involved in the regulation of cellulase production. Finally, the expression of cell wall integrity related genes, the expression of cellulase coding genes, cellulase production and biomass accumulation were compared between T. reesei Δtmk3 grown in solid state media and submerged media, showing a strong restoration effect in solid state media from defects resulted from tmk3 deletion. These results showed novel physiological processes that fungal Hog1-type MAPKs are involved in, and present the first experimental investigation of MAPK signaling pathways in T. reesei. Our observations on the restoration effect during solid state cultivation suggest that T. reesei

A mitogen-activated protein kinase Tmk3 participates in high osmolarity resistance, cell wall integrity maintenance and cellulase production regulation in Trichoderma reesei.

Directory of Open Access Journals (Sweden)

Mingyu Wang

Full Text Available The mitogen-activated protein kinase (MAPK pathways are important signal transduction pathways conserved in essentially all eukaryotes, but haven't been subjected to functional studies in the most important cellulase-producing filamentous fungus Trichoderma reesei. Previous reports suggested the presence of three MAPKs in T. reesei: Tmk1, Tmk2, and Tmk3. By exploring the phenotypic features of T. reesei Δtmk3, we first showed elevated NaCl sensitivity and repressed transcription of genes involved in glycerol/trehalose biosynthesis under higher osmolarity, suggesting Tmk3 participates in high osmolarity resistance via derepression of genes involved in osmotic stabilizer biosynthesis. We also showed significant downregulation of genes encoding chitin synthases and a β-1,3-glucan synthase, decreased chitin content, 'budded' hyphal appearance typical to cell wall defective strains, and increased sensitivity to calcofluor white/Congo red in the tmk3 deficient strain, suggesting Tmk3 is involved in cell wall integrity maintenance in T. reesei. We further observed the decrease of cellulase transcription and production in T. reesei Δtmk3 during submerged cultivation, as well as the presence of MAPK phosphorylation sites on known transcription factors involved in cellulase regulation, suggesting Tmk3 is also involved in the regulation of cellulase production. Finally, the expression of cell wall integrity related genes, the expression of cellulase coding genes, cellulase production and biomass accumulation were compared between T. reesei Δtmk3 grown in solid state media and submerged media, showing a strong restoration effect in solid state media from defects resulted from tmk3 deletion. These results showed novel physiological processes that fungal Hog1-type MAPKs are involved in, and present the first experimental investigation of MAPK signaling pathways in T. reesei. Our observations on the restoration effect during solid state cultivation suggest

GPI-anchored proteins are confined in subdiffraction clusters at the apical surface of polarized epithelial cells.

Science.gov (United States)

Paladino, Simona; Lebreton, Stéphanie; Lelek, Mickaël; Riccio, Patrizia; De Nicola, Sergio; Zimmer, Christophe; Zurzolo, Chiara

2017-12-01

Spatio-temporal compartmentalization of membrane proteins is critical for the regulation of diverse vital functions in eukaryotic cells. It was previously shown that, at the apical surface of polarized MDCK cells, glycosylphosphatidylinositol (GPI)-anchored proteins (GPI-APs) are organized in small cholesterol-independent clusters of single GPI-AP species (homoclusters), which are required for the formation of larger cholesterol-dependent clusters formed by multiple GPI-AP species (heteroclusters). This clustered organization is crucial for the biological activities of GPI-APs; hence, understanding the spatio-temporal properties of their membrane organization is of fundamental importance. Here, by using direct stochastic optical reconstruction microscopy coupled to pair correlation analysis (pc-STORM), we were able to visualize and measure the size of these clusters. Specifically, we show that they are non-randomly distributed and have an average size of 67 nm. We also demonstrated that polarized MDCK and non-polarized CHO cells have similar cluster distribution and size, but different sensitivity to cholesterol depletion. Finally, we derived a model that allowed a quantitative characterization of the cluster organization of GPI-APs at the apical surface of polarized MDCK cells for the first time. Experimental FRET (fluorescence resonance energy transfer)/FLIM (fluorescence-lifetime imaging microscopy) data were correlated to the theoretical predictions of the model. © 2017 The Author(s).

Epithelial cell polarity, stem cells and cancer

DEFF Research Database (Denmark)

Martin-Belmonte, Fernando; Perez-Moreno, Mirna

2011-01-01

, deregulation of adhesion and polarity proteins can cause misoriented cell divisions and increased self-renewal of adult epithelial stem cells. In this Review, we highlight some advances in the understanding of how loss of epithelial cell polarity contributes to tumorigenesis.......After years of extensive scientific discovery much has been learned about the networks that regulate epithelial homeostasis. Loss of expression or functional activity of cell adhesion and cell polarity proteins (including the PAR, crumbs (CRB) and scribble (SCRIB) complexes) is intricately related...

Active Polar Gels: a Paradigm for Cytoskeletal Dynamics

Science.gov (United States)

Julicher, Frank

2006-03-01

The cytoskeleton of eucaryotic cells is an intrinsically dynamic network of rod-like filaments. Active processes on the molecular scale such as the action of motor proteins and the polymerization and depolymerization of filaments drive active dynamic behaviors while consuming chemical energy in the form of a fuel. Such emergent dynamics is regulated by the cell and is important for many cellular processes such as cell locomotion and cell division. From a general point of view the cytoskeleton represents an active gel-like material with interesting material properties. We present a general theory of active viscoelastic materials made of polar filaments which is motivated by the the cytoskeleton. The continuous consumption of a fuel generates a non- equilibrium state characterized by the generation of flows and stresses. Our theory can be applied to experiments in which cytoskeletal patterns are set in motion by active processes such as those which are at work in cells. It can also capture generic aspects of the flows and stress profiles which occur during cell locomotion.

Mycobacterium avium subspecies paratuberculosis recombinant proteins modulate antimycobacterial functions of bovine macrophages

Science.gov (United States)

It has been shown that Mycobacterium avium subspecies paratuberculosis (M. paratuberculosis) activates the Mitogen Activated Protein Kinase (MAPK) p38 pathway, yet it is unclear which components of M. paratuberculosis are involved in the process. Therefore, a set of 42 M. paratuberculosis recombinan...

Slack sodium-activated potassium channel membrane expression requires p38 mitogen-activated protein kinase phosphorylation.

Science.gov (United States)

Gururaj, Sushmitha; Fleites, John; Bhattacharjee, Arin

2016-04-01

p38 MAPK has long been understood as an inducible kinase under conditions of cellular stress, but there is now increasing evidence to support its role in the regulation of neuronal function. Several phosphorylation targets have been identified, an appreciable number of which are ion channels, implicating the possible involvement of p38 MAPK in neuronal excitability. The KNa channel Slack is an important protein to be studied as it is highly and ubiquitously expressed in DRG neurons and is important in the maintenance of their firing accommodation. We sought to examine if the Slack channel could be a substrate of p38 MAPK activity. First, we found that the Slack C-terminus contains two putative p38 MAPK phosphorylation sites that are highly conserved across species. Second, we show via electrophysiology experiments that KNa currents and further, Slack currents, are subject to tonic modulation by p38 MAPK. Third, biochemical approaches revealed that Slack channel regulation by p38 MAPK occurs through direct phosphorylation at the two putative sites of interaction, and mutating both sites prevented surface expression of Slack channels. Based on these results, we conclude that p38 MAPK is an obligate regulator of Slack channel function via the trafficking of channels into the membrane. The present study identifies Slack KNa channels as p38 MAPK substrates. Copyright © 2015 Elsevier Ltd. All rights reserved.

Insulin-like growth factor (IGF)-I obliterates the pregnancy-associated protection against mammary carcinogenesis in rats: evidence that IGF-I enhances cancer progression through estrogen receptor-α activation via the mitogen-activated protein kinase pathway

International Nuclear Information System (INIS)

Thordarson, Gudmundur; Slusher, Nicole; Leong, Harriet; Ochoa, Dafne; Rajkumar, Lakshmanaswamy; Guzman, Raphael; Nandi, Satyabrata; Talamantes, Frank

2004-01-01

rats. We argue that tumor initiation (transformation and fixation of mutations) may be similar in parous and age-matched virgin animals, suggesting that the main differences in tumor formation lie in differences in tumor progression caused by the altered hormonal environment associated with parity. Furthermore, we provide evidence supporting the notion that tumor growth promotion seen in IGF-I-treated parous rats is caused by activation of estrogen receptor-α via the Raf/Ras/mitogen-activated protein kinase cascade

Mitogen-activated protein kinase kinase 1/2 inhibition and angiotensin II converting inhibition in mice with cardiomyopathy caused by lamin A/C gene mutation

Energy Technology Data Exchange (ETDEWEB)

Muchir, Antoine, E-mail: a.muchir@institut-myologie.org [Department of Medicine, College of Physicians and Surgeons, Columbia University, New York, NY (United States); Department of Pathology and Cell Biology, College of Physicians and Surgeons, Columbia University, New York, NY (United States); Wu, Wei [Department of Medicine, College of Physicians and Surgeons, Columbia University, New York, NY (United States); Department of Pathology and Cell Biology, College of Physicians and Surgeons, Columbia University, New York, NY (United States); Sera, Fusako; Homma, Shunichi [Department of Medicine, College of Physicians and Surgeons, Columbia University, New York, NY (United States); Worman, Howard J., E-mail: hjw14@columbia.edu [Department of Medicine, College of Physicians and Surgeons, Columbia University, New York, NY (United States); Department of Pathology and Cell Biology, College of Physicians and Surgeons, Columbia University, New York, NY (United States)

2014-10-03

Highlights: • Both ACE and MEK1/2 inhibition are beneficial on cardiac function in Lmna cardiomyopathy. • MEK1/2 inhibitor has beneficial effects beyond ACE inhibition for Lmna cardiomyopathy. • These results provide further preclinical rationale for a clinical trial of a MEK1/2 inhibitor. - Abstract: Background: Mutations in the LMNA gene encoding A-type nuclear lamins can cause dilated cardiomyopathy with or without skeletal muscular dystrophy. Previous studies have shown abnormally increased extracellular signal-regulated kinase 1/2 activity in hearts of Lmna{sup H222P/H222P} mice, a small animal model. Inhibition of this abnormal signaling activity with a mitogen-activated protein kinase kinase 1/2 (MEK1/2) inhibitor has beneficial effects on heart function and survival in these mice. However, such treatment has not been examined relative to any standard of care intervention for dilated cardiomyopathy or heart failure. We therefore examined the effects of an angiotensin II converting enzyme (ACE) inhibitor on left ventricular function in Lmna{sup H222P/H222P} mice and assessed if adding a MEK1/2 inhibitor would provide added benefit. Methods: Male Lmna{sup H222P/H222P} mice were treated with the ACE inhibitor benazepril, the MEK1/2 inhibitor selumetinib or both. Transthoracic echocardiography was used to measure left ventricular diameters and fractional shortening was calculated. Results: Treatment of Lmna{sup H222P/H222P} mice with either benazepril or selumetinib started at 8 weeks of age, before the onset of detectable left ventricular dysfunction, lead to statistically significantly increased fractional shortening compared to placebo at 16 weeks of age. There was a trend towards a great value for fractional shortening in the selumetinib-treated mice. When treatment was started at 16 weeks of age, after the onset of left ventricular dysfunction, the addition of selumetinib treatment to benazepril lead to a statistically significant increase in left

Mitogen-activated protein kinase kinase 1/2 inhibition and angiotensin II converting inhibition in mice with cardiomyopathy caused by lamin A/C gene mutation

International Nuclear Information System (INIS)

Muchir, Antoine; Wu, Wei; Sera, Fusako; Homma, Shunichi; Worman, Howard J.

2014-01-01

Highlights: • Both ACE and MEK1/2 inhibition are beneficial on cardiac function in Lmna cardiomyopathy. • MEK1/2 inhibitor has beneficial effects beyond ACE inhibition for Lmna cardiomyopathy. • These results provide further preclinical rationale for a clinical trial of a MEK1/2 inhibitor. - Abstract: Background: Mutations in the LMNA gene encoding A-type nuclear lamins can cause dilated cardiomyopathy with or without skeletal muscular dystrophy. Previous studies have shown abnormally increased extracellular signal-regulated kinase 1/2 activity in hearts of Lmna H222P/H222P mice, a small animal model. Inhibition of this abnormal signaling activity with a mitogen-activated protein kinase kinase 1/2 (MEK1/2) inhibitor has beneficial effects on heart function and survival in these mice. However, such treatment has not been examined relative to any standard of care intervention for dilated cardiomyopathy or heart failure. We therefore examined the effects of an angiotensin II converting enzyme (ACE) inhibitor on left ventricular function in Lmna H222P/H222P mice and assessed if adding a MEK1/2 inhibitor would provide added benefit. Methods: Male Lmna H222P/H222P mice were treated with the ACE inhibitor benazepril, the MEK1/2 inhibitor selumetinib or both. Transthoracic echocardiography was used to measure left ventricular diameters and fractional shortening was calculated. Results: Treatment of Lmna H222P/H222P mice with either benazepril or selumetinib started at 8 weeks of age, before the onset of detectable left ventricular dysfunction, lead to statistically significantly increased fractional shortening compared to placebo at 16 weeks of age. There was a trend towards a great value for fractional shortening in the selumetinib-treated mice. When treatment was started at 16 weeks of age, after the onset of left ventricular dysfunction, the addition of selumetinib treatment to benazepril lead to a statistically significant increase in left ventricular fractional

Normal mitogen-induced suppression of the interleukin-6 (IL-6) response and its deficiency in systemic lupus erythematosus

International Nuclear Information System (INIS)

Warrington, R.J.; Rutherford, W.J.

1990-01-01

A low-frequency suppressor-cell population in normal peripheral blood inhibits the B-cell CESS response to IL-6, following pokeweed mitogen stimulation. The suppression of IL-6 responsiveness is radiation sensitive, directed against CESS targets and not mediated by inhibition of IL-6 production, and associated with nonspecific cytotoxic activity against CESS targets. The generation of these cytolytic cells is also radiation sensitive. A correlation was found between PWM-induced cytotoxicity against CESS and the suppression of IL-6-dependent IgG production. But cytotoxicity toward CESS targets is not responsible for this suppression because IL-2 induces equivalent or greater nonspecific cytotoxicity against CESS in the total absence of suppression of CESS-derived IgG production and suppression is also induced by mitogen-activated PBL separated from CESS targets by a cell-impermeable membrane. This suppression was not mediated by TNF alpha/beta or IFN-gamma. In systemic lupus erythematosus, suppression of IL-6-dependent IgG production is impaired in patients with active disease (29.2 +/- 13.7%) compared to patients with inactive disease (70 +/- 19.5%) or normal controls (82.8 +/- 9.2%). There is also a defect in mitogen-induced nonspecific cytotoxicity in active SLE (specific lysis 15.1 +/- 3.5%, compared to 34 +/- 4% in normals). Pokeweed mitogen-activated PBL can therefore normally induce suppression of B-cell IL-6 responses and this response is deficient in lupus

Activation of Protein Kinase C and Protein Kinase D in Human Natural Killer Cells: Effects of Tributyltin, Dibutyltin, and Tetrabromobisphenol A

Science.gov (United States)

Rana, Krupa; Whalen, Margaret M.

2015-01-01

Up to now, the ability of target cells to activate protein kinase C (PKC) and protein kinase D (PKD) (which is often a downstream target of PKC) has not been examined in natural killer (NK) lymphocytes. Here we examined whether exposure of human NK cells to lysis sensitive tumor cells activated PKC and PKD. The results of these studies show for the first time that activation of PKC and PKD occurs in response to target cell binding to NK cells. Exposure of NK cells to K562 tumor cells for 10 and 30 minutes increased phosphorylation/activation of both PKC and PKD by roughly 2 fold. Butyltins (tributyltin (TBT); dibutyltin (DBT)) and brominated compounds (tetrabromobisphenol A (TBBPA)) are environmental contaminants that are found in human blood. Exposures of NK cells to TBT, DBT or TBBPA decrease NK cell lytic function in part by activating the mitogen activated protein kinases (MAPKs) that are part of the NK lytic pathway. We established that PKC and PKD are part of the lytic pathway upstream of MAPKs and thus we investigated whether DBT, TBT, and TBBPA exposures activated PKC and PKD. TBT activated PKC by 2–3 fold at 10 min at concentrations ranging from 50–300 nM while DBT caused a 1.3 fold activation at 2.5 μM at 10 min. Both TBT and DBT caused an approximately 2 fold increase in phosphorylation/activation of PKC. Exposures to TBBPA caused no statistically significant changes in either PKC or PKD activation. PMID:26228090

ZNF328, a novel human zinc-finger protein, suppresses transcriptional activities of SRE and AP-1

International Nuclear Information System (INIS)

Ou Ying; Wang Shenqiu; Cai Zhenyu; Wang Yuequn; Wang Canding; Li Yongqing; Li Fang; Yuan Wuzhou; Liu Bisheng; Wu Xiushan; Liu Mingyao

2005-01-01

The zinc finger proteins containing the Kruppel-associated box domain (KRAB-ZFPs) are the single largest class of transcription factors in human genome. Many of the KRAB-ZFPs are involved in cardiac development or cardiovascular diseases. Here, we have identified a novel human KRAB zinc finger gene, named ZNF328, from the human fetal heart cDNA library. The complete sequence of ZNF328 cDNA contains a 2376-bp open reading frame (ORF) and encodes a 792 amino acid protein with an N-terminal KRAB domain and classical zinc finger C 2 H 2 motifs in the C-terminus. Northern blot analysis indicates that the protein is expressed in most of the examined human adult and embryonic tissues. ZNF328 is a transcription suppressor when fused to Gal-4 DNA-binding domain and cotransfected with VP-16. Overexpression of ZNF328 in COS-7 cells inhibits the transcriptional activities of SRE and AP-1. Deletion analysis with a series of truncated fusion proteins indicates that the KRAB motif is a basal repression domain when cotransfected with VP-16. Similar results were obtained when the truncated fusion proteins were assayed for the transcriptional activities of SRE and AP-1. These results suggest that ZNF328 protein may act as a transcriptional repressor in mitogen-activated protein kinase (MAPK) signaling pathway to mediate cellular functions

PsMPK7, a stress-associated mitogen-activated protein kinase (MAPK) in Phytophthora sojae, is required for stress tolerance, reactive oxygenated species detoxification, cyst germination, sexual reproduction and infection of soybean.

Science.gov (United States)

Gao, Jian; Cao, Mingna; Ye, Wenwu; Li, Haiyang; Kong, Liang; Zheng, Xiaobo; Wang, Yuanchao

2015-01-01

The sensing of stress signals and their transduction into appropriate responses are crucial for the adaptation, survival and infection of phytopathogenic fungi and oomycetes. Amongst evolutionarily conserved pathways, mitogen-activated protein kinase (MAPK) cascades function as key signal transducers that use phosphorylation to convey information. In this study, we identified a gene, designated PsMPK7, one of 14 predicted genes encoding MAPKs in Phytophthora sojae. PsMPK7 was highly transcribed in each tested stage, but was up-regulated in the zoospore, cyst and cyst germination stages. Silencing of PsMPK7 affected the growth of germinated cysts, oospore production and the pathogenicity of soybean. PsMPK7 transcription was induced by stresses from sorbitol, NaCl and hydrogen peroxide. Transformants in which PsMPK7 expression was silenced (PsMPK7-silenced) were significantly more sensitive to osmotic and oxidative stress. Aniline blue and diaminobenzidine staining revealed that the silenced lines did not suppress the host reactive oxygen species (ROS) burst, indicating that either the inoculated plants activated stronger defence responses to the transformants and/or the PsMPK7-silenced transformants failed to overcome plant defences. In addition, extracellular secretion of laccase decreased in the silenced lines. Overall, our results indicate that the PsMPK7 gene encodes a stress-associated MAPK in P. sojae that is important not only for responses to various stresses, but also for ROS detoxification, cyst germination, sexual oospore production and infection of soybean. © 2014 BSPP AND JOHN WILEY & SONS LTD.

[G-protein potentiates the activation of TNF-alpha on calcium-activated potassium channel in ECV304].

Science.gov (United States)

Lin, L; Zheng, Y; Qu, J; Bao, G

2000-06-01

Observe the effect of tumor necrosis factor-alpha (TNF-alpha) on calcium-activated potassium channel in ECV304 and the possible involvement of G-protein mediation in the action of TNF-alpha. Using the cell-attached configuration of patch clamp technique. (1) the activity of high-conductance calcium-activated potassium channel (BKca) was recorded. Its conductance is (202.54 +/- 16.62) pS; (2) the activity of BKca was potentiated by 200 U/ml TNF-alpha; (3) G-protein would intensify this TNF-alpha activation. TNF-alpha acted on vascular endothelial cell ECV304 could rapidly activate the activity of BKca. Opening of BKca resulted in membrane hyper-polarization which could increase electro-chemical gradient for the resting Ca2+ influx and open leakage calcium channel, thus resting cytoplasmic free Ca2+ concentration could be elevated. G-protein may exert an important regulation in this process.

Do polar faculae terminate or commence an extended cycle of solar activity?

International Nuclear Information System (INIS)

Bumba, V.

1990-01-01

From the local as well as from the global points of view, polar magnetic fields are formed from successively developed trailing polarity fields expelled from the main activity zone. Polar faculae observed during the 20th and the 21st cycles of activity fill in the areas covered by the most intense unipolar fields distributed in the convection network. These polar regions formed from magnetic fields of an old activity cycle are sharply separated from low-latitude magnetic fields and from fields developed by a new cycle of activity. The polarity distribution in polar faculae seems to follow from unipolarity of their magnetic fields - the prevailing polarity becomes the main leading polarity. The greatest part of the main activity zone, the most intense faculae shifting equatorwards are connected with the zone of the prevailing leading polarity magnetic fields. Some of these faculae - the weak and inhomogeneously distributed ones, bordering the main faculae butterflies polewards - are related again to the trailing polarity fields shifting polewards. The main characteristic of the latitudinal distribution of solar faculae is the existence of their two latitudinal types: the polar faculae shifting polewards are related to the trailing polarity fields of the old cycle, the faculae of the main activity zone shifting equatorwards are related mainly (from the global point of view) to the leading polarity fields, and their activity ends several years earlier than that of the polar ones. The polar faculae with their magnetic fields represent the last phase of the magnetic activity cycle lasting 15-17 years. (author). 6 figs., 21 refs

Protein tyrosine phosphatase receptor type R deficient mice exhibit increased exploration in a new environment and impaired novel object recognition memory

NARCIS (Netherlands)

Erkens, M.; Bakker, B.; Duijn, L.M. van; Hendriks, W.J.A.J.; Zee, C.E.E.M. van der

2014-01-01

Mouse gene Ptprr encodes multiple protein tyrosine phosphatase receptor type R (PTPRR) isoforms that negatively regulate mitogen-activated protein kinase (MAPK) signaling pathways. In the mouse brain, PTPRR proteins are expressed in cerebellum, olfactory bulb, hippocampus, amygdala and perirhinal

Activation of p44/42 in Human Natural Killer Cells Decreases Cell-surface Protein Expression: Relationship to Tributyltin-induced alterations of protein expression

Science.gov (United States)

Dudimah, Fred D.; Abraha, Abraham; Wang, Xiaofei; Whalen, Margaret M.

2010-01-01

Tributyltin (TBT) activates the mitogen activated protein kinase (MAPK), p44/42 in human natural killer (NK) cells. TBT also reduces NK cytotoxic function and decreases the expression of several NK-cell proteins. To understand the role that p44/42 activation plays in TBT-induced loss of NK cell function, we have investigated how selective activation of p44/42 by phorbol 12-myristate 13-acetate (PMA) affects NK cells. Previously we showed that PMA caused losses of lytic function similar to those seen with TBT exposures. Here we examined activation of p44/42 in the regulation of NK-cell protein expression and how this regulation may explain the protein expression changes seen with TBT exposures. NK cells exposed to PMA were examined for levels of cell-surface proteins, granzyme mRNA, and perforin mRNA expression. The expression of CD11a, CD16, CD18, and CD56 were reduced, perforin mRNA levels were unchanged and granzyme mRNA levels were increased. To verify that activation of p44/42 was responsible for the alterations seen in CD11a, CD16, CD18, and CD56 with PMA, NK cells were treated with the p44/42 pathway inhibitor (PD98059) prior to PMA exposures. In the presence of PD98059, PMA caused no decreases in the expression of the cell-surface proteins. Results of these studies indicate that the activation of p44/42 may lead to the loss of NK cell cytotoxic function by decreasing the expression of CD11a, CD16, CD18, and CD56. Further, activation of p44/42 appears to be at least in part responsible for the TBT-induced decreases in expression of CD16, CD18, and CD56. PMID:20883105

Identity of the immunomodulatory proteins from garlic (Allium sativum) with the major garlic lectins or agglutinins.

Science.gov (United States)

Clement, Fatima; Pramod, Siddanakoppalu N; Venkatesh, Yeldur P

2010-03-01

Garlic (Allium sativum), an important medicinal spice, displays a plethora of biological effects including immunomodulation. Although some immunomodulatory proteins from garlic have been described, their identities are still unknown. The present study was envisaged to isolate immunomodulatory proteins from raw garlic, and examine their effects on certain cells of the immune system (lymphocytes, mast cells, and basophils) in relation to mitogenicity and hypersensitivity. Three protein components of approximately 13 kD (QR-1, QR-2, and QR-3 in the ratio 7:28:1) were separated by Q-Sepharose chromatography of 30 kD ultrafiltrate of raw garlic extract. All the 3 proteins exhibited mitogenic activity towards human peripheral blood lymphocytes, murine splenocytes and thymocytes. The mitogenicity of QR-2 was the highest among the three immunomodulatory proteins. QR-1 and QR-2 displayed hemagglutination and mannose-binding activities; QR-3 showed only mannose-binding activity. Immunoreactivity of rabbit anti-QR-1 and anti-QR-2 polyclonal antisera showed specificity for their respective antigens as well as mutual cross-reactivity; QR-3 was better recognized by anti-QR-2 (82%) than by anti-QR-1 (55%). QR-2 induced a 2-fold higher histamine release in vitro from leukocytes of atopic subjects compared to that of non-atopic subjects. In all functional studies, QR-2 was more potent compared to QR-1. Taken together, all these results indicate that the two major proteins QR-2 and QR-1 present in a ratio of 4:1 in raw garlic contribute to garlic's immunomodulatory activity, and their characteristics are markedly similar to the abundant Allium sativum agglutinins (ASA) I and II, respectively. Copyright 2010 Elsevier B.V. All rights reserved.

Role of nongenomic activation of phosphatidylinositol 3-kinase/Akt and mitogen-activated protein kinase/extracellular signal-regulated kinase kinase/extracellular signal-regulated kinase 1/2 pathways in 1,25D3-mediated apoptosis in squamous cell carcinoma cells.

Science.gov (United States)

Ma, Yingyu; Yu, Wei-Dong; Kong, Rui-Xian; Trump, Donald L; Johnson, Candace S

2006-08-15

Vitamin D is a steroid hormone that regulates calcium homeostasis and bone metabolism. The active form of vitamin D [1 alpha,25-dihydroxyvitamin D(3) (1,25D3)] acts through both genomic and nongenomic pathways. 1,25D3 has antitumor effects in a variety of cancers, including colorectal, prostate, breast, ovarian, and skin cancers. 1,25D3 exerts growth-inhibitory effects in cancer cells through the induction of apoptosis, cell cycle arrest, and differentiation. The mechanisms regulating 1,25D3-induced apoptosis remain unclear. We investigated the role of nongenomic signaling in 1,25D3-mediated apoptosis in squamous cell carcinoma (SCC) cells. 1,25D3 induced rapid and sustained activation of phosphatidylinositol 3-kinase/Akt and mitogen-activated protein kinase/extracellular signal-regulated kinase (ERK) 1/2 pathways in SCC cells. These effects were nongenomic: they occurred rapidly and were not inhibited by cycloheximide or actinomycin D. To examine whether the nongenomic activation of Akt and ERK1/2 plays a role in 1,25D3-mediated apoptosis, the expression of Akt or ERK1/2 was reduced by small interfering RNA (siRNA). siRNA-Akt significantly enhanced 1,25D3-induced apoptosis as indicated by increased levels of Annexin V-positive cells and increased sub-G(1) population and DNA fragmentation. In contrast, siRNA-ERK1/2 had no effects on 1,25D3-induced apoptosis. In addition, siRNA-Akt transfection followed by 1,25D3 treatment induced apoptosis much sooner than 1,25D3 alone. siRNA-Akt and 1,25D3 induced caspase-10 activation, suppressed the expression of c-IAP1 and XIAP, and promoted 1,25D3-induced caspase-3 activation. These results support a link between 1,25D3-induced nongenomic signaling and apoptosis. 1,25D3 induces the activation of phosphatidylinositol 3-kinase/Akt, which suppresses 1,25D3-mediated apoptosis and prolongs the survival of SCC cells.

Primary human polarized small intestinal epithelial barriers respond differently to a hazardous and an innocuous protein.

Science.gov (United States)

Eaton, A D; Zimmermann, C; Delaney, B; Hurley, B P

2017-08-01

An experimental platform employing human derived intestinal epithelial cell (IEC) line monolayers grown on permeable Transwell ® filters was previously investigated to differentiate between hazardous and innocuous proteins. This approach was effective at distinguishing these types of proteins and perturbation of monolayer integrity, particularly transepithelial electrical resistance (TEER), was the most sensitive indicator. In the current report, in vitro indicators of monolayer integrity, cytotoxicity, and inflammation were evaluated using primary (non-transformed) human polarized small intestinal epithelial barriers cultured on Transwell ® filters to compare effects of a hazardous protein (Clostridium difficile Toxin A [ToxA]) and an innocuous protein (bovine serum albumin [BSA]). ToxA exerted a reproducible decrease on barrier integrity at doses comparable to those producing effects observed from cell line-derived IEC monolayers, with TEER being the most sensitive indicator. In contrast, BSA, tested at concentrations substantially higher than ToxA, did not cause changes in any of the tested variables. These results demonstrate a similarity in response to certain proteins between cell line-derived polarized IEC models and a primary human polarized small intestinal epithelial barrier model, thereby reinforcing the potential usefulness of cell line-derived polarized IECs as a valid experimental platform to differentiate between hazardous and non-hazardous proteins. Copyright © 2017 The Authors. Published by Elsevier Ltd.. All rights reserved.

Application of empirical hydration distribution functions around polar atoms for assessing hydration structures of proteins

International Nuclear Information System (INIS)

Matsuoka, Daisuke; Nakasako, Masayoshi

2013-01-01

Highlights: ► Empirical distribution functions of water molecules in protein hydration are made. ► The functions measure how hydrogen-bond geometry in hydration deviate from ideal. ► The functions assess experimentally identified hydration structures of protein. - Abstract: To quantitatively characterize hydrogen-bond geometry in local hydration structures of proteins, we constructed a set of empirical hydration distribution functions (EHDFs) around polar protein atoms in the main and side chains of 11 types of hydrophilic amino acids (D. Matsuoka, M. Nakasako, Journal of Physical Chemistry B 113 (2009) 11274). The functions are the ensemble average of possible hydration patterns around the polar atoms, and describe the anisotropic deviations from ideal hydrogen bond geometry. In addition, we defined probability distribution function of hydration water molecules (PDFH) over the hydrophilic surface of a protein as the sum of EHDFs of solvent accessible polar protein atoms. The functions envelop most of hydration sites identified in crystal structures of proteins (D. Matsuoka, M. Nakasako, Journal of Physical Chemistry B 114 (2010) 4652). Here we propose the application of EHDFs and PDFHs for assessing crystallographically identified hydration structures of proteins. First, hydration water molecules are classified with respect to the geometry in hydrogen bonds in referring EHDFs. Difference Fourier electron density map weighted by PDFH of protein is proposed to identify easily density peaks as candidates of hydration water molecules. A computer program implementing those ideas was developed and used for assessing hydration structures of proteins

Mitogen-activated protein kinases mediate Mycobacterium

Indian Academy of Sciences (India)

CD44, an adhesion molecule, has been reported to be a binding site for Mycobacterium tuberculosis (M. tuberculosis) in macrophages and it also mediates mycobacterial phagocytosis, macrophage recruitment and protective immunity against pulmonary tuberculosis in vivo. However, the signalling pathways that are ...

The Ras GTPase-activating protein Rasal3 supports survival of naive T cells.

Directory of Open Access Journals (Sweden)

Ryunosuke Muro

Full Text Available The Ras-mitogen-activated protein kinase (MAPK pathway is crucial for T cell receptor (TCR signaling in the development and function of T cells. The significance of various modulators of the Ras-MAPK pathway in T cells, however, remains to be fully understood. Ras-activating protein-like 3 (Rasal3 is an uncharacterized member of the SynGAP family that contains a conserved Ras GTPase-activating protein (GAP domain, and is predominantly expressed in the T cell lineage. In the current study, we investigated the function and physiological roles of Rasal3. Our results showed that Rasal3 possesses RasGAP activity, but not Rap1GAP activity, and represses TCR-stimulated ERK phosphorylation in a T cell line. In systemic Rasal3-deficient mice, T cell development in the thymus including positive selection, negative selection, and β-selection was unaffected. However, the number of naive, but not effector memory CD4 and CD8 T cell in the periphery was significantly reduced in Rasal3-deficient mice, and associated with a marked increase in apoptosis of these cells. Indeed, survival of Rasal3 deficient naive CD4 T cells in vivo by adoptive transfer was significantly impaired, whereas IL-7-dependent survival of naive CD4 T cells in vitro was unaltered. Collectively, Rasal3 is required for in vivo survival of peripheral naive T cells, contributing to the maintenance of optimal T cell numbers.

Activation of the protein tyrosine phosphatase SHP2 via the interleukin-6 signal transducing receptor protein gp130 requires tyrosine kinase Jak1 and limits acute-phase protein expression.

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Schaper, F; Gendo, C; Eck, M; Schmitz, J; Grimm, C; Anhuf, D; Kerr, I M; Heinrich, P C

1998-11-01

Stimulation of the interleukin-6 (IL-6) signalling pathway occurs via the IL-6 receptor-glycoprotein 130 (IL-6R-gp130) receptor complex and results in the regulation of acute-phase protein genes in liver cells. Ligand binding to the receptor complex leads to tyrosine phosphorylation and activation of Janus kinases (Jak), phosphorylation of the signal transducing subunit gp130, followed by recruitment and phosphorylation of the signal transducer and activator of transcription factors STAT3 and STAT1 and the src homology domain (SH2)-containing protein tyrosine phosphatase (SHP2). The tyrosine phosphorylated STAT factors dissociate from the receptor, dimerize and translocate to the nucleus where they bind to enhancer sequences of IL-6 target genes. Phosphorylated SHP2 is able to bind growth factor receptor bound protein (grb2) and thus might link the Jak/STAT pathway to the ras/raf/mitogen-activated protein kinase pathway. Here we present data on the dose-dependence, kinetics and kinase requirements for SHP2 phosphorylation after the activation of the signal transducer, gp130, of the IL-6-type family receptor complex. When human fibrosarcoma cell lines deficient in Jak1, Jak2 or tyrosine kinase 2 (Tyk2) were stimulated with IL-6-soluble IL-6R complexes it was found that only in Jak1-, but not in Jak 2- or Tyk2-deficient cells, SHP2 activation was greatly impaired. It is concluded that Jak1 is required for the tyrosine phosphorylation of SHP2. This phosphorylation depends on Tyr-759 in the cytoplasmatic domain of gp130, since a Tyr-759-->Phe exchange abrogates SHP2 activation and in turn leads to elevated and prolonged STAT3 and STAT1 activation as well as enhanced acute-phase protein gene induction. Therefore, SHP2 plays an important role in acute-phase gene regulation.

TRIM45, a novel human RBCC/TRIM protein, inhibits transcriptional activities of ElK-1 and AP-1

International Nuclear Information System (INIS)

Wang Yuequn; Li Yongqing; Qi Xinzhu; Yuan Wuzhou; Ai Jianping; Zhu Chuanbing; Cao Lei; Yang Hong; Liu Fang; Wu Xiushan; Liu Mingyao

2004-01-01

The tripartite motif (TRIM) proteins play important roles in a variety of cellular functions including cell proliferation, differentiation, development, oncogenesis, and apoptosis. In this study, we report the identification and characterization of the human tripartite motif-containing protein 45 (TRIM45), a novel member of the TRIM family, from a human embryonic heart cDNA library. TRIM45 has a predicted 580 amino acid open reading frame, encoding a putative 64-kDa protein. The N-terminal region harbors a RING finger, two B-boxes, and a predicted α-helical coiled-coil domain, which together form the RBCC/TRIM motif found in a large family of proteins, whereas the C-terminal region contains a filamin-type immunoglobulin (IG-FLMN) domain. Northern blot analysis indicates that TRIM45 is expressed in a variety of human adult and embryonic tissues. In the cell, TRIM45 protein is expressed both in cytoplasm and in cell nucleus. Overexpression of TRIM45 in COS-7 cells inhibits the transcriptional activities of ElK-1 and AP-1. These results suggest that TRIM45 may act as a new transcriptional repressor in mitogen-activated protein kinase signaling pathway

Acidic pH stimulates the production of the angiogenic CXC chemokine, CXCL8 (interleukin-8), in human adult mesenchymal stem cells via the extracellular signal-regulated kinase, p38 mitogen-activated protein kinase, and NF-kappaB pathways.

Science.gov (United States)

Bischoff, David S; Zhu, Jian-Hua; Makhijani, Nalini S; Yamaguchi, Dean T

2008-07-01

Blood vessel injury results in limited oxygen tension and diffusion leading to hypoxia, increased anaerobic metabolism, and elevated production of acidic metabolites that cannot be easily removed due to the reduced blood flow. Therefore, an acidic extracellular pH occurs in the local microenvironment of disrupted bone. The potential role of acidic pH and glu-leu-arg (ELR(+)) CXC chemokines in early events in bone repair was studied in human mesenchymal stem cells (hMSCs) treated with medium of decreasing pH (7.4, 7.0, 6.7, and 6.4). The cells showed a reciprocal increase in CXCL8 (interleukin-8, IL-8) mRNA levels as extracellular pH decreased. At pH 6.4, CXCL8 mRNA was induced >60x in comparison to levels at pH 7.4. hMSCs treated with osteogenic medium (OGM) also showed an increase in CXCL8 mRNA with decreasing pH; although, at a lower level than that seen in cells grown in non-OGM. CXCL8 protein was secreted into the medium at all pHs with maximal induction at pH 6.7. Inhibition of the G-protein-coupled receptor alpha, G(alphai), suppressed CXCL8 levels in response to acidic pH; whereas phospholipase C inhibition had no effect on CXCL8. The use of specific mitogen-activated protein kinase (MAPK) signal transduction inhibitors indicated that the pH-dependent increase in CXCL8 mRNA is due to activation of ERK and p38 pathways. The JNK pathway was not involved. NF-kappaB inhibition resulted in a decrease in CXCL8 levels in hMSCs grown in non-OGM. However, OGM-differentiated hMSCs showed an increase in CXCL8 levels when treated with the NF-kappaB inhibitor PDTC, a pyrrolidine derivative of dithiocarbamate. 2008 Wiley-Liss, Inc.

[The mRNA expression of mitogen-activated protein kinase signal pathway related genes in the blood of arseniasis patients caused by burning coal].

Science.gov (United States)

Luo, Peng; Zhang, Ai-hua; Xiao, Yun; Pan, Xue-li; Dong, Xue-xin; Huang, Xiao-xin

2013-09-01

To detect the mRNA expression of ERK1, ERK2, JNK1 and P38 gene in mitogen-activated protein kinase(MAPK) path way in the arseniasis patients caused by burning coal. 70 arseniasis patients caused by burning coal at Jiaole village XingRen county in December 2006 were selected as case group, and another 30 villagers with similar living habits, matched gender and age, healthy physical condition without history of burning high arsenic coal were selected as control group from 12 km nearby the same village.Silver diethyl dithiocarbamate method (Ag-DDC) was taken to detect the arsenic contents in the environmental media, food, and arsenic level in the urine and hair of arseniasis patients.On the principle of informed consent, the peripheral blood was collected from the patients. The total RNA was extracted with Trizol method and cDNA was reversed from it. The mRNA expression of ERK1, ERK2, JNK1 and P38 gene in MAPK path way were tested by real-time fluorescent quantitative PCR (QT-PCR). A total of 70 cases of arseniasis patients (31 cases of mild, 25 cases of moderate and 14 cases of severe) and 30 cases of control were chosen. The median (quartile) of arsenic contents in the indoor air, outdoor air, coal, chili and corn were 0.079 (0.053-0.117) mg/m(3) ,0.007 (0.002-0.015) mg/m(3) , 93.010 (39.460-211.740) mg/kg, 3.460(0.550-16.760) mg/kg and 1.500(0.300-4.140) mg/kg respectively. They were above the national health standards. The median (quartile) of arsenic contents in the soil, rice and drinking water were separately 12.130(4.230-24.820) mg/kg, 0.650(0.300-0.980) mg/kg and 0.043(0.012-0.089)mg/kg, which were within the national health standards. Compared with the control group ((26.97 ± 9.71)µg/g Cr), arsenic level in the patients' urine ((71.48 ± 22.74)µg/g Cr) increased significantly, the differences were significant (F = 90.38, P coal.

An immunohistochemical study of retinol-binding protein (RBP) in livers of free-living polar bears (Ursus maritimus) from east Greenland.

Science.gov (United States)

Heier, Annabelle; Sonne, Christian; Gröne, Andrea; Leifsson, Pall S; Dietz, Rune; Born, Erik W; Bacciarini, Luca N

2005-09-01

From 1999 to 2002 samples from 114 free-ranging polar bears (Ursus maritimus) were collected in the municipality of Scoresby Sound, East Greenland, to detect levels of organochlorines and potential histopathologic changes. Livers of 16 female polar bears from this group were evaluated histologically and analyzed for hepatic retinol-binding protein by immunohistochemistry. Retinol-binding protein is the main transport protein for retinol, an important vitamin A metabolite in the polar bear. Only mild pathologic changes were noted on histologic evaluation of the livers. Small lymphocytic or lymphohistiocytic infiltrates were present in all the livers. Small lipid granulomas, mild periportal fibrosis, and bile duct proliferation were found in several cases. Immunohistochemistry for retinol-binding protein of hepatic tissue from free-ranging polar bears showed no distinct difference in staining intensity by a number of criteria: age, season (fasting and nonfasting), or lactation status. The staining was diffuse to finely stippled in the cytoplasm and showed very little variation among the animals. Because of the lack of macroscopic changes and the absence of severe histologic liver lesions, these polar bears were assumed to be healthy. The diffuse cytoplasmic retinol-binding protein staining in hepatocytes of free-ranging polar bears varies markedly from the prominent granular, less intense staining of captive polar bears investigated previously.

Runx expression is mitogenic and mutually linked to Wnt activity in blastula-stage sea urchin embryos.

Directory of Open Access Journals (Sweden)

Anthony J Robertson

Full Text Available The Runt homology domain (Runx defines a metazoan family of sequence-specific transcriptional regulatory proteins that are critical for animal development and causally associated with a variety of mammalian cancers. The sea urchin Runx gene SpRunt-1 is expressed throughout the blastula stage embryo, and is required globally during embryogenesis for cell survival and differentiation.Depletion of SpRunt-1 by morpholino antisense-mediated knockdown causes a blastula stage deficit in cell proliferation, as shown by bromodeoxyuridine (BrdU incorporation and direct cell counts. Reverse transcription coupled polymerase chain reaction (RT-PCR studies show that the cell proliferation deficit is presaged by a deficit in the expression of several zygotic wnt genes, including wnt8, a key regulator of endomesoderm development. In addition, SpRunt-1-depleted blastulae underexpress cyclinD, an effector of mitogenic Wnt signaling. Blastula stage cell proliferation is also impeded by knockdown of either wnt8 or cyclinD. Chromatin immunoprecipitation (ChIP indicates that Runx target sites within 5' sequences flanking cyclinD, wnt6 and wnt8 are directly bound by SpRunt-1 protein at late blastula stage. Furthermore, experiments using a green fluorescent protein (GFP reporter transgene show that the blastula-stage operation of a cis-regulatory module previously shown to be required for wnt8 expression (Minokawa et al., Dev. Biol. 288: 545-558, 2005 is dependent on its direct sequence-specific interaction with SpRunt-1. Finally, inhibitor studies and immunoblot analysis show that SpRunt-1 protein levels are negatively regulated by glycogen synthase kinase (GSK-3.These results suggest that Runx expression and Wnt signaling are mutually linked in a feedback circuit that controls cell proliferation during development.

Non-invasive detection of candidate pregnancy protein biomarkers in the feces of captive polar bears (Ursus maritimus).

Science.gov (United States)

Curry, E; Stoops, M A; Roth, T L

2012-07-15

Currently, there is no method of accurately and non-invasively diagnosing pregnancy in polar bears. Specific proteins may exhibit altered profiles in the feces of pregnant bears, but predicting appropriate candidate proteins to investigate is speculative at best. The objective of this study was to identify potential pregnancy biomarker proteins based on their increased abundance in the feces of pregnant polar bears compared to pseudopregnant females (controls) using two-dimensional in-gel electrophoresis (2D-DIGE) and mass spectrometry (MS). Three 2D-DIGE gels were performed to evaluate fecal protein profiles from controls (n=3) and pregnant polar bears (n=3). There were 2224.67±52.39 (mean±SEM) spots resolved per gel. Of these, only five proteins were elevated in the pregnant group (P99.9% confidence interval. The 11 spots represented seven distinct proteins, five of which were significantly more abundant in the pregnant group: IgGFc-binding protein, filamin-C, carboxypeptidase B, transthyretin, and immunoglobulin heavy chain variable region. To our knowledge, this was the first study that employed 2D-DIGE to identify differentially expressed proteins in fecal samples to characterize a physiological condition other than those related to gastrointestinal disorders. These promising results provided a strong foundation for ensuing efforts to develop a non-invasive pregnancy assay for use in both captive and wild polar bears. Copyright © 2012 Elsevier Inc. All rights reserved.

Mitogen activated protein kinase kinase kinase 3 (MAP3K3/MEKK3) overexpression is an early event in esophageal tumorigenesis and is a predictor of poor disease prognosis

International Nuclear Information System (INIS)

Hasan, Raghibul; Sharma, Rinu; Saraya, Anoop; Chattopadhyay, Tushar K; DattaGupta, Siddartha; Walfish, Paul G; Chauhan, Shyam S; Ralhan, Ranju

2014-01-01

Mitogen-activated protein kinase kinase kinase3 (MAP3K3/MEKK3) was identified to be differentially expressed in esophageal squamous cell carcinoma (ESCC) using cDNA microarrays by our laboratory. Here in we determined the clinical significance of MEKK3 in ESCC. Immunohistochemical analysis of MEKK3 expression was carried out in archived tissue sections from 93 ESCCs, 47 histologically normal and 61 dysplastic esophageal tissues and correlated with clinicopathological parameters and disease prognosis over up to 7.5 years for ESCC patients. MEKK3 expression was significantly increased in esophageal dysplasia and ESCC in comparison with normal mucosa (p trend < 0.001). Kaplan Meier survival analysis showed significantly reduced median disease free survival median DFS = 10 months in patients with MEKK3 positive ESCCs compared to patients with no immunopositivity (median DFS = 19 months, p = 0.04). ESCC patients with MEKK3 positive and lymph node positive tumors had median DFS = 9 months, as compared to median DFS = 21 months in patients who did not show the alterations (p = 0.01). In multivariate Cox regression analysis, combination of MEKK3 overexpression and node positivity [p = 0.015, hazard ratio (HR) = 2.082, 95% CI = 1.154 - 3.756] emerged as important predictor of reduced disease free survival and poor prognosticator for ESCC patients. Alterations in MEKK3 expression occur in early stages of development of ESCC and are sustained during disease progression; MEKK3 in combination with lymph node positivity has the potential to serve as adverse prognosticator in ESCC

AMP-activated protein kinase activation mediates CCL3-induced cell migration and matrix metalloproteinase-2 expression in human chondrosarcoma

Science.gov (United States)

2013-01-01

Chemokine (C-C motif) ligand 3 (CCL3), also known as macrophage inflammatory protein-1α, is a cytokine involved in inflammation and activation of polymorphonuclear leukocytes. CCL3 has been detected in infiltrating cells and tumor cells. Chondrosarcoma is a highly malignant tumor that causes distant metastasis. However, the effect of CCL3 on human chondrosarcoma metastasis is still unknown. Here, we found that CCL3 increased cellular migration and expression of matrix metalloproteinase (MMP)-2 in human chondrosarcoma cells. Pre-treatment of cells with the MMP-2 inhibitor or transfection with MMP-2 specific siRNA abolished CCL3-induced cell migration. CCL3 has been reported to exert its effects through activation of its specific receptor, CC chemokine receptor 5 (CCR5). The CCR5 and AMP-activated protein kinase (AMPK) inhibitor or siRNA also attenuated CCL3-upregulated cell motility and MMP-2 expression. CCL3-induced expression of MMP-2 and migration were also inhibited by specific inhibitors, and inactive mutants of AMPK, p38 mitogen activated protein kinase (p38 or p38-MAPK), and nuclear factor κB (NF-κB) cascades. On the other hand, CCL3 treatment demonstrably activated AMPK, p38, and NF-κB signaling pathways. Furthermore, the expression levels of CCL3, CCR5, and MMP-2 were correlated in human chondrosarcoma specimens. Taken together, our results indicate that CCL3 enhances the migratory ability of human chondrosarcoma cells by increasing MMP-2 expression via the CCR5, AMPK, p38, and NF-κB pathways. PMID:24047437

Polar transport in plants mediated by membrane transporters: focus on mechanisms of polar auxin transport.

Science.gov (United States)

Naramoto, Satoshi

2017-12-01

Directional cell-to-cell transport of functional molecules, called polar transport, enables plants to sense and respond to developmental and environmental signals. Transporters that localize to plasma membranes (PMs) in a polar manner are key components of these systems. PIN-FORMED (PIN) auxin efflux carriers, which are the most studied polar-localized PM proteins, are implicated in the polar transport of auxin that in turn regulates plant development and tropic growth. In this review, the regulatory mechanisms underlying polar localization of PINs, control of auxin efflux activity, and PIN abundance at PMs are considered. Up to date information on polar-localized nutrient transporters that regulate directional nutrient movement from soil into the root vasculature is also discussed. Copyright © 2017 Elsevier Ltd. All rights reserved.

Planar cell polarity proteins differentially regulate extracellular matrix organization and assembly during zebrafish gastrulation.

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Dohn, Michael R; Mundell, Nathan A; Sawyer, Leah M; Dunlap, Julie A; Jessen, Jason R

2013-11-01

Zebrafish gastrulation cell movements occur in the context of dynamic changes in extracellular matrix (ECM) organization and require the concerted action of planar cell polarity (PCP) proteins that regulate cell elongation and mediolateral alignment. Data obtained using Xenopus laevis gastrulae have shown that integrin-fibronectin interactions underlie the formation of polarized cell protrusions necessary for PCP and have implicated PCP proteins themselves as regulators of ECM. By contrast, the relationship between establishment of PCP and ECM assembly/remodeling during zebrafish gastrulation is unclear. We previously showed that zebrafish embryos carrying a null mutation in the four-pass transmembrane PCP protein vang-like 2 (vangl2) exhibit increased matrix metalloproteinase activity and decreased immunolabeling of fibronectin. These data implicated for the first time a core PCP protein in the regulation of pericellular proteolysis of ECM substrates and raised the question of whether other zebrafish PCP proteins also impact ECM organization. In Drosophila melanogaster, the cytoplasmic PCP protein Prickle binds Van Gogh and regulates its function. Here we report that similar to vangl2, loss of zebrafish prickle1a decreases fibronectin protein levels in gastrula embryos. We further show that Prickle1a physically binds Vangl2 and regulates both the subcellular distribution and total protein level of Vangl2. These data suggest that the ability of Prickle1a to impact fibronectin organization is at least partly due to effects on Vangl2. In contrast to loss of either Vangl2 or Prickle1a function, we find that glypican4 (a Wnt co-receptor) and frizzled7 mutant gastrula embryos with disrupted non-canonical Wnt signaling exhibit the opposite phenotype, namely increased fibronectin assembly. Our data show that glypican4 mutants do not have decreased proteolysis of ECM substrates, but instead have increased cell surface cadherin protein expression and increased intercellular

Probing intracellular motor protein activity using an inducible cargo trafficking assay.

Science.gov (United States)

Kapitein, Lukas C; Schlager, Max A; van der Zwan, Wouter A; Wulf, Phebe S; Keijzer, Nanda; Hoogenraad, Casper C

2010-10-06

Although purified cytoskeletal motor proteins have been studied extensively with the use of in vitro approaches, a generic approach to selectively probe actin and microtubule-based motor protein activity inside living cells is lacking. To examine specific motor activity inside living cells, we utilized the FKBP-rapalog-FRB heterodimerization system to develop an in vivo peroxisomal trafficking assay that allows inducible recruitment of exogenous and endogenous kinesin, dynein, and myosin motors to drive specific cargo transport. We demonstrate that cargo rapidly redistributes with distinct dynamics for each respective motor, and that combined (antagonistic) actions of more complex motor combinations can also be probed. Of importance, robust cargo redistribution is readily achieved by one type of motor protein and does not require the presence of opposite-polarity motors. Simultaneous live-cell imaging of microtubules and kinesin or dynein-propelled peroxisomes, combined with high-resolution particle tracking, revealed that peroxisomes frequently pause at microtubule intersections. Titration and washout experiments furthermore revealed that motor recruitment by rapalog-induced heterodimerization is dose-dependent but irreversible. Our assay directly demonstrates that robust cargo motility does not require the presence of opposite-polarity motors, and can therefore be used to characterize the motile properties of specific types of motor proteins. Copyright © 2010 Biophysical Society. Published by Elsevier Inc. All rights reserved.

Hippocampal mitogen-activated protein kinase activation is associated with intermittent hypoxia in a rat model of obstructive sleep apnea syndrome.

Science.gov (United States)

Zhao, Ya-Ning; Wang, Hong-Yang; Li, Jian-Min; Chen, Bao-Yuan; Xia, Guo; Zhang, Pan-Pan; Ge, Yan-Lei

2016-01-01

Obstructive sleep apnea syndrome (OSAS), characterized by intermittent hypoxia/re‑oxygenation, may impair the cerebral system. Although mitogen‑activated protein kinase (MAPK) signaling was observed to have a key role in hypoxia‑induced brain injury, the intracellular events and their underlying mechanisms for intermittent hypoxia/re‑oxygenation-associated damage to hippocamal MAPKs, including extracellular signal‑regulated kinase (ERK)1/2, P38MAPK and c‑Jun N‑terminal kinase (JNK) remain to be elucidated and require further investigation. A total of five rats in each sub‑group were exposed to intermittent hypoxia or continued hypoxia for 2, 4, 6 or 8 weeks. Histological, immunohistochemical and biological analyses were performed to assess nerve cell injury in the hippocampus. Surviving CA1 pyramidal cells were identified by hematoxylin and eosin staining. The levels of phosphorylated ERK1/2, P38MAPK and JNK were detected by western blotting. B‑cell lymphoma 2 (Bcl‑2) and Bcl‑2‑associated X protein (Bax) in neural cells were examined by immunohistochemistry. The malondialdehyde (MDA) contents and superoxide dismutase (SOD) activities were measured by thiobarbituric acid and xanthine oxidation methods, respectively. Under continued hypoxia, the levels of phospho‑ERK1/2 peaked at the fourth week and then declined, whereas phospho‑P38MAPK and JNK were detected only in the late stages. By contrast, under intermittent hypoxia, ERK1/2, P38MAPK and JNK were activated at all time-points assessed (2, 4, 6 and 8 weeks). The levels of phospho‑ERK1/2, P38MAPK and JNK were all higher in the intermittent hypoxia groups than those in the corresponding continued hypoxia groups. Bcl‑2 was mainly increased and reached the highest level at six weeks in the continued hypoxia group. Of note, Bcl‑2 rapidly increased to the peak level at four weeks, followed by a decrease to the lowest level at the eighth week in the intermittent hypoxia group. Bax was

A Nitrogen Response Pathway Regulates Virulence Functions in Fusarium oxysporum via the Protein Kinase TOR and the bZIP Protein MeaB

OpenAIRE

López-Berges, Manuel S.; Rispail, Nicolas; Prados-Rosales, Rafael C.; Pietro, Antonio D.

2010-01-01

During infection, fungal pathogens activate virulence mechanisms, such as host adhesion, penetration and invasive growth. In the vascular wilt fungus Fusarium oxysporum, the mitogen-activated protein kinase Fmk1 is required for plant infection and controls processes such as cellophane penetration, vegetative hyphal fusion, or root adhesion. Here, we show that these virulence-related functions are repressed by the preferred nitrogen source ammonium and restored by treatment with l-methionine s...

Active polarization imaging system based on optical heterodyne balanced receiver

Science.gov (United States)

Xu, Qian; Sun, Jianfeng; Lu, Zhiyong; Zhou, Yu; Luan, Zhu; Hou, Peipei; Liu, liren

2017-08-01

Active polarization imaging technology has recently become the hot research field all over the world, which has great potential application value in the military and civil area. By introducing active light source, the Mueller matrix of the target can be calculated according to the incident light and the emitted or reflected light. Compared with conventional direct detection technology, optical heterodyne detection technology have higher receiving sensitivities, which can obtain the whole amplitude, frequency and phase information of the signal light. In this paper, an active polarization imaging system will be designed. Based on optical heterodyne balanced receiver, the system can acquire the horizontal and vertical polarization of reflected optical field simultaneously, which contain the polarization characteristic of the target. Besides, signal to noise ratio and imaging distance can be greatly improved.

Role of Bioavailable Iron in Coal Dust-Induced Activation of Activator Protein-1 and Nuclear Factor of Activated T Cells

Science.gov (United States)

Huang, Chuanshu; Li, Jingxia; Zhang, Qi; Huang, Xi

2010-01-01

Activator protein-1 (AP-1) and nuclear factor of activated T cells (NFAT) are two important transcription factors responsible for the regulation of cytokines, which are involved in cell proliferation and inflammation. Coal workers’ pneumoconiosis (CWP) is an occupational lung disease that may be related to chronic inflammation caused by coal dust exposure. In the present study, we demonstrate that coal from the Pennsylvania (PA) coalmine region, which has a high prevalence of CWP, can activate both AP-1 and NFAT in JB6 mouse epidermal cells. In contrast, coal from the Utah (UT) coalmine region, which has a low prevalence of CWP, has no such effects. The PA coal stimulates mitogen-activated protein kinase (MAPK) family members of extracellular signal-regulated kinases (ERKs) and p38 MAPK but not c-Jun-NH2-terminal kinases, as determined by the phosphorylation assay. The increase in AP-1 by the PA coal was completely eliminated by the pretreatment of cells with PD98059, a specific MAPK kinase inhibitor, and SB202190, a p38 kinase inhibitor, further confirming that the PA coal-induced AP-1 activation is mediated through ERKs and p38 MAPK pathways. Deferoxamine (DFO), an iron chelator, synergistically enhanced the PA coal-induced AP-1 activity, but inhibited NFAT activity. For comparison, cells were treated with ferrous sulfate and/or DFO. We have found that iron transactivated both AP-1 and NFAT, and DFO further enhanced iron-induced AP-1 activation but inhibited NFAT. These results indicate that activation of AP-1 and NFAT by the PA coal is through bioavailable iron present in the coal. These data are in agreement with our previous findings that the prevalence of CWP correlates well with levels of bioavailable iron in coals from various mining regions. PMID:12397016

Mitogenic stimuli and phosphatidylinositol (PI) turnover in cultured 3T3 fibroblasts

International Nuclear Information System (INIS)

Kohler, C.; Petersen, R.

1986-01-01

The hydrolysis of PI and polyphosphoinositides by phopholipase C is an early and rapid response to cell activation by a variety of neurotransmitters, hormones, growth factors and pharmacological agonists. The authors have examined the role of PI turnover and the generation of second messengers (diacylglycerol and inositol trisphosphate) in the mitogenic response of cultured Balb/c and Swiss 3T3 cells to polypeptide growth factors. Cells were prelabelled with 3 H inositol for 18-20 hours, washed and suspended in Herpes + Li + buffer, and stimulated with platelet-derived growth factor (PDGF), vasopressin, insulin, and other growth factors. PI turnover was measured as the increase in total inositol phosphate (IP) production. IP1, IP2, and IP3 were characterized by sequential elution from a Dowex column. Partially purified PDGF produced a 2-4 fold stimulation of total IP production. This was seen as early as 30 seconds after stimulation and increased for up to 1-2 hours. Balb/c cells were more sensitive than Swiss cells to the mitogenic and PI effects of PDGF. Other mitogenic stimuli had differential effects on PI turnover. Vasopressin (4-400 ng/ml) markedly stimulated PI turnover (3-6 fold) in Swiss, but not Balb/c cells. Insulin (100 ng/ml - 10 μg/ml) increased total IP to a greater degree in Balb/c cells. Epidermal growth factor (10 ng/ml - 10 μg/ml) had no effect on PI turnover and fibroblast growth factor (10 ng/ml - 10 μg/ml) only stimulated at the higher concentrations in Swiss cells. Thrombin (1U/ml - 10 U/ml) produced a 1.5 - 2 fold stimulation in Balb/c cells. Thus, various polypeptide growth factors have differential effects on PI turnover depending on their mitogenic potential and the effector cell type

Active Polar Two-Fluid Macroscopic Dynamics

Science.gov (United States)

Pleiner, Harald; Svensek, Daniel; Brand, Helmut R.

2014-03-01

We study the dynamics of systems with a polar dynamic preferred direction. Examples include the pattern-forming growth of bacteria (in a solvent, shoals of fish (moving in water currents), flocks of birds and migrating insects (flying in windy air). Because the preferred direction only exists dynamically, but not statically, the macroscopic variable of choice is the macroscopic velocity associated with the motion of the active units. We derive the macroscopic equations for such a system and discuss novel static, reversible and irreversible cross-couplings connected to this second velocity. We find a normal mode structure quite different compared to the static descriptions, as well as linear couplings between (active) flow and e.g. densities and concentrations due to the genuine two-fluid transport derivatives. On the other hand, we get, quite similar to the static case, a direct linear relation between the stress tensor and the structure tensor. This prominent ``active'' term is responsible for many active effects, meaning that our approach can describe those effects as well. In addition, we also deal with explicitly chiral systems, which are important for many active systems. In particular, we find an active flow-induced heat current specific for the dynamic chiral polar order.

Polarized excitons and optical activity in single-wall carbon nanotubes

Science.gov (United States)

Chang, Yao-Wen; Jin, Bih-Yaw

2018-05-01

The polarized excitons and optical activity of single-wall carbon nanotubes (SWNTs) are studied theoretically by π -electron Hamiltonian and helical-rotational symmetry. By taking advantage of the symmetrization, the single-particle energy and properties of a SWNT are characterized with the corresponding helical band structure. The dipole-moment matrix elements, magnetic-moment matrix elements, and the selection rules can also be derived. Based on different selection rules, the optical transitions can be assigned as the parallel-polarized, left-handed circularly-polarized, and right-handed circularly-polarized transitions, where the combination of the last two gives the cross-polarized transition. The absorption and circular dichroism (CD) spectra are simulated by exciton calculation. The calculated results are well comparable with the reported measurements. Built on the foundation, magnetic-field effects on the polarized excitons and optical activity of SWNTs are studied. Dark-bright exciton splitting and interband Faraday effect in the CD spectrum of SWNTs under an axial magnetic field are predicted. The Faraday rotation dispersion can be analyzed according to the selection rules of circular polarizations and the helical band structure.

Analysis of the Yeast Kinome Reveals a Network of Regulated Protein Localization during Filamentous Growth

OpenAIRE

Bharucha, Nikë; Ma, Jun; Dobry, Craig J.; Lawson, Sarah K.; Yang, Zhifen; Kumar, Anuj

2008-01-01

The subcellular distribution of kinases and other signaling proteins is regulated in response to cellular cues; however, the extent of this regulation has not been investigated for any gene set in any organism. Here, we present a systematic analysis of protein kinases in the budding yeast, screening for differential localization during filamentous growth. Filamentous growth is an important stress response involving mitogen-activated protein kinase and cAMP-dependent protein kinase signaling m...

Comparison of selected immunological parameters in silver and polar foxes

International Nuclear Information System (INIS)

Kostro, K.; Woloszyn, S.

1996-01-01

The aim of the work was to compare selected nonspecific immunological parameters in silver and polar foxes. It was found that out of three mitogens (Con A, La and PWM) only Con A stimulated the strongest lymphocyte proliferation in both species of foxes. Mean stimulation indices and the absolute values of incorporated thymidine were significantly higher in polar foxes. The average percentages of phagocytes and the fatal indices of neutrophils were comparable in both species of foxes, while the mean value of the phagocyte index was much higher in the silver fox. However, in polar foxes a higher concentration of lysozyme in the blood serum was found. The differences in the parameters may influence the susceptibility of these foxes to some infections already at the level of nonspecific primary response. (author)

Planar Cell Polarity Breaks the Symmetry of PAR Protein Distribution prior to Mitosis in Drosophila Sensory Organ Precursor Cells.

Science.gov (United States)

Besson, Charlotte; Bernard, Fred; Corson, Francis; Rouault, Hervé; Reynaud, Elodie; Keder, Alyona; Mazouni, Khalil; Schweisguth, François

2015-04-20

During development, cell-fate diversity can result from the unequal segregation of fate determinants at mitosis. Polarization of the mother cell is essential for asymmetric cell division (ACD). It often involves the formation of a cortical domain containing the PAR complex proteins Par3, Par6, and atypical protein kinase C (aPKC). In the fly notum, sensory organ precursor cells (SOPs) divide asymmetrically within the plane of the epithelium and along the body axis to generate two distinct cells. Fate asymmetry depends on the asymmetric localization of the PAR complex. In the absence of planar cell polarity (PCP), SOPs divide with a random planar orientation but still asymmetrically, showing that PCP is dispensable for PAR asymmetry at mitosis. To study when and how the PAR complex localizes asymmetrically, we have used a quantitative imaging approach to measure the planar polarization of the proteins Bazooka (Baz, fly Par3), Par6, and aPKC in living pupae. By using imaging of functional GFP-tagged proteins with image processing and computational modeling, we find that Baz, Par6, and aPKC become planar polarized prior to mitosis in a manner independent of the AuroraA kinase and that PCP is required for the planar polarization of Baz, Par6, and aPKC during interphase. This indicates that a "mitosis rescue" mechanism establishes asymmetry at mitosis in PCP mutants. This study therefore identifies PCP as the initial symmetry-breaking signal for the planar polarization of PAR proteins in asymmetrically dividing SOPs. Copyright © 2015 Elsevier Ltd. All rights reserved.

Immune Enhancing Activity of β-(1,3)-Glucan Isolated from Genus Agrobacterium in Bone-Marrow Derived Macrophages and Mice Splenocytes.

Science.gov (United States)

Byun, Eui-Baek; Jang, Beom-Su; Byun, Eui-Hong; Sung, Nak-Yun

2016-01-01

An effective method for activating macrophages and deriving a Th1 immune response could be used to improve the defenses of hosts. In this study, we investigated the immunomodulation effect and the related signaling mechanism of [Formula: see text]-(1,3)-glucan, isolated from the Agrobacterium species. Here, we found that [Formula: see text]-(1,3)-glucan predominantly induced the tumor necrosis factor (TNF)-[Formula: see text], interleukin (IL)-1[Formula: see text], IL-6, IL-12p70, and nitric oxide, which was dependent on mitogen-activated protein kinases (MAPK) and nuclear factor (NF)-[Formula: see text]B signaling. Additionally, [Formula: see text]-(1,3)-glucan treatment significantly up-regulated the expression of the co-stimulatory molecules CD80 and CD86, and also significantly increased the expression of iNOS and Dectin-1, which is a transmembrane protein that binds [Formula: see text]-glucan and associates with macrophage activation. Importantly, the splenic T cells co-cultured with [Formula: see text]-(1,3)-glucan-treated macrophages produced the a Th1 cytokine profile that includes high levels of IFN-[Formula: see text], but not IL-4 (Th2 cytokine), indicating that [Formula: see text]-(1,3)-glucan contributes to Th1 polarization of the immune response. Taken together, our results suggest that [Formula: see text]-(1,3)-glucan isolated from Agrobacterium species can induce macrophage activation through the MAPK and NF-[Formula: see text]B signaling pathway, as well as Th1 polarization.

Aldose Reductase Inhibitor Protects against Hyperglycemic Stress by Activating Nrf2-Dependent Antioxidant Proteins.

Science.gov (United States)

Shukla, Kirtikar; Pal, Pabitra Bikash; Sonowal, Himangshu; Srivastava, Satish K; Ramana, Kota V

2017-01-01

We have shown earlier that pretreatment of cultured cells with aldose reductase (AR) inhibitors prevents hyperglycemia-induced mitogenic and proinflammatory responses. However, the effects of AR inhibitors on Nrf2-mediated anti-inflammatory responses have not been elucidated yet. We have investigated how AR inhibitor fidarestat protects high glucose- (HG-) induced cell viability changes by increasing the expression of Nrf2 and its dependent phase II antioxidant enzymes. Fidarestat pretreatment prevents HG (25 mM)-induced Thp1 monocyte viability. Further, treatment of Thp1 monocytes with fidarestat caused a time-dependent increase in the expression as well as the DNA-binding activity of Nrf2. In addition, fidarestat augmented the HG-induced Nrf2 expression and activity and also upregulated the expression of Nrf2-dependent proteins such as hemeoxygenase-1 (HO1) and NQO1 in Thp1 cells. Similarly, treatment with AR inhibitor also induced the expression of Nrf2 and HO1 in STZ-induced diabetic mice heart and kidney tissues. Further, AR inhibition increased the HG-induced expression of antioxidant enzymes such as SOD and catalase and activation of AMPK- α 1 in Thp1 cells. Our results thus suggest that pretreatment with AR inhibitor prepares the monocytes against hyperglycemic stress by overexpressing the Nrf2-dependent antioxidative proteins.

Enhanced Expression of WD Repeat-Containing Protein 35 via Nuclear Factor-Kappa B Activation in Bupivacaine-Treated Neuro2a Cells

Science.gov (United States)

Huang, Lei; Kondo, Fumio; Harato, Misako; Feng, Guo-Gang; Ishikawa, Naoshisa; Fujiwara, Yoshihiro; Okada, Shoshiro

2014-01-01

The family of WD repeat proteins comprises a large number of proteins and is involved in a wide variety of cellular processes such as signal transduction, cell growth, proliferation, and apoptosis. Bupivacaine is a sodium channel blocker administered for local infiltration, nerve block, epidural, and intrathecal anesthesia. Recently, we reported that bupivacaine induces reactive oxygen species (ROS) generation and p38 mitogen-activated protein kinase (MAPK) activation, resulting in an increase in the expression of WD repeat-containing protein 35 (WDR35) in mouse neuroblastoma Neuro2a cells. It has been shown that ROS activate MAPK through phosphorylation, followed by activation of nuclear factor-kappa B (NF-κB) and activator protein 1 (AP-1). The present study was undertaken to test whether NF-κB and c-Jun/AP-1 are involved in bupivacaine-induced WDR35 expression in Neuro2a cells. Bupivacaine activated both NF-κB and c-Jun in Neuro2a cells. APDC, an NF-κB inhibitor, attenuated the increase in NF-κB activity and WDR35 protein expression in bupivacaine-treated Neuro2a cells. GW9662, a selective peroxisome proliferator-activated receptor-γ antagonist, enhanced the increase in NF-κB activity and WDR35 protein expression in bupivacaine-treated Neuro2a cells. In contrast, c-Jun siRNA did not inhibit the bupivacaine-induced increase in WDR35 mRNA expression. These results indicate that bupivacaine induces the activation of transcription factors NF-κB and c-Jun/AP-1 in Neuro2a cells, while activation of NF-κB is involved in bupivacaine-induced increases in WDR35 expression. PMID:24466034

Autotaxin: A protein with two faces

Energy Technology Data Exchange (ETDEWEB)

Tania, Mousumi; Khan, Md. Asaduzzaman; Zhang, Huaiyuan; Li, Jinhua [Department of Biochemistry, School of Biological Science and Technology, Central South University, Changsha, Hunan 410013 (China); Song, Yuanda, E-mail: yuanda_song@hotmail.com [Department of Biochemistry, School of Biological Science and Technology, Central South University, Changsha, Hunan 410013 (China)

2010-10-29

Research highlights: {yields} Autotaxin (ATX) has lysophospholipase D activity. {yields} ATX catalyzes the formation of lysophosphatidic acid (LPA). {yields} LPA is a mitogen, and thus is responsible for cancer. {yields} ATX also catalyzes the formation of anti-cancerous cyclic phosphatidic acid. {yields} Autotaxin is a novel target of cancer therapy research. -- Abstract: Autotaxin (ATX) is a catalytic protein, which possesses lysophospholipase D activity, and thus involved in cellular membrane lipid metabolism and remodeling. Primarily, ATX was thought as a culprit protein for cancer, which potently stimulates cancer cell proliferation and tumor cell motility, augments the tumorigenicity and induces angiogenic responses. The product of ATX catalyzed reaction, lysophosphatidic acid (LPA) is a potent mitogen, which facilitates cell proliferation and migration, neurite retraction, platelet aggregation, smooth muscle contraction, actin stress formation and cytokine and chemokine secretion. In addition to LPA formation, later ATX has been found to catalyze the formation of cyclic phosphatidic acid (cPA), which have antitumor role by antimitogenic regulation of cell cycle, inhibition of cancer invasion and metastasis. Furthermore, the very attractive information to the scientists is that the LPA/cPA formation can be altered at different physiological conditions. Thus the dual role of ATX with the scope of product manipulation has made ATX a novel target for cancer treatment.

Nicotine as a mitogenic stimulus for pancreatic acinar cell proliferation

Institute of Scientific and Technical Information of China (English)

Parimal Chowdhury; Kodetthoor B Udupa

2006-01-01

Cell proliferation is an important process in life for growth of normal and cancer cells. The signal transduction pathways activated during this process are strictly regulated. This editorial focuses on the role of nicotine,a mitogen, in the induction of signaling pathways resulting in proliferation of pancreatic tumor cells and compares these events with those in normal acinar cells isolated from the rat pancreas. The data shows striking similarities between these two cellular systems.In addition, the editorial reviews very recent literature of the contribution of MAPK signaling in cell lines associated with human diseases. A prospective cellular model of nicotine induced activation of MAPK cascade is presented.

SOcK, MiSTs, MASK and STicKs: the GCKIII (germinal centre kinase III) kinases and their heterologous protein-protein interactions.

Science.gov (United States)

Sugden, Peter H; McGuffin, Liam J; Clerk, Angela

2013-08-15

The GCKIII (germinal centre kinase III) subfamily of the mammalian Ste20 (sterile 20)-like group of serine/threonine protein kinases comprises SOK1 (Ste20-like/oxidant-stress-response kinase 1), MST3 (mammalian Ste20-like kinase 3) and MST4. Initially, GCKIIIs were considered in the contexts of the regulation of mitogen-activated protein kinase cascades and apoptosis. More recently, their participation in multiprotein heterocomplexes has become apparent. In the present review, we discuss the structure and phosphorylation of GCKIIIs and then focus on their interactions with other proteins. GCKIIIs possess a highly-conserved, structured catalytic domain at the N-terminus and a less-well conserved C-terminal regulatory domain. GCKIIIs are activated by tonic autophosphorylation of a T-loop threonine residue and their phosphorylation is regulated primarily through protein serine/threonine phosphatases [especially PP2A (protein phosphatase 2A)]. The GCKIII regulatory domains are highly disorganized, but can interact with more structured proteins, particularly the CCM3 (cerebral cavernous malformation 3)/PDCD10 (programmed cell death 10) protein. We explore the role(s) of GCKIIIs (and CCM3/PDCD10) in STRIPAK (striatin-interacting phosphatase and kinase) complexes and their association with the cis-Golgi protein GOLGA2 (golgin A2; GM130). Recently, an interaction of GCKIIIs with MO25 has been identified. This exhibits similarities to the STRADα (STE20-related kinase adaptor α)-MO25 interaction (as in the LKB1-STRADα-MO25 heterotrimer) and, at least for MST3, the interaction may be enhanced by cis-autophosphorylation of its regulatory domain. In these various heterocomplexes, GCKIIIs associate with the Golgi apparatus, the centrosome and the nucleus, as well as with focal adhesions and cell junctions, and are probably involved in cell migration, polarity and proliferation. Finally, we consider the association of GCKIIIs with a number of human diseases, particularly

Rachycentron canadum (cobia) lectin promoted mitogenic response in mice BALB/c splenocytes.

Science.gov (United States)

Coriolano, M C; de Melo, C M L; Santos, A J G; Pereira, V R A; Coelho, L C B B

2012-12-01

The mitogenic lectins are invaluable tools to study the biochemical changes associated with lymphocyte activation and proliferation of various immune cells. Rachycentron canadum lectin (RcaL) was detected and purified from serum of cobia fish. The aim of this study was to evaluate the proliferative response and cytokine production in splenocytes of mice in vitro stimulated with RcaL lectin; Canavalia ensiformis lectin (Con A) was used as positive control. A high proliferation index was induced by RcaL in relation to control cells. Furthermore, RcaL induced higher IL-2 and IL-6 production in relation to control. The cell viability was 90% in splenocytes treated with RcaL lectin, but RcaL promoted significant late apoptosis after 24 and 48 h in relation to control. RcaL induced proliferative responses suggesting that this lectin can be used as a mitogenic agent in immunostimulatory assays. © 2012 The Authors. Scandinavian Journal of Immunology © 2012 Blackwell Publishing Ltd.

Mitogenic activity of a water-soluble adjuvant (Bu-WSA) obtained from Bacterionema matruchotii. IV. Synergistic effects of Bu-WSA on Concanavalin A-induced proliferative response of human peripheral blood lymphocytes.

Science.gov (United States)

Nitta, T; Okumura, S; Tsushi, M; Nakano, M

1982-01-01

Butanol-extracted water-soluble adjuvant (Bu-WSA) obtained from Bacterionema matruchotii was cultured with peripheral blood mononuclear cells (PBM) in the presence of sub- and/or supra-optimal mitogenic concentrations of concanavalin A (Con A). The addition of Bu-WSA resulted in increased tritiated thymidine incorporation above that produced by Con A alone. Bu-WSA by itself is not mitogenic for PBM and in fact produced a decrease in thymidine uptake compared to the control. We investigated the response of subpopulation(s) of PBM to Bu-WSA, Con A and a mixture of Bu-WSA and Con A. Separation of PBM into purified T cells, B cells and macrophages showed that cell-cell cooperation of T cells with B cells or macrophages is necessary for the observed synergistic effect of Bu-WSA with Con A. A marked increase in thymidine incorporation by the mixture of T and B cell populations occurred, while only a small amount of thymidine was incorporated when the B cell population was absent. Mitomycin treatment revealed that the response could be ascribed to the T-cell response with a B-cell helper effect. Moreover, Con A and Bu-WSA appeared to act on the same T cell population. This model may provide unique information about the activation of human peripheral blood T cells compared with the activation of these cells by other mitogens.

A novel role for inhibitor of apoptosis (IAP) proteins as regulators of endothelial barrier function by mediating RhoA activation.

Science.gov (United States)

Hornburger, Michael C; Mayer, Bettina A; Leonhardt, Stefanie; Willer, Elisabeth A; Zahler, Stefan; Beyerle, Andrea; Rajalingam, Krishnaraj; Vollmar, Angelika M; Fürst, Robert

2014-04-01

Inhibitor of apoptosis (IAP) proteins, such as XIAP or cIAP1/2, are important regulators of apoptosis in cancer cells, and IAP antagonists are currently evaluated as antitumor agents. Beyond their function in cancer cells, this study demonstrates a novel role of IAPs as regulators of vascular endothelial permeability. Two structurally different IAP antagonists, ABT and Smac085, as well as silencing of IAPs, reduced the thrombin receptor-activating peptide (TRAP)-induced barrier dysfunction in human endothelial cells as assessed by measuring macromolecular permeability or transendothelial electrical resistance. ABT diminished thrombin-evoked stress fiber formation, activation of myosin light chain 2, and disassembly of adherens junctions independent of calcium signaling, protein kinase C, and mitogen-activated protein kinases. Interestingly, ABT and silencing of IAPs, in particular XIAP, reduced the TRAP-evoked RhoA activation, whereas Rac1 was not affected. XIAP and, to a lesser extent, cIAP1 were found to directly interact with RhoA independently of the RhoA activation status. Under cell-free conditions, XIAP did not induce an ubiquitination of RhoA. In summary, our work discloses IAPs as crucial regulators of endothelial permeability and suggests IAP inhibition as interesting approach for the prevention of endothelial barrier dysfunction.

Mitogenic activation of B cells in vitro: the properties of adherent accessory cells as revealed by partition analysis

Energy Technology Data Exchange (ETDEWEB)

Kettman, J.R.; Soederberg, A.; Lefkovits, I.

1986-08-15

The requirement of B cells activated by mitogen (dextran sulfate plus lipopolysaccharide) for accessory cells was studied by partition analysis. Small numbers of splenic B cells were activated to clonal growth, as determined by visual inspection, and to immunoglobulin (Ig) synthesis, as determined by release of Ig into the culture fluid. By placing irradiated adherent cells in the periphery of the microculture wells and forcing responding cells to different areas of the well (slant experiments), it was observed that no cell contact was necessary for B cell activation, and that promoted contact (Rock and Roll experiments) does not increase the efficiency of activation. Sequential microcultures suggest that only some irradiated adherent cells act as accessory cells, but they can perform this function to more than one B cell. Attempts to perform limiting dilution analysis by varying irradiated adherent cell input showed non-single-hit behavior. When the data were rearranged, taking into account the distribution of irradiated adherent cells, then single-hit behavior with about 1 to 5% of irradiated adherent cells acting as an accessory cells for B cell clonal activation was observed. The evidence suggests that an uncommon irradiated adherent cell releases a soluble factor necessary for B cell activation and/or clonal proliferation.

Mitogenic activation of B cells in vitro: the properties of adherent accessory cells as revealed by partition analysis

International Nuclear Information System (INIS)

Kettman, J.R.; Soederberg, A.; Lefkovits, I.

1986-01-01

The requirement of B cells activated by mitogen (dextran sulfate plus lipopolysaccharide) for accessory cells was studied by partition analysis. Small numbers of splenic B cells were activated to clonal growth, as determined by visual inspection, and to immunoglobulin (Ig) synthesis, as determined by release of Ig into the culture fluid. By placing irradiated adherent cells in the periphery of the microculture wells and forcing responding cells to different areas of the well (slant experiments), it was observed that no cell contact was necessary for B cell activation, and that promoted contact (Rock and Roll experiments) does not increase the efficiency of activation. Sequential microcultures suggest that only some irradiated adherent cells act as accessory cells, but they can perform this function to more than one B cell. Attempts to perform limiting dilution analysis by varying irradiated adherent cell input showed non-single-hit behavior. When the data were rearranged, taking into account the distribution of irradiated adherent cells, then single-hit behavior with about 1 to 5% of irradiated adherent cells acting as an accessory cells for B cell clonal activation was observed. The evidence suggests that an uncommon irradiated adherent cell releases a soluble factor necessary for B cell activation and/or clonal proliferation

The double bromodomain protein Brd2 promotes B cell expansion and mitogenesis.

Science.gov (United States)

Belkina, Anna C; Blanton, Wanda P; Nikolajczyk, Barbara S; Denis, Gerald V

2014-03-01

Bromodomain-containing transcriptional regulators represent new epigenetic targets in different hematologic malignancies. However, bromodomain-mediated mechanisms that couple histone acetylation to transcription in lymphopoiesis and govern mature lymphocyte mitogenesis are poorly understood. Brd2, a transcriptional coregulator that contains dual bromodomains and an extraterminal domain (the BET family), couples chromatin to cell-cycle progression. We reported previously the first functional characterization of a BET protein as an effector of mammalian mitogenic signal transduction: Eμ-Brd2 Tg mice develop "activated B cell" diffuse large B cell lymphoma. No other animal models exist for genetic or lentiviral expression of BET proteins, hampering testing of novel anti-BET anticancer drugs, such as JQ1. We transduced HSCs with Brd2 lentivirus and reconstituted recipient mice to test the hypothesis that Brd2 regulates hematopoiesis in BM and mitogenesis in the periphery. Forced expression of Brd2 provides an expansion advantage to the donor-derived B cell compartment in BM and increases mature B cell mitogenic responsiveness in vitro. Brd2 binds the cyclin A promoter in B cells, shown by ChIP, and increases cyclin A mRNA and protein levels, and S-phase progression in vitro in mitogen-stimulated primary B cells, but not T cells, reinforcing results from Eμ-Brd2 mice. The small molecule BET inhibitor JQ1 reduces B cell mitogenesis, consistent with the interpretation that BET inhibitors are antiproliferative. Brd2-specific knockdown experiments show that Brd2 is also required for hematopoiesis. We conclude that Brd2 plays a critical, independent role in regulation of mitogenic response genes, particularly cyclin A, in B cells.

Radiosensitivity of angiogenic and mitogenic factors in human amniotic membrane

International Nuclear Information System (INIS)

Deocaris, Custer C.; De Guzman, Zenaida M.; Deocaris, Chester C.; Jacinto, Sonia D.

2003-01-01

Amniotic membrane as a temporary biological dressing remains as a beneficial and cost-effective means of treating burns in developing countries. This medical application is attributed mainly to placental structural and biochemical features that are important for maintaining proper embryonic development. Since fresh amnions are nevertheless for straightforward clinical use and for preservation, radiation-sterilization is been performed to improve the safety of this placental material. However, like any other sterilization method, gamma-radiation may induce physical and chemical changes that may influence the biological property of the material. Thus, the aim of this study is to compare the effects of various levels of radiation-sterilization protocols for human amnions on angiogenic (neovascularization) and epithelial-mitogenic activities, both of which are physiological processes fundamental to wound healing. Water-soluble extract of non-irradiated amnions demonstrates a strong stimulatory effect on both cell proliferation and angiogenesis. No change in biological activity is seen in amnions irradiated at 25 kGy, the sterilization dose used by the Philippine Nuclear Research Institute (PNRI) for the production of radiation-sterilized human amniotic membranes (RSHAM). However, it appears that amniotic angiogenic factors are more radiosensitive than its mitogenic components, evident from the depressed vascularization of the chorioallantoic membrane (CAM) exposed to 35 kGy-irradiated amnions. The dose of 35 kGy is at present the medical sterilization dose used at the Central Tissue Bank in Warsaw (Poland) for the preparation of their amnion allografts. (Authors)

Effects of 17β-estradiol on the release of monocyte chemotactic protein-1 and MAPK activity in monocytes stimulated with peritoneal fluid from endometriosis patients.

Science.gov (United States)

Lee, Dong-Hyung; Kim, Seung-Chul; Joo, Jong-Kil; Kim, Hwi-Gon; Na, Young-Jin; Kwak, Jong-Young; Lee, Kyu-Sup

2012-03-01

Hormones and inflammation have been implicated in the pathological process of endometriosis; therefore, we investigated the combined effects of 17β-estradiol (E2) and peritoneal fluid obtained from patients with endometriosis (ePF) or a control peritoneal fluid (cPF) obtained from patients without endometriosis on the release of monocyte chemotactic protein-1 (MCP-1) by monocytes and the role of signaling pathways. Monocytes were cultured with ePF and cPF in the presence of E2; the MCP-1 levels in the supernatants were then measured by ELISA. In addition, mitogen activated protein kinase (MAPK) activation was measured by Western blotting of phosphorylated proteins. E2 down-regulated MCP-1 release by lipopolysaccharide- or cPF-treated monocytes, but failed to suppress its release by ePF-treated monocytes. The release of MCP-1 by ePF- and cPF-treated monocytes was efficiently abrogated by p38 mitogen activated protein kinase (MAPK) inhibitors; however, the MCP-1 release by cPF-treated monocytes, but not by ePF-treated monocytes, was blocked by a MAPK kinase inhibitor. In addition, ePF and cPF induced the phosphorylation of extracellular stress regulated kinase (ERK)1/2, p38 MAPK and c-Jun N-terminal kinase (JNK). E2 decreased the phosphorylation of p38 MAPK, but not ERK1/2 in ePF-treated monocytes; however, E2 decreased the phosphorylation of p38 MAPK, ERK1/2 and JNK in cPF-treated monocytes. The ability of E2 to modulate MCP-1 production is impaired in ePF-treated monocytes, which may be related to regulation of MAPK activity. These findings suggest that the failure of E2 to suppress ePF-treated production of MCP-1 may be involved in the pathogenesis of endometriosis. © 2012 The Authors. Journal of Obstetrics and Gynaecology Research © 2012 Japan Society of Obstetrics and Gynecology.

Effects of polycationic compounds on mitogen stimulation

DEFF Research Database (Denmark)

Heron, I; Larsen, B; Hokland, M

1981-01-01

The effects of polycations added to phytomitogen stimulated human lymphocyte cultures have been studied. Within certain dose ranges all polycations tested gave rise to augmented thymidine uptake in mitogen stimulated cultures. The optimum enhancing concentrations of polycations was depending on t...

Inhibition of Curcumin on ZAKα Activity Resultant in Apoptosis and Anchorage-Independent Growth in Cancer Cells.

Science.gov (United States)

Lee, Jin-Sun; Wang, Tsu-Shing; Lin, Ming Cheng; Lin, Wei-Wen; Yang, Jaw-Ji

2017-10-31

Curcumin, a popular yellow pigment of the dietary spice turmeric, has been reported to inhibit cell growth and to induce apoptosis in a wide variety of cancer cells. Although numerous studies have investigated anticancer effects of curcumin, the precise molecular mechanism of action remains unidentified. Whereas curcumin mediates cell survival and apoptosis through mitogen-activated protein kinase (MAPK) and nuclear factor-kappa B (NF-κB) signaling cascades, its impact on the upstream regulation of MAPK is unclear. The leucine-zipper and sterile-α motif kinase alpha (ZAKα), a mitogen-activated protein kinase kinase kinase (MAP3K), activates the c-Jun N-terminal kinase (JNK) and NF-κB pathway. This paper investigated the prospective involvement of ZAKα in curcumin-induced effects on cancer cells. Our results suggest that the antitumor activity of curcumin is mediated via a mechanism involving inhibition of ZAKα activity.

A Classroom Activity for Teaching Electric Polarization of Insulators and Conductors

Science.gov (United States)

Deligkaris, Christos

2018-04-01

The phenomenon of electric polarization is crucial to student understanding of forces exerted between charged objects and insulators or conductors, the process of charging by induction, and the behavior of electroscopes near charged objects. In addition, polarization allows for microscopic-level models of everyday-life macroscopic-level phenomena. Textbooks may adequately discuss polarization, but there is little material in active learning labs and tutorials on this topic. Since polarization of materials is a microscopic phenomenon, instructors often use diagrams and figures on the classroom board to explain the process in a lecture setting. In this paper I will describe a classroom activity where the students play the role of electrons as an alternative option.

Mitogen-activated protein kinase phosphatase-3 (MKP-3 in the surgical wound is necessary for the resolution of postoperative pain in mice

Directory of Open Access Journals (Sweden)

Skopelja-Gardner S

2017-03-01

Full Text Available Sladjana Skopelja-Gardner,1,* Madhurima Saha,1,* Perla Abigail Alvarado-Vazquez,2 Brenna S Liponis,1 Elena Martinez,1 E Alfonso Romero-Sandoval2 1Department of Anesthesiology, Geisel School of Medicine at Dartmouth, Lebanon, NH, 2Department of Pharmaceutical and Administrative Sciences, Presbyterian College School of Pharmacy, Clinton, SC, USA *These authors contributed equally to this work. Abstract: Mitogen-activated protein kinase (MAPK phosphatase-3 (MKP-3 and its substrates (extracellular signal-regulated kinase [ERK] and p38 play an important role in pathophysiological mechanisms of acute postoperative and chronic neuropathic pain in the spinal cord. This study aimed to understand the role of MKP-3 and its target MAPKs at the site of surgical incision in nociceptive behavior. Wild-type (WT and MKP-3 knockout (KO mice underwent unilateral plantar hind paw incision. Mechanical allodynia was assessed by using von Frey filaments. Peripheral ERK-1/2 and p38 phosphorylation were measured by Western blot. Cell infiltration was determined using hematoxylin and eosin histological staining. Peripheral phosphorylated ERK-1/2 (p-ERK-1/2 inhibition was performed in MKP-3 KO mice. In WT mice, mechanical hypersensitivity was observed on postoperative day 1 (0.69±0.17 g baseline vs 0.13±0.08 g day 1, which resolved normally by postoperative day 12 (0.46±0.08 g, N=6. In MKP-3 KO mice, this hypersensitivity persisted at least 12 days after surgery (0.19±0.06 g; N=6. KO mice displayed higher numbers of infiltrating cells (51.4±6 cells/0.1 mm2 than WT mice (8.7±1.2 cells/0.1 mm2 on postoperative day 1 (vs 5–6 cells/0.1 mm2 at baseline that returned to baseline 12 days after surgery (10–12 cells/0.1 mm2. In WT mice, peripheral p-p38 and p-ERK-1/2 expression increased (5- and 3-fold, respectively on postoperative days 1 and 5, and returned to basal levels 7–12 days after surgery (N=3 per group. Peripheral p-p38 levels in MKP-3 KO mice followed

Aldose Reductase Inhibitor Protects against Hyperglycemic Stress by Activating Nrf2-Dependent Antioxidant Proteins

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Kirtikar Shukla

2017-01-01

Full Text Available We have shown earlier that pretreatment of cultured cells with aldose reductase (AR inhibitors prevents hyperglycemia-induced mitogenic and proinflammatory responses. However, the effects of AR inhibitors on Nrf2-mediated anti-inflammatory responses have not been elucidated yet. We have investigated how AR inhibitor fidarestat protects high glucose- (HG- induced cell viability changes by increasing the expression of Nrf2 and its dependent phase II antioxidant enzymes. Fidarestat pretreatment prevents HG (25 mM-induced Thp1 monocyte viability. Further, treatment of Thp1 monocytes with fidarestat caused a time-dependent increase in the expression as well as the DNA-binding activity of Nrf2. In addition, fidarestat augmented the HG-induced Nrf2 expression and activity and also upregulated the expression of Nrf2-dependent proteins such as hemeoxygenase-1 (HO1 and NQO1 in Thp1 cells. Similarly, treatment with AR inhibitor also induced the expression of Nrf2 and HO1 in STZ-induced diabetic mice heart and kidney tissues. Further, AR inhibition increased the HG-induced expression of antioxidant enzymes such as SOD and catalase and activation of AMPK-α1 in Thp1 cells. Our results thus suggest that pretreatment with AR inhibitor prepares the monocytes against hyperglycemic stress by overexpressing the Nrf2-dependent antioxidative proteins.

Mitogenic Activity of a Water-Soluble Adjuvant (Bu-WSA) Obtained from Bacterionema matruchotii: IV. Synergistic Effects of Bu-WSA on Concanavalin A-Induced Proliferative Response of Human Peripheral Blood Lymphocytes.

Science.gov (United States)

Nitta, Toshimasa; Okumura, Seiichi; Tsushi, Masao; Nakano, Masayasu

1982-07-01

Butanol-extracted water-soluble adjuvant (Bu-WSA) obtained from Bacterionema matruchotii was cultured with peripheral blood mononuclear cells (PBM) in the presence of sub- and/or supra-optimal mitogenic concentrations of concanavalin A (Con A). The addition of Bu-WSA resulted in increased tritiated thymidine incorporation above that produced by Con A alone. Bu-WSA by itself is not mitogenic for PBM and in fact produced a decrease in thymidine uptake compared to the control. We investigated the response of subpopulation(s) of PBM to Bu-WSA, Con A and a mixture of Bu-WSA and Con A. Separation of PBM into purified T cells, B cells and macrophages showed that cell-cell cooperation of T cells with B cells or macrophages is necessary for the observed synergistic effect of Bu-WSA with Con A. A marked increase in thymidine incorporation by the mixture of T and B cell populations occurred, while only a small amount of thymidine was incorporated when the B cell population was absent. Mitomycin treatment revealed that the response could be ascribed to the T-cell response with a B-cell helper effect. Moreover, Con A and Bu-WSA appeared to act on the same T cell population. This model may provide unique information about the activation of human peripheral blood T cells compared with the activation of these cells by other mitogens. © owned by Center for Academic Publications Japan (Publisher).

In vitro induction of lymphocyte responsiveness by a Strongylus vulgaris-derived mitogen.

Science.gov (United States)

Bailey, M; Lloyd, S; Martin, S C; Soulsby, E J

1984-01-01

Proliferation in vitro of peripheral blood lymphocytes both from horses infected with Strongylus vulgaris and from helminth-free ponies was observed in the presence of extracts of the fourth and fifth stage larvae and adults of S. vulgaris. In addition, S. vulgaris extracts induced transformation in cultures of peripheral blood lymphocytes from sheep and dogs and in mouse spleen cell cultures. Nylon wool non-adherent, T cell enriched fractions of lymphocytes from both mice and horses were stimulated by the S. vulgaris larval mitogen while no proliferation was observed in cultures containing nylon wool adherent, B cell enriched fractions. Macrophage co-operation appeared not to be necessary for S. vulgaris mitogen-induced transformation of spleen cells. The S. vulgaris mitogen stimulated a subpopulation of mouse spleen cells different from those responsive to PHA, Con A and LPS. These cells might be T helper cells since B cells were stimulated to proliferate in the presence of both T cells and S. vulgaris larval mitogen. In addition, the supernatant of in vitro cultured larvae of S. vulgaris induced slight, but significant transformation of equine peripheral blood lymphocytes. Therefore, it is possible that the S. vulgaris mitogen released by both viable parasites and degenerating larvae might induce T cell dependent production of immunoglobulin in vivo and account for the beta-globulinaemia, of which IgG(T) is a major component, in S vulgaris infected horses.

Activin and TGFβ use diverging mitogenic signaling in advanced colon cancer

OpenAIRE

Bauer, Jessica; Ozden, Ozkan; Akagi, Naomi; Carroll, Timothy; Principe, Daniel R.; Staudacher, Jonas J.; Spehlmann, Martina E.; Eckmann, Lars; Grippo, Paul J.; Jung, Barbara

2015-01-01

Background Understanding cell signaling pathways that contribute to metastatic colon cancer is critical to risk stratification in the era of personalized therapeutics. Here, we dissect the unique involvement of mitogenic pathways in a TGFβ or activin-induced metastatic phenotype of colon cancer. Method Mitogenic signaling/growth factor receptor status and p21 localization were correlated in primary colon cancers and intestinal tumors from either AOM/DSS treated ACVR2A (activin receptor 2) −/−...

Nonequilibrium phase transitions, fluctuations and correlations in an active contractile polar fluid.

Science.gov (United States)

Gowrishankar, Kripa; Rao, Madan

2016-02-21

We study the patterning, fluctuations and correlations of an active polar fluid consisting of contractile polar filaments on a two-dimensional substrate, using a hydrodynamic description. The steady states generically consist of arrays of inward pointing asters and show a continuous transition from a moving lamellar phase, a moving aster street, to a stationary aster lattice with no net polar order. We next study the effect of spatio-temporal athermal noise, parametrized by an active temperature TA, on the stability of the ordered phases. In contrast to its equilibrium counterpart, we find that the active crystal shows true long range order at low TA. On increasing TA, the asters dynamically remodel, concomitantly we find novel phase transitions characterized by bond-orientational and polar order upon "heating".

Inhibition of stress-activated MAP kinases induces clinical improvement in moderate to severe Crohn's disease

NARCIS (Netherlands)

Hommes, Daan; van den Blink, Bernt; Plasse, Terry; Bartelsman, Joep; Xu, Cuiping; Macpherson, Bret; Tytgat, Guido; Peppelenbosch, Mailkel; van Deventer, Sander

2002-01-01

Background & Aims: We investigated if inhibition of mitogen-activated protein kinases (MAPKs) was beneficial in Crohn's disease. Methods: Inhibition of JNK and p38 MAPK activation with CNI-1493, a guanylhydrazone, was tested in vitro. Twelve patients with severe Crohn's disease (mean baseline, CDAI

Multiscale modeling and simulation of microtubule–motor-protein assemblies

Science.gov (United States)

Gao, Tong; Blackwell, Robert; Glaser, Matthew A.; Betterton, M. D.; Shelley, Michael J.

2016-01-01

Microtubules and motor proteins self-organize into biologically important assemblies including the mitotic spindle and the centrosomal microtubule array. Outside of cells, microtubule-motor mixtures can form novel active liquid-crystalline materials driven out of equilibrium by adenosine triphosphate–consuming motor proteins. Microscopic motor activity causes polarity-dependent interactions between motor proteins and microtubules, but how these interactions yield larger-scale dynamical behavior such as complex flows and defect dynamics is not well understood. We develop a multiscale theory for microtubule-motor systems in which Brownian dynamics simulations of polar microtubules driven by motors are used to study microscopic organization and stresses created by motor-mediated microtubule interactions. We identify polarity-sorting and crosslink tether relaxation as two polar-specific sources of active destabilizing stress. We then develop a continuum Doi-Onsager model that captures polarity sorting and the hydrodynamic flows generated by these polar-specific active stresses. In simulations of active nematic flows on immersed surfaces, the active stresses drive turbulent flow dynamics and continuous generation and annihilation of disclination defects. The dynamics follow from two instabilities, and accounting for the immersed nature of the experiment yields unambiguous characteristic length and time scales. When turning off the hydrodynamics in the Doi-Onsager model, we capture formation of polar lanes as observed in the Brownian dynamics simulation. PMID:26764729

Multiscale modeling and simulation of microtubule-motor-protein assemblies.

Science.gov (United States)

Gao, Tong; Blackwell, Robert; Glaser, Matthew A; Betterton, M D; Shelley, Michael J

2015-01-01

Microtubules and motor proteins self-organize into biologically important assemblies including the mitotic spindle and the centrosomal microtubule array. Outside of cells, microtubule-motor mixtures can form novel active liquid-crystalline materials driven out of equilibrium by adenosine triphosphate-consuming motor proteins. Microscopic motor activity causes polarity-dependent interactions between motor proteins and microtubules, but how these interactions yield larger-scale dynamical behavior such as complex flows and defect dynamics is not well understood. We develop a multiscale theory for microtubule-motor systems in which Brownian dynamics simulations of polar microtubules driven by motors are used to study microscopic organization and stresses created by motor-mediated microtubule interactions. We identify polarity-sorting and crosslink tether relaxation as two polar-specific sources of active destabilizing stress. We then develop a continuum Doi-Onsager model that captures polarity sorting and the hydrodynamic flows generated by these polar-specific active stresses. In simulations of active nematic flows on immersed surfaces, the active stresses drive turbulent flow dynamics and continuous generation and annihilation of disclination defects. The dynamics follow from two instabilities, and accounting for the immersed nature of the experiment yields unambiguous characteristic length and time scales. When turning off the hydrodynamics in the Doi-Onsager model, we capture formation of polar lanes as observed in the Brownian dynamics simulation.

Multiscale modeling and simulation of microtubule-motor-protein assemblies

Science.gov (United States)

Gao, Tong; Blackwell, Robert; Glaser, Matthew A.; Betterton, M. D.; Shelley, Michael J.

2015-12-01

Microtubules and motor proteins self-organize into biologically important assemblies including the mitotic spindle and the centrosomal microtubule array. Outside of cells, microtubule-motor mixtures can form novel active liquid-crystalline materials driven out of equilibrium by adenosine triphosphate-consuming motor proteins. Microscopic motor activity causes polarity-dependent interactions between motor proteins and microtubules, but how these interactions yield larger-scale dynamical behavior such as complex flows and defect dynamics is not well understood. We develop a multiscale theory for microtubule-motor systems in which Brownian dynamics simulations of polar microtubules driven by motors are used to study microscopic organization and stresses created by motor-mediated microtubule interactions. We identify polarity-sorting and crosslink tether relaxation as two polar-specific sources of active destabilizing stress. We then develop a continuum Doi-Onsager model that captures polarity sorting and the hydrodynamic flows generated by these polar-specific active stresses. In simulations of active nematic flows on immersed surfaces, the active stresses drive turbulent flow dynamics and continuous generation and annihilation of disclination defects. The dynamics follow from two instabilities, and accounting for the immersed nature of the experiment yields unambiguous characteristic length and time scales. When turning off the hydrodynamics in the Doi-Onsager model, we capture formation of polar lanes as observed in the Brownian dynamics simulation.

The Flavonoid Quercetin Ameliorates Liver Inflammation and Fibrosis by Regulating Hepatic Macrophages Activation and Polarization in Mice

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Xi Li

2018-02-01

Full Text Available At present, there are no effective antifibrotic drugs for patients with chronic liver disease; hence, the development of antifibrotic therapies is urgently needed. Here, we performed an experimental and translational study to investigate the potential and underlying mechanism of quercetin treatment in liver fibrosis, mainly focusing on the impact of quercetin on macrophages activation and polarization. BALB/c mice were induced liver fibrosis by carbon tetrachloride (CCl4 for 8 weeks and concomitantly treated with quercetin (50 mg/kg or vehicle by daily gavage. Liver inflammation, fibrosis, and hepatic stellate cells (HSCs activation were examined. Moreover, massive macrophages accumulation, M1 macrophages and their related markers, such as tumor necrosis factor (TNF-α, interleukin (IL-1β, IL-6, and monocyte chemotactic protein-1 (MCP-1 in livers were analyzed. In vitro, we used Raw 264.7 cells to examine the effect of quercetin on M1-polarized macrophages activation. Our results showed that quercetin dramatically ameliorated liver inflammation, fibrosis, and inhibited HSCs activation. These results were attributed to the reductive recruitment of macrophages (F4/80+ and CD68+ into the liver in quercetin-treated fibrotic mice confirmed by immunostaining and expression levels of marker molecules. Importantly, quercetin strongly inhibited M1 polarization and M1-related inflammatory cytokines in fibrotic livers when compared with vehicle-treated mice. In vitro, studies further revealed that quercetin efficiently inhibited macrophages activation and M1 polarization, as well as decreased the mRNA expression of M1 macrophage markers such as TNF-α, IL-1β, IL-6, and nitric oxide synthase 2. Mechanistically, the inhibition of M1 macrophages by quercetin was associated with the decreased levels of Notch1 expression on macrophages both in vivo and in vitro. Taken together, our data indicated that quercetin attenuated CCl4-induced liver inflammation and

Schistosome-derived omega-1 drives Th2 polarization by suppressing protein synthesis following internalization by the mannose receptor

Science.gov (United States)

Everts, Bart; Hussaarts, Leonie; Driessen, Nicole N.; Meevissen, Moniek H.J.; Schramm, Gabriele; van der Ham, Alwin J.; van der Hoeven, Barbara; Scholzen, Thomas; Burgdorf, Sven; Mohrs, Markus; Pearce, Edward J.; Hokke, Cornelis H.; Haas, Helmut; Smits, Hermelijn H.

2012-01-01

Omega-1, a glycosylated T2 ribonuclease (RNase) secreted by Schistosoma mansoni eggs and abundantly present in soluble egg antigen, has recently been shown to condition dendritic cells (DCs) to prime Th2 responses. However, the molecular mechanisms underlying this effect remain unknown. We show in this study by site-directed mutagenesis of omega-1 that both the glycosylation and the RNase activity are essential to condition DCs for Th2 polarization. Mechanistically, we demonstrate that omega-1 is bound and internalized via its glycans by the mannose receptor (MR) and subsequently impairs protein synthesis by degrading both ribosomal and messenger RNA. These experiments reveal an unrecognized pathway involving MR and interference with protein synthesis that conditions DCs for Th2 priming. PMID:22966004

Muscle Stem Cell Fate Is Controlled by the Cell-Polarity Protein Scrib

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Yusuke Ono

2015-02-01

Full Text Available Satellite cells are resident skeletal muscle stem cells that supply myonuclei for homeostasis, hypertrophy, and repair in adult muscle. Scrib is one of the major cell-polarity proteins, acting as a potent tumor suppressor in epithelial cells. Here, we show that Scrib also controls satellite-cell-fate decisions in adult mice. Scrib is undetectable in quiescent cells but becomes expressed during activation. Scrib is asymmetrically distributed in dividing daughter cells, with robust accumulation in cells committed to myogenic differentiation. Low Scrib expression is associated with the proliferative state and preventing self-renewal, whereas high Scrib levels reduce satellite cell proliferation. Satellite-cell-specific knockout of Scrib in mice causes a drastic and insurmountable defect in muscle regeneration. Thus, Scrib is a regulator of tissue stem cells, controlling population expansion and self-renewal with Scrib expression dynamics directing satellite cell fate.

Protein kinase D stabilizes aldosterone-induced ERK1/2 MAP kinase activation in M1 renal cortical collecting duct cells to promote cell proliferation.

LENUS (Irish Health Repository)

McEneaney, Victoria

2010-01-01

Aldosterone elicits transcriptional responses in target tissues and also rapidly stimulates the activation of protein kinase signalling cascades independently of de novo protein synthesis. Here we investigated aldosterone-induced cell proliferation and extra-cellular regulated kinase 1 and 2 (ERK1\\/2) mitogen activated protein (MAP) kinase signalling in the M1 cortical collecting duct cell line (M1-CCD). Aldosterone promoted the proliferative growth of M1-CCD cells, an effect that was protein kinase D1 (PKD1), PKCdelta and ERK1\\/2-dependent. Aldosterone induced the rapid activation of ERK1\\/2 with peaks of activation at 2 and 10 to 30 min after hormone treatment followed by sustained activation lasting beyond 120 min. M1-CCD cells suppressed in PKD1 expression exhibited only the early, transient peaks in ERK1\\/2 activation without the sustained phase. Aldosterone stimulated the physical association of PKD1 with ERK1\\/2 within 2 min of treatment. The mineralocorticoid receptor (MR) antagonist RU28318 inhibited the early and late phases of aldosterone-induced ERK1\\/2 activation, and also aldosterone-induced proliferative cell growth. Aldosterone induced the sub-cellular redistribution of ERK1\\/2 to the nuclei at 2 min and to cytoplasmic sites, proximal to the nuclei after 30 min. This sub-cellular distribution of ERK1\\/2 was inhibited in cells suppressed in the expression of PKD1.

Protein Kinases in Shaping Plant Architecture.

Science.gov (United States)

Wu, Juan; Wang, Bo; Xin, Xiaoyun; Ren, Dongtao

2018-02-13

Plant architecture, the three-dimensional organization of the plant body, includes the branching pattern and the size, shape, and position of organs. Plant architecture is genetically controlled and is influenced by environmental conditions. The regulations occur at most of the stages from the first division of the fertilized eggs to the final establishment of plant architecture. Among the various endogenous regulators, protein kinases and their associated signaling pathways have been shown to play important roles in regulating the process of plant architecture establishment. In this review, we summarize recent progress in the understanding of the mechanisms by which plant architecture formation is regulated by protein kinases, especially mitogen-activated protein kinase (MAPK). Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

PDGF activates K-Cl cotransport through phosphoinositide 3-kinase and protein phosphatase-1 in primary cultures of vascular smooth muscle cells.

Science.gov (United States)

Zhang, Jing; Lauf, Peter K; Adragna, Norma C

2005-07-15

K-Cl cotransport (K-Cl COT, KCC) is an electroneutrally coupled movement of K and Cl present in most cells. In this work, we studied the pathways of regulation of K-Cl COT by platelet-derived growth factor (PDGF) in primary cultures of vascular smooth muscle cells (VSMCs). Wortmannin and LY 294002 blocked the PDGF-induced K-Cl COT activation, indicating that the phosphoinositide 3-kinase (PI 3-K) pathway is involved. However, PD 98059 had no effect on K-Cl COT activation by PDGF, suggesting that the mitogen-activated protein kinase pathway is not involved under the experimental conditions tested. Involvement of phosphatases was also examined. Sodium orthovanadate, cyclosporin A and okadaic acid had no effect on PDGF-stimulated K-Cl COT. Calyculin A blocked the PDGF-stimulated K-Cl COT by 60%, suggesting that protein phosphatase-1 (PP-1) is a mediator in the PDGF signaling pathway/s. In conclusion, our results indicate that the PDGF-mediated pathways of K-Cl COT regulation involve the signaling molecules PI 3-K and PP-1.

Antimicrobial Activity Investigation on Wuyiencin Fractions of Different Polarity

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Zengjie Cui

2010-04-01

Full Text Available The aim of this study was to evaluate the antimicrobial activity of Wuyiencin fractions with different polarities against six indicator microorganisms: Rhodotorula rubra, Bacillus subtilis, Bacillus megaterium, Escherichia coli, Cladosporium fulvum and Staphylococcus aureus. The fermentation broth of Wuyiencin was submitted to AB-8 macroporous adsorptive resin and fractionated with solvents of different polarity. The fraction eluted with water had remarkably antimicrobial activity against all the microorganisms investigated except for C. fulvum and S. aureus (MIC ≤ 0.0625 mg/mL, probably due to the presence of active components. The fraction eluted with methanol showed potential antimicrobial activity against all the test microorganisms except for R.rubra, with MIC values of0.5 and 2 mg/mL. In conclusion, fractions eluted with water and methanol, respectively, represent the main active-part of Wuyiencin, and could be emphasized for agricultural applications in the future.

A Role for Mitogen- and Stress-Activated Kinase 1 in L-DOPA-Induced Dyskinesia and ∆FosB Expression

DEFF Research Database (Denmark)

Feyder, Michael; Södersten, Erik; Santini, Emanuela

2014-01-01

BACKGROUND: Abnormal regulation of extracellular signal-regulated kinases 1 and 2 has been implicated in 3,4-dihydroxy-l-phenylalanine (L-DOPA)-induced dyskinesia (LID), a motor complication affecting Parkinson's disease patients subjected to standard pharmacotherapy. We examined the involvement...... of mitogen- and stress-activated kinase 1 (MSK1), a downstream target of extracellular signal-regulated kinases 1 and 2, and an important regulator of transcription in LID. METHODS: 6-Hydroxydopamine was used to produce a model of Parkinson's disease in MSK1 knockout mice and in ∆FosB- or ∆c......Jun-overexpressing transgenic mice, which were assessed for LID following long-term L-DOPA administration. Biochemical processes were evaluated by Western blotting or immunofluorescence. Histone H3 phosphorylation was analyzed by chromatin immunoprecipitation followed by promotor-specific quantitative polymerase chain reaction...

The active metabolite of leflunomide, A77 1726, protects rat hepatocytes against bile acid-induced apoptosis.

NARCIS (Netherlands)

Vrenken, T.E.; Buist-Homan, M.; Kalsbeek, A.J.; Faber, K.N.; Moshage, H.J.

2008-01-01

BACKGROUND/AIMS: Leflunomide is used in the treatment of autoimmune diseases as an anti-inflammatory agent. Leflunomide and its active metabolite A77 1726 modulate mitogen-activated protein kinases (MAPK), Src kinases, the phosphoinositide-3 kinase (PI3K)/Akt-pathway and nuclear factor (NF)-kappaB

The active metabolite of leflunomide, A77 1726, protects rat hepatocytes against bile acid-induced apoptosis

NARCIS (Netherlands)

Vrenken, Titia E.; Buist-Homan, Manon; Kalsbeek, Allard Jan; Faber, Klaas Nico; Moshage, Han

Background/Aims: Leflunomide is used in the treatment of autoimmune diseases as an anti-inflammatory agent. Leflunomide and its active metabolite A77 1726 modulate mitogen-activated protein kinases (MAPK), Src kinases, the phosphoinositide-3 kinase (PI3K)/Akt-pathway and nuclear factor (NF)-kappa B

AMP-activated protein kinase induces actin cytoskeleton reorganization in epithelial cells

International Nuclear Information System (INIS)

Miranda, Lisa; Carpentier, Sarah; Platek, Anna; Hussain, Nusrat; Gueuning, Marie-Agnes; Vertommen, Didier; Ozkan, Yurda; Sid, Brice; Hue, Louis; Courtoy, Pierre J.; Rider, Mark H.; Horman, Sandrine

2010-01-01

AMP-activated protein kinase (AMPK), a known regulator of cellular and systemic energy balance, is now recognized to control cell division, cell polarity and cell migration, all of which depend on the actin cytoskeleton. Here we report the effects of A769662, a pharmacological activator of AMPK, on cytoskeletal organization and signalling in epithelial Madin-Darby canine kidney (MDCK) cells. We show that AMPK activation induced shortening or radiation of stress fibers, uncoupling from paxillin and predominance of cortical F-actin. In parallel, Rho-kinase downstream targets, namely myosin regulatory light chain and cofilin, were phosphorylated. These effects resembled the morphological changes in MDCK cells exposed to hyperosmotic shock, which led to Ca 2+ -dependent AMPK activation via calmodulin-dependent protein kinase kinase-β(CaMKKβ), a known upstream kinase of AMPK. Indeed, hypertonicity-induced AMPK activation was markedly reduced by the STO-609 CaMKKβ inhibitor, as was the increase in MLC and cofilin phosphorylation. We suggest that AMPK links osmotic stress to the reorganization of the actin cytoskeleton.

A sensitive fluorescent probe for the polar solvation dynamics at protein-surfactant interfaces.

Science.gov (United States)

Singh, Priya; Choudhury, Susobhan; Singha, Subhankar; Jun, Yongwoong; Chakraborty, Sandipan; Sengupta, Jhimli; Das, Ranjan; Ahn, Kyo-Han; Pal, Samir Kumar

2017-05-17

Relaxation dynamics at the surface of biologically important macromolecules is important taking into account their functionality in molecular recognition. Over the years it has been shown that the solvation dynamics of a fluorescent probe at biomolecular surfaces and interfaces account for the relaxation dynamics of polar residues and associated water molecules. However, the sensitivity of the dynamics depends largely on the localization and exposure of the probe. For noncovalent fluorescent probes, localization at the region of interest in addition to surface exposure is an added challenge compared to the covalently attached probes at the biological interfaces. Here we have used a synthesized donor-acceptor type dipolar fluorophore, 6-acetyl-(2-((4-hydroxycyclohexyl)(methyl)amino)naphthalene) (ACYMAN), for the investigation of the solvation dynamics of a model protein-surfactant interface. A significant structural rearrangement of a model histone protein (H1) upon interaction with anionic surfactant sodium dodecyl sulphate (SDS) as revealed from the circular dichroism (CD) studies is nicely corroborated in the solvation dynamics of the probe at the interface. The polarization gated fluorescence anisotropy of the probe compared to that at the SDS micellar surface clearly reveals the localization of the probe at the protein-surfactant interface. We have also compared the sensitivity of ACYMAN with other solvation probes including coumarin 500 (C500) and 4-(dicyanomethylene)-2-methyl-6-(p-dimethylamino-styryl)-4H-pyran (DCM). In comparison to ACYMAN, both C500 and DCM fail to probe the interfacial solvation dynamics of a model protein-surfactant interface. While C500 is found to be delocalized from the protein-surfactant interface, DCM becomes destabilized upon the formation of the interface (protein-surfactant complex). The timescales obtained from this novel probe have also been compared with other femtosecond resolved studies and molecular dynamics simulations.

Enhanced Expression of WD Repeat-Containing Protein 35 via CaMKK/AMPK Activation in Bupivacaine-Treated Neuro2a Cells

Science.gov (United States)

Huang, Lei; Kondo, Fumio; Gosho, Masahiko; Feng, Guo-Gang; Harato, Misako; Xia, Zhong-yuan; Ishikawa, Naohisa; Fujiwara, Yoshihiro; Okada, Shoshiro

2014-01-01

We previously reported that bupivacaine induces reactive oxygen species (ROS) generation, p38 mitogen-activated protein kinase (MAPK) activation and nuclear factor-kappa B activation, resulting in an increase in expression of WD repeat-containing protein 35 (WDR35) in mouse neuroblastoma Neuro2a cells. However, the identity of signaling upstream of p38 MAPK pathways to WDR35 expression remains unclear. It has been shown that AMP-activated protein kinase (AMPK) can activate p38 MAPK through diverse mechanisms. In addition, several kinases acting upstream of AMPK have been identified including Ca2+/calmodulin-dependent protein kinase kinase (CaMKK). Recent studies reported that AMPK may be involved in bupivacaine-induced cytotoxicity in Schwann cells and in human neuroblastoma SH-SY5Y cells. The present study was undertaken to test whether CaMKK and AMPK are involved in bupivacaine-induced WDR35 expression in Neuro2a cells. Our results showed that bupivacaine induced activation of AMPK and p38 MAPK in Neuro2a cells. The AMPK inhibitors, compound C and iodotubercidin, attenuated the bupivacaine-induced activation of AMPK and p38 MAPK, resulting in an inhibition of the bupivacaine-induced increase in WDR35 expression. Treatment with the CaMKK inhibitor STO-609 also attenuated the bupivacaine-induced activation of AMPK and p38 MAPK, resulting in an inhibition of the bupivacaine-induced increase in WDR35 expression. These results suggest that bupivacaine activates AMPK and p38 MAPK via CaMKK in Neuro2a cells, and that the CaMKK/AMPK/p38 MAPK pathway is involved in regulating WDR35 expression. PMID:24859235

Precipitin response of the mitogen produced by Strongylus vulgaris arterial larvae.

Science.gov (United States)

Adeyefa, C A

1992-07-01

The precipitin response of the mitogen produced by Strongylus vulgaris arterial larvae was investigated. IgG (T) from the sera of horses naturally infected with S. vulgaris adults and arterial larvae recognised the presence of two antigenic components of the mitogenic fractions. The results obtained seem to confirm that these antigens are immunogenic in stimulating the production of increased levels of IgG(T) in infected animals, and showed that the procedures could be used as immunological tools in the diagnosis of S. vulgaris infection.

Changes in aerobic performance, body composition, and physical activity in polar explorers during a year-long stay at the polar station in the Arctic

Science.gov (United States)

Maciejczyk, Marcin; Araźny, Andrzej; Opyrchał, Marta

2017-04-01

The aim of this study was to evaluate changes in physical activity, aerobic performance, and body composition in polar explorers during a 1-year stay at the polar station. The study group consisted of 10 people, including 8 men and 2 women. Aerobic performance (maximal oxygen uptake), physical activity, body mass, and composition were evaluated for the polar explores of the Polish Polar Station prior to departure, and then during their stay at the station for a period of 1 year. The measurements were performed every 3 months. Compared to the measurements taken before going to the polar station, aerobic performance significantly ( p = 0.02) increased in the first 3 months of residing at the polar station and then remained relatively stable for the following duration of the stay. In the first 3 months of the stay, we also observed the highest level of physical activity in participants. In the polar explorers, no significant ( p > 0.05) body fatness changes were noted. Nonetheless, lean body mass, body mass, and BMI significantly increased compared to the measurements taken before departure to the polar station. The greatest changes in aerobic performance, physical activity, and body composition were observed during the first 3 months after arrival to the Arctic and then, despite changing biometeorological conditions, they remained stable for the next months of the stay. We recommend the introduction of a physical preparation program before departing to the polar station to improve explorers' physical fitness, so that they can meet the physical challenges they are faced with immediately after arrival to the polar station.

PRO40 is a scaffold protein of the cell wall integrity pathway, linking the MAP kinase module to the upstream activator protein kinase C.

Directory of Open Access Journals (Sweden)

Ines Teichert

2014-09-01

Full Text Available Mitogen-activated protein kinase (MAPK pathways are crucial signaling instruments in eukaryotes. Most ascomycetes possess three MAPK modules that are involved in key developmental processes like sexual propagation or pathogenesis. However, the regulation of these modules by adapters or scaffolds is largely unknown. Here, we studied the function of the cell wall integrity (CWI MAPK module in the model fungus Sordaria macrospora. Using a forward genetic approach, we found that sterile mutant pro30 has a mutated mik1 gene that encodes the MAPK kinase kinase (MAPKKK of the proposed CWI pathway. We generated single deletion mutants lacking MAPKKK MIK1, MAPK kinase (MAPKK MEK1, or MAPK MAK1 and found them all to be sterile, cell fusion-deficient and highly impaired in vegetative growth and cell wall stress response. By searching for MEK1 interaction partners via tandem affinity purification and mass spectrometry, we identified previously characterized developmental protein PRO40 as a MEK1 interaction partner. Although fungal PRO40 homologs have been implicated in diverse developmental processes, their molecular function is currently unknown. Extensive affinity purification, mass spectrometry, and yeast two-hybrid experiments showed that PRO40 is able to bind MIK1, MEK1, and the upstream activator protein kinase C (PKC1. We further found that the PRO40 N-terminal disordered region and the central region encompassing a WW interaction domain are sufficient to govern interaction with MEK1. Most importantly, time- and stress-dependent phosphorylation studies showed that PRO40 is required for MAK1 activity. The sum of our results implies that PRO40 is a scaffold protein for the CWI pathway, linking the MAPK module to the upstream activator PKC1. Our data provide important insights into the mechanistic role of a protein that has been implicated in sexual and asexual development, cell fusion, symbiosis, and pathogenicity in different fungal systems.

Polar bears experience skeletal muscle atrophy in response to food deprivation and reduced activity in winter and summer

Science.gov (United States)

Whiteman, John P.; Harlow, Henry J.; Durner, George M.; Regehr, Eric V.; Rourke, Bryan C.; Robles, Manuel; Amstrup, Steven C.; Ben-David, Merav

2017-01-01

When reducing activity and using stored energy during seasonal food shortages, animals risk degradation of skeletal muscles, although some species avoid or minimize the resulting atrophy while experiencing these conditions during hibernation. Polar bears may be food deprived and relatively inactive during winter (when pregnant females hibernate and hunting success declines for other demographic groups) as well as summer (when sea ice retreats from key foraging habitats). We investigated muscle atrophy in samples of biceps femoris collected from free-ranging polar bears in the Southern Beaufort Sea (SBS) throughout their annual cycle. Atrophy was most pronounced in April–May as a result of food deprivation during the previous winter, with muscles exhibiting reduced protein concentration, increased water content, and lower creatine kinase mRNA. These animals increased feeding and activity in spring (when seal prey becomes more available), initiating a period of muscle recovery. During the following ice melt of late summer, ~30% of SBS bears abandon retreating sea ice for land; in August, these ‘shore’ bears exhibited no muscle atrophy, indicating that they had fully recovered from winter food deprivation. These individuals subsequently scavenged whale carcasses deposited by humans and by October, had retained good muscle condition. In contrast, ~70% of SBS bears follow the ice north in late summer, into deep water with less prey. These ‘ice’ bears fast; by October, they exhibited muscle protein loss and rapid changes in myosin heavy-chain isoforms in response to reduced activity. These findings indicate that, unlike other bears during winter hibernation, polar bears without food in summer cannot mitigate atrophy. Consequently, prolonged summer fasting resulting from climate change-induced ice loss creates a risk of greater muscle atrophy and reduced abilities to travel and hunt.

Polar bears experience skeletal muscle atrophy in response to food deprivation and reduced activity in winter and summer

Science.gov (United States)

Harlow, Henry J.; Durner, George M.; Regehr, Eric V.; Rourke, Bryan C.; Robles, Manuel; Amstrup, Steven C.; Ben-David, Merav

2017-01-01

Abstract When reducing activity and using stored energy during seasonal food shortages, animals risk degradation of skeletal muscles, although some species avoid or minimize the resulting atrophy while experiencing these conditions during hibernation. Polar bears may be food deprived and relatively inactive during winter (when pregnant females hibernate and hunting success declines for other demographic groups) as well as summer (when sea ice retreats from key foraging habitats). We investigated muscle atrophy in samples of biceps femoris collected from free-ranging polar bears in the Southern Beaufort Sea (SBS) throughout their annual cycle. Atrophy was most pronounced in April–May as a result of food deprivation during the previous winter, with muscles exhibiting reduced protein concentration, increased water content, and lower creatine kinase mRNA. These animals increased feeding and activity in spring (when seal prey becomes more available), initiating a period of muscle recovery. During the following ice melt of late summer, ~30% of SBS bears abandon retreating sea ice for land; in August, these ‘shore’ bears exhibited no muscle atrophy, indicating that they had fully recovered from winter food deprivation. These individuals subsequently scavenged whale carcasses deposited by humans and by October, had retained good muscle condition. In contrast, ~70% of SBS bears follow the ice north in late summer, into deep water with less prey. These ‘ice’ bears fast; by October, they exhibited muscle protein loss and rapid changes in myosin heavy-chain isoforms in response to reduced activity. These findings indicate that, unlike other bears during winter hibernation, polar bears without food in summer cannot mitigate atrophy. Consequently, prolonged summer fasting resulting from climate change-induced ice loss creates a risk of greater muscle atrophy and reduced abilities to travel and hunt. PMID:28835844

Polar bears experience skeletal muscle atrophy in response to food deprivation and reduced activity in winter and summer.

Science.gov (United States)

Whiteman, John P; Harlow, Henry J; Durner, George M; Regehr, Eric V; Rourke, Bryan C; Robles, Manuel; Amstrup, Steven C; Ben-David, Merav

2017-01-01

When reducing activity and using stored energy during seasonal food shortages, animals risk degradation of skeletal muscles, although some species avoid or minimize the resulting atrophy while experiencing these conditions during hibernation. Polar bears may be food deprived and relatively inactive during winter (when pregnant females hibernate and hunting success declines for other demographic groups) as well as summer (when sea ice retreats from key foraging habitats). We investigated muscle atrophy in samples of biceps femoris collected from free-ranging polar bears in the Southern Beaufort Sea (SBS) throughout their annual cycle. Atrophy was most pronounced in April-May as a result of food deprivation during the previous winter, with muscles exhibiting reduced protein concentration, increased water content, and lower creatine kinase mRNA. These animals increased feeding and activity in spring (when seal prey becomes more available), initiating a period of muscle recovery. During the following ice melt of late summer, ~30% of SBS bears abandon retreating sea ice for land; in August, these 'shore' bears exhibited no muscle atrophy, indicating that they had fully recovered from winter food deprivation. These individuals subsequently scavenged whale carcasses deposited by humans and by October, had retained good muscle condition. In contrast, ~70% of SBS bears follow the ice north in late summer, into deep water with less prey. These 'ice' bears fast; by October, they exhibited muscle protein loss and rapid changes in myosin heavy-chain isoforms in response to reduced activity. These findings indicate that, unlike other bears during winter hibernation, polar bears without food in summer cannot mitigate atrophy. Consequently, prolonged summer fasting resulting from climate change-induced ice loss creates a risk of greater muscle atrophy and reduced abilities to travel and hunt.

Regulation of death induction and chemosensitizing action of 3-bromopyruvate in myeloid leukemia cells: energy depletion, oxidative stress, and protein kinase activity modulation.

Science.gov (United States)

Calviño, Eva; Estañ, María Cristina; Sánchez-Martín, Carlos; Brea, Rocío; de Blas, Elena; Boyano-Adánez, María del Carmen; Rial, Eduardo; Aller, Patricio

2014-02-01

3-Bromopyruvate (3-BrP) is an alkylating, energy-depleting drug that is of interest in antitumor therapies, although the mechanisms underlying its cytotoxicity are ill-defined. We show here that 3-BrP causes concentration-dependent cell death of HL60 and other human myeloid leukemia cells, inducing both apoptosis and necrosis at 20-30 μM and a pure necrotic response at 60 μM. Low concentrations of 3-BrP (10-20 μM) brought about a rapid inhibition of glycolysis, which at higher concentrations was followed by the inhibition of mitochondrial respiration. The combination of these effects causes concentration-dependent ATP depletion, although this cannot explain the lethality at intermediate 3-BrP concentrations (20-30 μM). The oxidative stress caused by exposure to 3-BrP was evident as a moderate overproduction of reactive oxygen species and a concentration-dependent depletion of glutathione, which was an important determinant of 3-BrP toxicity. In addition, 3-BrP caused glutathione-dependent stimulation of p38 mitogen-activated protein kinase (MAPK), mitogen-induced extracellular kinase (MEK)/extracellular signal-regulated kinase (ERK), and protein kinase B (Akt)/mammalian target of rapamycin/p70S6K phosphorylation or activation, as well as rapid LKB-1/AMP kinase (AMPK) activation, which was later followed by Akt-mediated inactivation. Experiments with pharmacological inhibitors revealed that p38 MAPK activation enhances 3-BrP toxicity, which is conversely restrained by ERK and Akt activity. Finally, 3-BrP was seen to cooperate with antitumor agents like arsenic trioxide and curcumin in causing cell death, a response apparently mediated by both the generation of oxidative stress induced by 3-BrP and the attenuation of Akt and ERK activation by curcumin. In summary, 3-BrP cytotoxicity is the result of several combined regulatory mechanisms that might represent important targets to improve therapeutic efficacy.

Zeno inhibition of polarization rotation in an optically active medium

International Nuclear Information System (INIS)

Gonzalo, Isabel; Luis, Alfredo; Porras, Miguel A

2015-01-01

We describe an experiment in which the rotation of the polarization of light propagating in an optically active water solution of D-fructose tends to be inhibited by frequent monitoring whether the polarization remains unchanged. This is an example of the Zeno effect that has remarkable pedagogical interest because of its conceptual simplicity, easy implementation, low cost, and because the same the Zeno effect holds at classical and quantum levels. An added value is the demonstration of the Zeno effect beyond typical idealized assumptions in a practical setting with real polarizers. (paper)

Exercise and Glycemic Control: Focus on Redox Homeostasis and Redox-Sensitive Protein Signaling

Science.gov (United States)

Parker, Lewan; Shaw, Christopher S.; Stepto, Nigel K.; Levinger, Itamar

2017-01-01

Physical inactivity, excess energy consumption, and obesity are associated with elevated systemic oxidative stress and the sustained activation of redox-sensitive stress-activated protein kinase (SAPK) and mitogen-activated protein kinase signaling pathways. Sustained SAPK activation leads to aberrant insulin signaling, impaired glycemic control, and the development and progression of cardiometabolic disease. Paradoxically, acute exercise transiently increases oxidative stress and SAPK signaling, yet postexercise glycemic control and skeletal muscle function are enhanced. Furthermore, regular exercise leads to the upregulation of antioxidant defense, which likely assists in the mitigation of chronic oxidative stress-associated disease. In this review, we explore the complex spatiotemporal interplay between exercise, oxidative stress, and glycemic control, and highlight exercise-induced reactive oxygen species and redox-sensitive protein signaling as important regulators of glucose homeostasis. PMID:28529499

A study of auroral activity in the nightside polar cap

International Nuclear Information System (INIS)

Wu, Q.

1989-01-01

Using various ground observations at South Pole, Antarctica (invariant magnetic latitude -74 degree) and its conjugate point, Frobisher Bay, Canada, the author has studied the following aspects of nightside polar cap auroral activity: the appearance and disappearance of polar cap auroras (diffuse and discrete) associated with substorms and interplanetary magnetic field (IMF) variations; auroral optical emission line intensities; and the seasonal variation of auroral conjugacy. The observations show that the polar cap auroras usually fade away before the expansive phase of a substorm and bright auroral arcs reach high latitude (-74 degree) near the recovery phase. Just before the auroras fade away the discrete polar cap auroral arcs, which are usually on the poleward boundary of the diffuse aurora, intensify for 1 to 2 minutes. The observations also indicate the IMF may have stronger control over polar cap auroral activity than do substorms. A search for energy spectral variation of precipitating electrons using the intensities of 630.0 nm (0) and 427 nm (N 2 + ) auroral emission lines reveals no dramatic changes in the energy spectrum; instead, the data show possible atmospheric scattering and geometric effects on the photometric measurements while the bright auroral arc is moving into the polar cap. The conjugate observations show that the stormtime auroral electrojet current, which is associated with the bright auroral arc, in most cases reaches higher (lower) latitudes in the winter (summer) hemisphere. An asymmetric plasma sheet (with respect to the neutral sheet) is proposed, which expands deeper into the winter lobe, under a tilted geomagnetic dipole. Accordingly, the winter polar cap would have smaller area and the auroral electrojet would be at higher latitude

A cytoskeletal activator and inhibitor are downstream targets of the frizzled/starry night planar cell polarity pathway in the Drosophila epidermis.

Science.gov (United States)

Adler, Paul N

2018-04-10

The frizzled pathway regulates the planar polarity of epithelial cells. In insects this is manifested by the polarity of cuticular structures such as hairs (trichomes) and sensory bristles. A variety of evidence has established that this is achieved by regulating the subcellular location for activating the cytoskeleton in the epithelial cells. How this is accomplished is still poorly understood. In the best-studied tissue, the Drosophila pupal wing two important cytoskeletal regulators have been identified. One, shavenoid (sha), appears to be an activator while the second multiple wing hairs (mwh), appears to be an inhibitor. In vitro biochemistry has confirmed that the Multiple Wing Hairs protein inhibits the elongation of F-actin chains and surprisingly that it also bundles F-actin. These two activities can explain the multifaceted mwh mutant phenotype. Copyright © 2018. Published by Elsevier Ltd.

Pervanadate induces Mammalian Ste20 Kinase 3 (MST3) tyrosine phosphorylation but not activation.

Science.gov (United States)

Kan, Wei-Chih; Lu, Te-Ling; Ling, Pin; Lee, Te-Hsiu; Cho, Chien-Yu; Huang, Chi-Ying F; Jeng, Wen-Yih; Weng, Yui-Ping; Chiang, Chun-Yen; Wu, Jin Bin; Lu, Te-Jung

2016-07-01

The yeast Ste20 (sterile) protein kinase, which is a serine/threonine kinase, responds to the stimulation of the G proteincoupled receptor (GPCR) pheromone receptor. Ste20 protein kinase serves as the critical component that links signaling from the GPCR/G proteins to the mitogen-activated protein kinase (MAPK) cascade in yeast. The yeast Ste20p functions as a MAP kinase kinase kinase kinase (MAP4K) in the pheromone response. Ste20-like kinases are structurally conserved from yeast to mammals. The mechanism by which MAP4K links GPCR to the MAPK pathway is less clearly defined in vertebrates. In addition to MAP4K, the tyrosine kinase cascade bridges G proteins and the MAPK pathway in vertebrate cells. Mammalian Ste20 Kinase 3 (MST3) has been categorized into the Ste20 family and has been reported to function in the regulation of cell polarity and migration. However, whether MST3 tyrosine phosphorylation regulates diverse signaling pathways is unknown. In this study, the tyrosine phosphatase inhibitor pervanadate was found to induce MST3 tyrosine phosphorylation in intact cells, and the activity of tyrosine-phosphorylated MST3 was measured. This tyrosine-directed phosphorylation was independent of MST3 activity. Parameters including protein conformation, Triton concentration and ionic concentration influenced the sensitivity of MST3 activity. Taken together, our data suggests that the serine/threonine kinase MST3 undergoes tyrosinedirected phosphorylation. The tyrosine-phosphorylated MST3 may create a docking site for the structurally conserved SH2/SH3 (Src Homology 2 and 3) domains within the Src oncoprotein. The unusual tyrosinephosphorylated MST3 may recruit MST3 to various signaling components. Copyright © 2016. Published by Elsevier Inc.

Inhibition of cerebrovascular raf activation attenuates cerebral blood flow and prevents upregulation of contractile receptors after subarachnoid hemorrhage

DEFF Research Database (Denmark)

Ansar, Saema; Maddahi, Aida; Edvinsson, Lars

2011-01-01

of mitogen-activated protein kinase (MAPK) of the extracellular signal-regulated kinase (ERK)1/2 signal pathway. We hypothesize that SAH initiates cerebrovascular ERK1/2 activation, resulting in receptor upregulation. The raf inhibitor will inhibit the molecular events upstream ERK1/2 and may provide...

Poxviral protein A52 stimulates p38 mitogen-activated protein kinase (MAPK) activation by causing tumor necrosis factor receptor-associated factor 6 (TRAF6) self-association leading to transforming growth factor β-activated kinase 1 (TAK1) recruitment.

Science.gov (United States)

Stack, Julianne; Hurst, Tara P; Flannery, Sinead M; Brennan, Kiva; Rupp, Sebastian; Oda, Shun-ichiro; Khan, Amir R; Bowie, Andrew G

2013-11-22

Vaccinia virus encodes a number of proteins that inhibit and manipulate innate immune signaling pathways that also have a role in virulence. These include A52, a protein shown to inhibit IL-1- and Toll-like receptor-stimulated NFκB activation, via interaction with interleukin-1 receptor-associated kinase 2 (IRAK2). Interestingly, A52 was also found to activate p38 MAPK and thus enhance Toll-like receptor-dependent IL-10 induction, which was TRAF6-dependent, but the manner in which A52 manipulates TRAF6 to stimulate p38 activation was unclear. Here, we show that A52 has a non-canonical TRAF6-binding motif that is essential for TRAF6 binding and p38 activation but dispensable for NFκB inhibition and IRAK2 interaction. Wild-type A52, but not a mutant defective in p38 activation and TRAF6 binding (F154A), caused TRAF6 oligomerization and subsequent TRAF6-TAK1 association. The crystal structure of A52 shows that it adopts a Bcl2-like fold and exists as a dimer in solution. Residue Met-65 was identified as being located in the A52 dimer interface, and consistent with that, A52-M65E was impaired in its ability to dimerize. A52-M65E although capable of interacting with TRAF6, was unable to cause either TRAF6 self-association, induce the TRAF6-TAK1 association, or activate p38 MAPK. The results suggest that an A52 dimer causes TRAF6 self-association, leading to TAK1 recruitment and p38 activation. This reveals a molecular mechanism whereby poxviruses manipulate TRAF6 to activate MAPKs (which can be proviral) without stimulating antiviral NFκB activation.

Caffeic Acid Phenethyl Ester Inhibits Oral Cancer Cell Metastasis by Regulating Matrix Metalloproteinase-2 and the Mitogen-Activated Protein Kinase Pathway

Directory of Open Access Journals (Sweden)

Chih-Yu Peng

2012-01-01

Full Text Available Caffeic acid phenethyl ester (CAPE, an active component extracted from honeybee hives, exhibits anti-inflammatory and anticancer activities. However, the molecular mechanism by which CAPE affects oral cancer cell metastasis has yet to be elucidated. In this study, we investigated the potential mechanisms underlying the effects of CAPE on the invasive ability of SCC-9 oral cancer cells. Results showed that CAPE attenuated SCC-9 cell migration and invasion at noncytotoxic concentrations (0 μM to 40 μM. Western blot and gelatin zymography analysis findings further indicated that CAPE downregulated matrix metalloproteinase-2 (MMP-2 protein expression and inhibited its enzymatic activity. CAPE exerted its inhibitory effects on MMP-2 expression and activity by upregulating tissue inhibitor of metalloproteinase-2 (TIMP-2 and potently decreased migration by reducing focal adhesion kinase (FAK phosphorylation and the activation of its downstream signaling molecules p38/MAPK and JNK. These data indicate that CAPE could potentially be used as a chemoagent to prevent oral cancer metastasis.

Apical Polarity Protein PrkCi Is Necessary for Maintenance of Spinal Cord Precursors in Zebrafish

OpenAIRE

Roberts, Randolph K.; Appel, Bruce

2009-01-01

During development, neural precursors divide to produce new precursors and cells that differentiate as neurons and glia. In Drosophila, apicobasal polarity and orientation of the mitotic spindle play important roles in specifying the progeny of neural precursors for different fates. We examined orientation of zebrafish spinal cord precursors using time-lapse imaging and tested the function of protein kinase C, iota (PrkCi), a member of the Par complex of proteins necessary for apicobasal pola...

Receptor-like protein-tyrosine phosphatase alpha specifically inhibits insulin-increased prolactin gene expression

DEFF Research Database (Denmark)

Jacob, K K; Sap, J; Stanley, F M

1998-01-01

A physiologically relevant response to insulin, stimulation of prolactin promoter activity in GH4 pituitary cells, was used as an assay to study the specificity of protein-tyrosine phosphatase function. Receptor-like protein-tyrosine phosphatase alpha (RPTPalpha) blocks the effect of insulin...... is specific by two criteria. A number of potential RPTPalpha targets were ruled out by finding (a) that they are not affected or (b) that they are not on the pathway to insulin-increased prolactin-CAT activity. The negative effect of RPTPalpha on insulin activation of the prolactin promoter is not due...... to reduced phosphorylation or kinase activity of the insulin receptor or to reduced phosphorylation of insulin receptor substrate-1 or Shc. Inhibitor studies suggest that insulin-increased prolactin gene expression is mediated by a Ras-like GTPase but is not mitogen-activated protein kinase dependent...

Polarization ray tracing in anisotropic optically active media. I. Algorithms

International Nuclear Information System (INIS)

McClain, S.C.; Hillman, L.W.; Chipman, R.A.

1993-01-01

Procedures for performing polarization ray tracing through birefringent media are presented in a form compatible with the standard methods of geometrical ray tracing. The birefringent materials treated include the following: anisotropic optically active materials such as quartz, non-optically active uniaxial materials such as calcite, and isotropic optically active materials such as mercury sulfide and organic liquids. Refraction and reflection algorithms are presented that compute both ray directions and wave directions. Methods for computing polarization modes, refractive indices, optical path lengths, and Fresnel transmission and reflection coefficients are also specified. A numerical example of these algorithms is given for analyzing the field of view of a quartz rotator. 37 refs., 3 figs

Polarization of migrating monocytic cells is independent of PI 3-kinase activity.

Directory of Open Access Journals (Sweden)

Silvia Volpe

Full Text Available BACKGROUND: Migration of mammalian cells is a complex cell type and environment specific process. Migrating hematopoietic cells assume a rapid amoeboid like movement when exposed to gradients of chemoattractants. The underlying signaling mechanisms remain controversial with respect to localization and distribution of chemotactic receptors within the plasma membrane and the role of PI 3-kinase activity in cell polarization. METHODOLOGY/PRINCIPAL FINDINGS: We present a novel model for the investigation of human leukocyte migration. Monocytic THP-1 cells transfected with the alpha(2A-adrenoceptor (alpha(2AAR display comparable signal transduction responses, such as calcium mobilization, MAP-kinase activation and chemotaxis, to the noradrenaline homologue UK 14'304 as when stimulated with CCL2, which binds to the endogenous chemokine receptor CCR2. Time-lapse video microscopy reveals that chemotactic receptors remain evenly distributed over the plasma membrane and that their internalization is not required for migration. Measurements of intramolecular fluorescence resonance energy transfer (FRET of alpha(2AAR-YFP/CFP suggest a uniform activation of the receptors over the entire plasma membrane. Nevertheless, PI 3-kinase activation is confined to the leading edge. When reverting the gradient of chemoattractant by moving the dispensing micropipette, polarized monocytes--in contrast to neutrophils--rapidly flip their polarization axis by developing a new leading edge at the previous posterior side. Flipping of the polarization axis is accompanied by re-localization of PI-3-kinase activity to the new leading edge. However, reversal of the polarization axis occurs in the absence of PI 3-kinase activation. CONCLUSIONS/SIGNIFICANCE: Accumulation and internalization of chemotactic receptors at the leading edge is dispensable for cell migration. Furthermore, uniformly distributed receptors allow the cells to rapidly reorient and adapt to changes in the

Temperature thresholds for chlorine activation and ozone loss in the polar stratosphere

Energy Technology Data Exchange (ETDEWEB)

Drdla, K. [NASA Ames Research Center, Moffett Field, CA (United States); Mueller, R. [Forschungszentrum Juelich (DE). Inst. of Energy and Climate Research (IEK-7)

2012-07-01

Low stratospheric temperatures are known to be responsible for heterogeneous chlorine activation that leads to polar ozone depletion. Here, we discuss the temperature threshold below which substantial chlorine activation occurs. We suggest that the onset of chlorine activation is dominated by reactions on cold binary aerosol particles, without the formation of polar stratospheric clouds (PSCs), i.e. without any significant uptake of HNO{sub 3} from the gas phase. Using reaction rates on cold binary aerosol in a model of stratospheric chemistry, a chlorine activation threshold temperature, T{sub ACL}, is derived. At typical stratospheric conditions, T{sub ACL} is similar in value to T{sub NAT} (within 1-2 K), the highest temperature at which nitric acid trihydrate (NAT) can exist. T{sub NAT} is still in use to parameterise the threshold temperature for the onset of chlorine activation. However, perturbations can cause T{sub ACL} to differ from T{sub NAT}: T{sub ACL} is dependent upon H{sub 2} O and potential temperature, but unlike T{sub NAT} is not dependent upon HNO3. Furthermore, in contrast to T{sub NAT}, T{sub ACL} is dependent upon the stratospheric sulfate aerosol loading and thus provides a means to estimate the impact on polar ozone of strong volcanic eruptions and some geo-engineering options, which are discussed. A parameterisation of T{sub ACL} is provided here, allowing it to be calculated for low solar elevation (or high solar zenith angle) over a comprehensive range of stratospheric conditions. Considering T{sub ACL} as a proxy for chlorine activation cannot replace a detailed model calculation, and polar ozone loss is influenced by other factors apart from the initial chlorine activation. However, T{sub ACL} provides a more accurate description of the temperature conditions necessary for chlorine activation and ozone loss in the polar stratosphere than T{sub NAT}. (orig.)

Time-dependent enhancement of lymphocyte activation by mitogens after exposure to isolation or water scheduling.

Science.gov (United States)

Jessop, J J; Gale, K; Bayer, B M

1988-01-01

The effects of isolation and water scheduling on mitogen induced lymphocyte proliferation were investigated. Isolated rats were animals which had been raised in group-housed conditions and then transferred to individual cages with ad lib access to water for a 1 or 2 week period. Water scheduled rats were maintained in group housing (5 rats per cage) with ad lib access to food but with access to water for a single 30 minute session each day. Responses of these groups were compared to those of animals which had been continuously group-housed with ad lib access to food and water. No differences in lymphocyte responses to phytohemagglutinin (PHA) were found 1 week after exposure to isolation. However, after 2 weeks, splenic and blood T lymphocytes from isolated animals demonstrated an increased proliferative response to suboptimum and maximum concentrations of PHA. Splenic B lymphocyte responses to lipopolysaccharide (LPS) from isolated animals were also increased by 2- to 3-fold compared to group-housed controls. Two weeks of exposure of animals to daily water scheduling similarly increased the splenic lymphocyte proliferation. This increased responsiveness to PHA was not accompanied by a significant change in the sensitivity of the lymphocytes to PHA, in the total number of white blood cells, or the proportion of splenic T or T helper lymphocytes. Our results show that the increase in lymphocyte proliferation is time-dependent, requires greater than 1 week of exposure to isolation and is due to factors other than changes in sensitivity to mitogen or T lymphocyte number.

G protein abnormalities in pituitary adenomas.

Science.gov (United States)

Spada, A; Lania, A; Ballarè, E

1998-07-25

It has been demonstrated that the majority of secreting and nonsecreting adenomas is monoclonal in origin suggesting that these neoplasia arise from the replication of a single mutated cell, in which growth advantage results from either activation of protooncogenes or inactivation of antioncogenes. Although a large number of genes has been screened for mutations, only few genetic abnormalities have been found in pituitary tumors such as allelic deletion of chromosome 11q13 where the MEN-1 gene has been localised, and mutations in the gene encoding the alpha subunit of the stimulatory Gs and Gi2 protein. These mutations constitutively activate the alpha subunit of the Gs and Gi2 protein by inhibiting their intrinsic GTPase activity. Both Gs alpha and Gi2alpha can be considered products of protooncogenes (gsp and gip2, respectively) since gain of function mutations that activate mitogenic signals have been recognized in human tumors. Gsp oncogene is found in 30-40% of GH-secreting adenomas, in a low percentage of nonfunctioning and ACTH-secreting pituitary adenomas, in toxic thyroid adenomas and differentiated thyroid carcinomas. The same mutations, occurred early in embriogenesis, have been also identified in tissues from patients affected with the McCune Albright syndrome. These mutations result in an increased cAMP production and in the subsequent overactivation of specific pathways involved in both cell growth and specific programmes of cell differentiation. By consequence, the endocrine tumors expressing gsp oncogene retain differentiated functions. The gip2 oncogene has been identified in about 10% of nonfunctioning pituitary adenomas, in tumors of the ovary and the adrenal cortex. However, it remains to be established whether Gi proteins activate mitogenic signals in pituitary cells. Since Gi proteins are involved in mediating the effect of inhibitory neurohormones on intracellular effectors, it has been proposed that in pituitary tumors the low expression of

The polarity protein Par6 is coupled to the microtubule network during molluscan early embryogenesis

Energy Technology Data Exchange (ETDEWEB)

Homma, Taihei [Department of Biophysics and Biochemistry, Graduate School of Science, The University of Tokyo, Hongo, Bunkyo-ku, Tokyo 113-0033 (Japan); Shimizu, Miho [Kuroda Chiromorphology Team, ERATO-SORST, JST, Komaba, Meguro-ku, Tokyo 153-8902 (Japan); Kuroda, Reiko, E-mail: ckuroda@mail.ecc.u-tokyo.ac.jp [Department of Biophysics and Biochemistry, Graduate School of Science, The University of Tokyo, Hongo, Bunkyo-ku, Tokyo 113-0033 (Japan); Kuroda Chiromorphology Team, ERATO-SORST, JST, Komaba, Meguro-ku, Tokyo 153-8902 (Japan); Department of Life Sciences, Graduate School of Arts and Sciences, The University of Tokyo, Komaba, Meguro-ku, Tokyo 153-8902 (Japan)

2011-01-07

Research highlights: {yields} The cDNAs encoding Par6 and aPKC homologues were cloned from the snail Lymnaea stagnalis. {yields} L. stagnalis Par6 directly interacts with tubulin and microtubules and localizes to the microtubule cytoskeleton during the early embryogenesis. {yields} Identical sequence and localization of LsPar6 for the dextral and the sinistral snails exclude the possibility of the gene being the primary determinant of body handedness. -- Abstract: Cell polarity, which directs the orientation of asymmetric cell division and segregation of fate determinants, is a fundamental feature of development and differentiation. Regulators of polarity have been extensively studied, and the critical importance of the Par (partitioning-defective) complex as the polarity machinery is now recognized in a wide range of eukaryotic systems. The Par polarity module is evolutionarily conserved, but its mechanism and cooperating factors vary among different systems. Here we describe the cloning and characterization of a pond snail Lymnaea stagnalis homologue of partitioning-defective 6 (Lspar6). The protein product LsPar6 shows high affinity for microtubules and localizes to the mitotic apparatus during embryonic cell division. In vitro assays revealed direct binding of LsPar6 to tubulin and microtubules, which is the first evidence of the direct interaction between the two proteins. The interaction is mediated by two distinct regions of LsPar6 both located in the N-terminal half. Atypical PKC, a functional partner of Par6, was also found to localize to the mitotic spindle. These results suggest that the L. stagnalis Par complex employs the microtubule network in cell polarity processes during the early embryogenesis. Identical sequence and localization of LsPar6 for the dextral and the sinistral snails exclude the possibility of the gene being the primary determinant of handedness.

The polarity protein Par6 is coupled to the microtubule network during molluscan early embryogenesis

International Nuclear Information System (INIS)

Homma, Taihei; Shimizu, Miho; Kuroda, Reiko

2011-01-01

Research highlights: → The cDNAs encoding Par6 and aPKC homologues were cloned from the snail Lymnaea stagnalis. → L. stagnalis Par6 directly interacts with tubulin and microtubules and localizes to the microtubule cytoskeleton during the early embryogenesis. → Identical sequence and localization of LsPar6 for the dextral and the sinistral snails exclude the possibility of the gene being the primary determinant of body handedness. -- Abstract: Cell polarity, which directs the orientation of asymmetric cell division and segregation of fate determinants, is a fundamental feature of development and differentiation. Regulators of polarity have been extensively studied, and the critical importance of the Par (partitioning-defective) complex as the polarity machinery is now recognized in a wide range of eukaryotic systems. The Par polarity module is evolutionarily conserved, but its mechanism and cooperating factors vary among different systems. Here we describe the cloning and characterization of a pond snail Lymnaea stagnalis homologue of partitioning-defective 6 (Lspar6). The protein product LsPar6 shows high affinity for microtubules and localizes to the mitotic apparatus during embryonic cell division. In vitro assays revealed direct binding of LsPar6 to tubulin and microtubules, which is the first evidence of the direct interaction between the two proteins. The interaction is mediated by two distinct regions of LsPar6 both located in the N-terminal half. Atypical PKC, a functional partner of Par6, was also found to localize to the mitotic spindle. These results suggest that the L. stagnalis Par complex employs the microtubule network in cell polarity processes during the early embryogenesis. Identical sequence and localization of LsPar6 for the dextral and the sinistral snails exclude the possibility of the gene being the primary determinant of handedness.

A microRNA signature associated with chondrogenic lineage ...

Indian Academy of Sciences (India)

peroxisome proliferator-activated receptor gamma. PPAR signalling hsa-mir-143. MAP3K7 mitogen-activated protein kinase kinase kinase 7. MAPK signalling. CHST10 carbohydrate sulfotransferase 10. Sulfation of chondroitin. MAPK7 mitogen-activated protein kinase 7. MAPK signalling. COL1A1 collagen, type I, alpha 1.

Toward an effective adsorbent for polar pollutants: Formaldehyde adsorption by activated carbon

International Nuclear Information System (INIS)

Lee, Kyung Jin; Miyawaki, Jin; Shiratori, Nanako; Yoon, Seong-Ho; Jang, Jyongsik

2013-01-01

Highlights: • Activated carbon fiber with mild activation condition is useful as adsorbent for polar pollutants. • Diverse variations are investigated for developing an effective adsorbent. • Surface functional group is the most important factor for capacity as a adsorbent. • Surface functional groups on ACFs are investigated using micro-ATR FTIR. -- Abstract: Due to increasing concerns about environmental pollutants, the development of an effective adsorbent or sensitive sensor has been pursued in recent years. Diverse porous materials have been selected as promising candidates for detecting and removing harmful materials, but the most appropriate pore structure and surface functional groups, both important factors for effective adsorbency, have not yet been fully elucidated. In particular, there is limited information relating to the use of activated carbon materials for effective adsorbent of specific pollutants. Here, the pore structure and surface functionality of polyacrylonitrile-based activated carbon fibers were investigated to develop an efficient adsorbent for polar pollutants. The effect of pore structure and surface functional groups on removal capability was investigated. The activated carbons with higher nitrogen content show a great ability to absorb formaldehyde because of their increased affinity with polar pollutants. In particular, nitrogen functional groups that neighbor oxygen atoms play an important role in maximizing adsorption capability. However, because there is also a similar increase in water affinity in adsorbents with polar functional groups, there is a considerable decrease in adsorption ability under humid conditions because of preferential adsorption of water to adsorbents. Therefore, it can be concluded that pore structures, surface functional groups and the water affinity of any adsorbent should be considered together to develop an effective and practical adsorbent for polar pollutants. These studies can provide vital

Role of bioavailable iron in coal dust-induced activation of activator protein-1 and nuclear factor of activated T cells: difference between Pennsylvania and Utah coal dusts.

Science.gov (United States)

Huang, Chuanshu; Li, Jingxia; Zhang, Qi; Huang, Xi

2002-11-01

Activator protein-1 (AP-1) and nuclear factor of activated T cells (NFAT) are two important transcription factors responsible for the regulation of cytokines, which are involved in cell proliferation and inflammation. Coal workers' pneumoconiosis (CWP) is an occupational lung disease that may be related to chronic inflammation caused by coal dust exposure. In the present study, we demonstrate that coal from the Pennsylvania (PA) coalmine region, which has a high prevalence of CWP, can activate both AP-1 and NFAT in JB6 mouse epidermal cells. In contrast, coal from the Utah (UT) coalmine region, which has a low prevalence of CWP, has no such effects. The PA coal stimulates mitogen-activated protein kinase (MAPK) family members of extracellular signal-regulated kinases (ERKs) and p38 MAPK but not c-Jun-NH(2)-terminal kinases, as determined by the phosphorylation assay. The increase in AP-1 by the PA coal was completely eliminated by the pretreatment of cells with PD98059, a specific MAPK kinase inhibitor, and SB202190, a p38 kinase inhibitor, further confirming that the PA coal-induced AP-1 activation is mediated through ERKs and p38 MAPK pathways. Deferoxamine (DFO), an iron chelator, synergistically enhanced the PA coal-induced AP-1 activity, but inhibited NFAT activity. For comparison, cells were treated with ferrous sulfate and/or DFO. We have found that iron transactivated both AP-1 and NFAT, and DFO further enhanced iron-induced AP-1 activation but inhibited NFAT. These results indicate that activation of AP-1 and NFAT by the PA coal is through bioavailable iron present in the coal. These data are in agreement with our previous findings that the prevalence of CWP correlates well with levels of bioavailable iron in coals from various mining regions.

Enhancement in irradiated mononuclear cells in culture of mitogen-induced incorporation of [3H]thymidine by homologous conditioned medium

International Nuclear Information System (INIS)

Sandru, G.; Greiner, R.

1994-01-01

Incorporation of [ 3 H]thymidine in irradiated peripheral blood mononuclear cell cultures irradiated in vitro was stimulated significantly by either concanavalin A or phytohemagglutinin only in the presence of homologous conditioned medium. Production of this activity by mononuclear cells was enhanced by irradiation and/or pulsed exposure to puromycin but was abolished by actinomycin D. Addition of anti-interleukin 1 or anti-interleukin 2 monoclonal antibodies to the conditioned medium before assay did not influence the stimulatory action. A similar significant stimulation of mononuclear cell cultures irradiated with 6 Gy by concanavalin A was obtained when purified preparations of homologous conditioned medium were used in the assay. Purification was done by ultrafiltration and concentration, heparin agarose chromatography, ammonium sulfate precipitation, concanavalin A agarose chromatography, DEAE-ion exchange chromatography and HPLC gel filtration chromatography. With SDS-PAGE and silver staining, the active HPLC fraction gave one band of 50 kDa, suggesting that this protein is responsible for the co-stimulatory effect of homologous conditioned medium for both mitogen-induced irradiated and nonirradiated mononuclear cell cultures. 42 refs., 9 figs., 3 tabs

COMPARISON OF CHAOTIC AND FRACTAL PROPERTIES OF POLAR FACULAE WITH SUNSPOT ACTIVITY

Energy Technology Data Exchange (ETDEWEB)

Deng, L. H.; Xiang, Y. Y.; Dun, G. T. [Yunnan Observatories, Chinese Academy of Sciences, Kunming 650216 (China); Li, B., E-mail: wooden@escience.cn [Shandong Provincial Key Laboratory of Optical Astronomy and Solar-Terrestrial Environment, School of Space Science and Physics, Shandong University at Weihai, Weihai 264209 (China)

2016-01-15

The solar magnetic activity is governed by a complex dynamo mechanism and exhibits a nonlinear dissipation behavior in nature. The chaotic and fractal properties of solar time series are of great importance to understanding the solar dynamo actions, especially with regard to the nonlinear dynamo theories. In the present work, several nonlinear analysis approaches are proposed to investigate the nonlinear dynamical behavior of the polar faculae and sunspot activity for the time interval from 1951 August to 1998 December. The following prominent results are found: (1) both the high- and the low-latitude solar activity are governed by a three-dimensional chaotic attractor, and the chaotic behavior of polar faculae is the most complex, followed by that of the sunspot areas, and then the sunspot numbers; (2) both the high- and low-latitude solar activity exhibit a high degree of persistent behavior, and their fractal nature is due to such long-range correlation; (3) the solar magnetic activity cycle is predictable in nature, but the high-accuracy prediction should only be done for short- to mid-term due to its intrinsically dynamical complexity. With the help of the Babcock–Leighton dynamo model, we suggest that the nonlinear coupling of the polar magnetic fields with strong active-region fields exhibits a complex manner, causing the statistical similarities and differences between the polar faculae and the sunspot-related indicators.

Overexpression of the mitogen-activated protein kinase gene ...

Indian Academy of Sciences (India)

1Department of Agricultural Biotechnology, National Academy of Agricultural Science, ... 4Department of Biological Sciences, Seoul National University, Seoul ... Supplementary figures pertaining to this article are available on the Journal of Biosciences Website at .... International Rice Research Institute (Los Baños, Philip-.

Aqueous exposure to Aroclor 1254 modulates the mitogenic response of Atlantic salmon anterior kidney T-cells: Indications of short- and long-term immunomodulation

International Nuclear Information System (INIS)

Iwanowicz, Luke R.; Lerner, Darren T.; Blazer, Vicki S.; McCormick, Stephen D.

2005-01-01

Polychlorinated biphenyls (PCBs) exist as persistent organic pollutants in numerous river systems in the United States. Unfortunately, some of these rivers are sites of active Atlantic salmon restoration programs, and polychlorinated biphenyls have been implicated as ancillary factors contributing to failed salmon restoration. Here, we investigate the immediate and chronic effects of intermediate duration aqueous PCB exposure (1 or 10 μg L -1 Aroclor 1254) on the mitogen-stimulated lymphoproliferative response of Atlantic salmon anterior kidney leukocytes (AKLs). A short-term study was designed to examine immunomodulation in Atlantic salmon smolts immediately following 21 days of aqueous exposure, while a long-term study evaluated chronic impacts in the mitogen response in parr 15 months post-exposure as larvae. The proliferative response of AKLs to the mitogens concanavalin A (CON A), phytohemaglutinnin-P (PHA-P), pokeweed mitogen (PWM), and lipopolysaccharide were used as an indice of immunomodulation. The proliferative response to the T-cell mitogens CON A and PHA-P was significantly increased in the 10 μg L -1 group (n = 10; P = 0.043 and 0.002, respectively) immediately following exposure of smolts. Additionally, The PHA-P response was significantly increased in the 1 μg L -1 exposure group (n = 10, P = 0.036). In fish treated as larvae and tested 15 months later, the PHA-P sensitive populations exhibited elevated proliferation in the 1 and 10 μg L -1 groups (n = 12, P -1 treated groups. These results demonstrate an immunomodulatory effect of PCBs on T-cell mitogen sensitive populations of lymphocytes in Atlantic salmon as well as long-term immunomodulation in PHA-P and PWM sensitive populations

Primate Torpor: Regulation of Stress-activated Protein Kinases During Daily Torpor in the Gray Mouse Lemur, Microcebus murinus

Directory of Open Access Journals (Sweden)

Kyle K. Biggar

2015-04-01

Full Text Available Very few selected species of primates are known to be capable of entering torpor. This exciting discovery means that the ability to enter a natural state of dormancy is an ancestral trait among primates and, in phylogenetic terms, is very close to the human lineage. To explore the regulatory mechanisms that underlie primate torpor, we analyzed signal transduction cascades to discover those involved in coordinating tissue responses during torpor. The responses of mitogen-activated protein kinase (MAPK family members to primate torpor were compared in six organs of control (aroused versus torpid gray mouse lemurs, Microcebus murinus. The proteins examined include extracellular signal-regulated kinases (ERKs, c-jun NH2-terminal kinases (JNKs, MAPK kinase (MEK, and p38, in addition to stress-related proteins p53 and heat shock protein 27 (HSP27. The activation of specific MAPK signal transduction pathways may provide a mechanism to regulate the expression of torpor-responsive genes or the regulation of selected downstream cellular processes. In response to torpor, each MAPK subfamily responded differently during torpor and each showed organ-specific patterns of response. For example, skeletal muscle displayed elevated relative phosphorylation of ERK1/2 during torpor. Interestingly, adipose tissues showed the highest degree of MAPK activation. Brown adipose tissue displayed an activation of ERK1/2 and p38, whereas white adipose tissue showed activation of ERK1/2, p38, MEK, and JNK during torpor. Importantly, both adipose tissues possess specialized functions that are critical for torpor, with brown adipose required for non-shivering thermogenesis and white adipose utilized as the primary source of lipid fuel for torpor. Overall, these data indicate crucial roles of MAPKs in the regulation of primate organs during torpor.

Hypoxia-induced mitogenic factor enhances angiogenesis by promoting proliferation and migration of endothelial cells

International Nuclear Information System (INIS)

Tong Qiangsong; Zheng Liduan; Li Bo; Wang Danming; Huang Chuanshu; Matuschak, George M.; Li Dechun

2006-01-01

Our previous studies have indicated that hypoxia-induced mitogenic factor (HIMF) has angiogenic properties in an in vivo matrigel plug model and HIMF upregulates expression of vascular endothelial growth factor (VEGF) in mouse lungs and cultured lung epithelial cells. However, whether HIMF exerts angiogenic effects through modulating endothelial cell function remains unknown. In this study, mouse aortic rings cultured with recombinant HIMF protein resulted in enhanced vascular sprouting and increased endothelial cell spreading as confirmed by Dil-Ac-LDL uptake, von Willebrand factor and CD31 staining. In cultured mouse endothelial cell line SVEC 4-10, HIMF dose-dependently enhanced cell proliferation, in vitro migration and tubulogenesis, which was not attenuated by SU1498, a VEGFR2/Flk-1 receptor tyrosine kinase inhibitor. Moreover, HIMF stimulation resulted in phosphorylation of Akt, p38 and ERK1/2 kinases in SVEC 4-10 cells. Treatment of mouse aortic rings and SVEC 4-10 cells with LY294002, but not SB203580, PD098059 or U0126, abolished HIMF-induced vascular sprouting and angiogenic responses. In addition, transfection of a dominant-negative mutant of phosphatidylinositol 3-kinase (PI-3K), Δp85, blocked HIMF-induced phosphorylation of Akt, endothelial activation and tubulogenesis. These results indicate that HIMF enhances angiogenesis by promoting proliferation and migration of endothelial cells via activation of the PI-3K/Akt pathways

Expression of human choline kinase in NIH 3T3 fibroblasts increases the mitogenic potential of insulin and insulin-like growth factor I.

Science.gov (United States)

Chung, T; Huang, J S; Mukherjee, J J; Crilly, K S; Kiss, Z

2000-05-01

In mammalian cells, growth factors, oncogenes, and carcinogens stimulate phosphocholine (PCho) synthesis by choline kinase (CK), suggesting that PCho may regulate cell growth. To validate the role of PCho in mitogenesis, we determined the effects of insulin, insulin-like growth factor I (IGF-I), and other growth factors on DNA synthesis in NIH 3T3 fibroblast sublines highly expressing human choline kinase (CK) without increasing phosphatidylcholine synthesis. In serum-starved CK expressor cells, insulin and IGF-I stimulated DNA synthesis, p70 S6 kinase (p70 S6K) activity, phosphatidylinositol 3-kinase (PI3K) activity, and activating phosphorylation of p42/p44 mitogen-activated protein kinases (MAPK) to greater extents than in the corresponding vector control cells. Furthermore, the CK inhibitor hemicholinium-3 (HC-3) inhibited insulin- and IGF-I-induced DNA synthesis in the CK overexpressors, but not in the vector control cells. The results indicate that high cellular levels of PCho potentiate insulin- and IGF-I-induced DNA synthesis by MAPK- and p70 S6K-regulated mechanisms.

Novel Antiplatelet Activity of Minocycline Involves Inhibition of MLK3-p38 Mitogen Activated Protein Kinase Axis.

Science.gov (United States)

Jackson, Joseph W; Singh, Meera V; Singh, Vir B; Jones, Letitia D; Davidson, Gregory A; Ture, Sara; Morrell, Craig N; Schifitto, Giovanni; Maggirwar, Sanjay B

2016-01-01

Platelets play an essential role in hemostasis and wound healing by facilitating thrombus formation at sites of injury. Platelets also mediate inflammation and contain several pro-inflammatory molecules including cytokines and chemokines that mediate leukocyte recruitment and activation. Not surprisingly, platelet dysfunction is known to contribute to several inflammatory disorders. Antiplatelet therapies, such as aspirin, adenosine diphosphate (ADP) antagonists, glycoprotein IIb/IIIa (GPIIb/IIIa) inhibitors, and anticoagulants such as warfarin, dampen platelet activity at the risk of unwarranted bleeding. Thus, the development of drugs that reduce platelet-mediated inflammation without interfering with thrombus formation is of importance to combat platelet-associated disorders. We have shown here for the first time that the tetracycline antibiotic, minocycline, administered to HIV-infected individuals reduces plasma levels of soluble CD40L and platelet factor 4 levels, host molecules predominately released by platelets. Minocycline reduced the activation of isolated platelets in the presence of the potent platelet activator, thrombin, as measured by ELISA and flow cytometry. Platelet degranulation was reduced upon exposure to minocycline as shown by mepacrine retention and flow cytometry. However, minocycline had no effect on spreading, aggregation, GPIIb/IIIa activation, or in vivo thrombus formation. Lastly, immunoblot analysis suggests that the antiplatelet activity of minocycline is likely mediated by inhibition of mixed lineage kinase 3 (MLK3)-p38 MAPK signaling axis and loss of p38 activity. Our findings provide a better understanding of platelet biology and a novel repurposing of an established antibiotic, minocycline, to specifically reduce platelet granule release without affecting thrombosis, which may yield insights in generating novel, specific antiplatelet therapies.

Cytokine production by porcine mononuclear leukocytes stimulated by mitogens

Czech Academy of Sciences Publication Activity Database

Rašková, G.; Kovářů, František; Bártová, J.

2005-01-01

Roč. 74, - (2005), s. 521-525 ISSN 0001-7213 R&D Projects: GA ČR GA524/05/0267 Institutional research plan: CEZ:AV0Z50450515 Keywords : cytokine * ELISpot * mitogen Subject RIV: ED - Physiology Impact factor: 0.353, year: 2005

Cell-type-specific activation of mitogen-activated protein kinases in PAN-induced progressive renal disease in rats

International Nuclear Information System (INIS)

Park, Sang-Joon; Jeong, Kyu-Shik

2004-01-01

We examined the time-course activation and the cell-type specific role of MAP kinases in puromycin aminonucleoside (PAN)-induced renal disease. The maximal activation of c-Jun-NH 2 -terminal kinase (JNK), extracellular signal regulated kinase (ERK), and p38 MAP kinase was detected on Days 52, 38, and 38 after PAN-treatment, respectively. p-JNK was localized in mesangial and proximal tubular cells at the early renal injury. It was expressed, therefore, in the inflammatory cells of tubulointerstitial lesions. While, p-ERK was markedly increased in the glomerular regions and macrophages p-p38 was observed in glomerular endothelial cells, tubular cells, and some inflammatory cells. The results show that the activation of MAP kinases in the early renal injury by PAN-treatment involves cellular changes such as cell proliferation or apoptosis in renal native cells. The activation of MAP kinases in infiltrated inflammatory cells and fibrotic cells plays an important role in destructive events such as glomerulosclerosis and tubulointerstitial fibrosis

Mycobacterium tuberculosis Latent Antigen Rv2029c from the Multistage DNA Vaccine A39 Drives TH1 Responses via TLR-mediated Macrophage Activation

Directory of Open Access Journals (Sweden)

Haibo Su

2017-11-01

Full Text Available Targeting of Mycobacterium tuberculosis (MTB latent antigens comprises a crucial strategy for the development of alternative tuberculosis (TB vaccine(s that protects against TB reactivation. Here, we generated a multistage DNA vaccine, A39, containing the early antigens Ag85A and Rv3425 as well as the latency-associated protein Rv2029c, which conferred protective immunity in a pre-exposure mouse model. Moreover, administration of the A39 vaccination after MTB exposure inhibited reactivation and resulted in significantly lower bacterial loads in the lungs and spleen of mice, compared to those in the control population. Subsequently, we investigated the effect of Rv2029c on innate immunity and characterized the molecular details of the interaction of this protein with the host via iTRAQ proteomic and biochemical assay analyses. Rv2029c activated macrophages, triggered the production of pro-inflammatory cytokines, and promoted toll-like receptor/mitogen-activated protein kinase (TLR/MAPK-dependent macrophage apoptosis. Furthermore, Rv2029c treatment enhanced the ability of Mycobacterium bovis Bacillus Calmette-Guérin (BCG-infected macrophages to present antigens to CD4+ T cells in vitro, which correlated with an increase in MHC-II expression. Lastly, Rv2029c-treated macrophages activated T cells, effectively polarized CD4+ and CD8+ T cells to secrete IFN-γ and IL-2, and specifically expanded a population of CD44highCD62LlowCD4+/CD8+ effector/memory cells, indicating that Rv2029c, as a specific recall antigen, contributes to Th1 polarization in T cell immunity. These results suggest that Rv2029c and A39 comprise promising targets for the development of next-generation clinical TB therapeutic vaccines.

The effect of G protein-coupled receptor kinase 2 (GRK2) on lactation and on proliferation of mammary epithelial cells from dairy cows.

Science.gov (United States)

Hou, Xiaoming; Hu, Hongliu; Lin, Ye; Qu, Bo; Gao, Xuejun; Li, Qingzhang

2016-07-01

Milk protein is an important component of milk and a nutritional source for human consumption. To better understand the molecular events underlying synthesis of milk proteins, the global gene expression patterns in mammary glands of dairy cow with high-quality milk (>3% milk protein; >3.5% milk fat) and low-quality milk (milk protein; milk fat) were examined via digital gene expression study. A total of 139 upregulated and 66 downregulated genes were detected in the mammary tissues of lactating cows with high-quality milk compared with the tissues of cows with low-quality milk. A pathway enrichment study of these genes revealed that the top 5 pathways that were differentially affected in the tissues of cows with high- versus low-quality milk involved metabolic pathways, cancer, cytokine-cytokine receptor interactions, regulation of the actin cytoskeleton, and insulin signaling. We also found that the G protein-coupled receptor kinase 2 (GRK2) was one of the most highly upregulated genes in lactating mammary tissue with low-quality milk compared with tissue with high-quality milk. The knockdown of GRK2 in cultured bovine mammary epithelial cells enhanced CSN2 expression and activated signaling molecules related to translation, including protein kinase B, mammalian target of rapamycin, and p70 ribosomal protein S6 kinase 1 (S6K1), whereas overexpression of GRK2 had the opposite effects. However, expression of genes involved in the mitogen-activated protein kinase pathway was positively regulated by GRK2. Therefore, GRK2 seems to act as a negative mediator of milk-protein synthesis via the protein kinase B-mammalian target of rapamycin signaling axis. Furthermore, GRK2 may negatively control milk-protein synthesis by activating the mitogen-activated protein kinase pathway in dairy cow mammary epithelial cells. Copyright © 2016 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.

Aqueous exposure to Aroclor 1254 modulates the mitogenic response of Atlantic salmon anterior kidney T-cells: indications of short- and long-term immunomodulation.

Science.gov (United States)

Iwanowicz, Luke R; Lerner, Darren T; Blazer, Vicki S; McCormick, Stephen D

2005-05-15

Polychlorinated biphenyls (PCBs) exist as persistent organic pollutants in numerous river systems in the United States. Unfortunately, some of these rivers are sites of active Atlantic salmon restoration programs, and polychlorinated biphenyls have been implicated as ancillary factors contributing to failed salmon restoration. Here, we investigate the immediate and chronic effects of intermediate duration aqueous PCB exposure (1 or 10 microgL-1 Aroclor 1254) on the mitogen-stimulated lymphoproliferative response of Atlantic salmon anterior kidney leukocytes (AKLs). A short-term study was designed to examine immunomodulation in Atlantic salmon smolts immediately following 21 days of aqueous exposure, while a long-term study evaluated chronic impacts in the mitogen response in parr 15 months post-exposure as larvae. The proliferative response of AKLs to the mitogens concanavalin A (CON A), phytohemaglutinnin-P (PHA-P), pokeweed mitogen (PWM), and lipopolysaccharide were used as an indice of immunomodulation. The proliferative response to the T-cell mitogens CON A and PHA-P was significantly increased in the 10 microgL-1 group (n=10; P=0.043 and 0.002, respectively) immediately following exposure of smolts. Additionally, The PHA-P response was significantly increased in the 1 microgL-1 exposure group (n=10, P=0.036). In fish treated as larvae and tested 15 months later, the PHA-P sensitive populations exhibited elevated proliferation in the 1 and 10 microgL-1 groups (n=12, P<0.04) relative to the vehicle control while the PWM response was significantly increased (n=12, P=0.036) only in the 10 microgL-1 treated groups. These results demonstrate an immunomodulatory effect of PCBs on T-cell mitogen sensitive populations of lymphocytes in Atlantic salmon as well as long-term immunomodulation in PHA-P and PWM sensitive populations.

Solitons in proteins at non-zero temperatures with allowance for the fluctuations of polarization

International Nuclear Information System (INIS)

Simo, E.; Caputo, J.G.

2007-06-01

A model for the nonlinear transfer of vibrational energy in molecular chains is derived at temperatures of realistic interest for transport in proteins. The study includes the influence of the fluctuations of polarization in the chain. This theory exhibits a new form of temperature-dependence in intrinsic parameters of alpha-helix, and consequently in the coefficients of the nonlinear Schroedinger equation governing the system and in the solitons' parameters. Thermal fluctuations are analysed in the basis of the non-Gaussian approximation and the total free-energy of the alpha-helix is determined to elucidate the denaturation process of the protein. (author)

Cell adhesion and EGFR activation regulate EphA2 expression in cancer

DEFF Research Database (Denmark)

Larsen, Alice Bjerregaard; Stockhausen, Marie-Thérése; Poulsen, Hans Skovgaard

2010-01-01

family kinases (SRC). Moreover, the results show that adhesion-induced EGFR activation and EphA2 expression is affected by interactions with extracellular matrix (ECM) proteins working as integrin ligands. Stimulation with the EphA2 ligand, ephrinA1 inhibited ERK phosphorylation and cancer cell viability...... largely unknown. Here we show that the expression of EphA2 in in vitro cultured cells, is restricted to cells growing adherently and that adhesion-induced EphA2 expression is dependent upon activation of the epidermal growth factor receptor (EGFR), mitogen activated protein kinase kinase (MEK) and Src...

Regulation of p21ras activity

DEFF Research Database (Denmark)

Lowy, D R; Zhang, K; DeClue, J E

1992-01-01

The ras genes encode GTP/GDP-binding proteins that participate in mediating mitogenic signals from membrane tyrosine kinases to downstream targets. The activity of p21ras is determined by the concentration of GTP-p21ras, which is tightly regulated by a complex array of positive and negative control...... mechanisms. GAP and NF1 can negatively regulate p21ras activity by stimulating hydrolysis of GTP bound to p21ras. Other cellular factors can positively regulate p21ras by stimulating GDP/GTP exchange....