WorldWideScience

Sample records for adhesion kinase functions

  1. FAK dimerization controls its kinase-dependent functions at focal adhesions

    KAUST Repository

    Brami-Cherrier, Karen

    2014-01-30

    Focal adhesion kinase (FAK) controls adhesion-dependent cell motility, survival, and proliferation. FAK has kinase-dependent and kinase-independent functions, both of which play major roles in embryogenesis and tumor invasiveness. The precise mechanisms of FAK activation are not known. Using x-ray crystallography, small angle x-ray scattering, and biochemical and functional analyses, we show that the key step for activation of FAK\\'s kinase-dependent functions-autophosphorylation of tyrosine-397-requires site-specific dimerization of FAK. The dimers form via the association of the N-terminal FERM domain of FAK and are stabilized by an interaction between FERM and the C-terminal FAT domain. FAT binds to a basic motif on FERM that regulates co-activation and nuclear localization. FAK dimerization requires local enrichment, which occurs specifically at focal adhesions. Paxillin plays a dual role, by recruiting FAK to focal adhesions and by reinforcing the FAT:FERM interaction. Our results provide a structural and mechanistic framework to explain how FAK combines multiple stimuli into a site-specific function. The dimer interfaces we describe are promising targets for blocking FAK activation. © 2014 The Authors.

  2. Conformational Dynamics of the Focal Adhesion Targeting Domain Control Specific Functions of Focal Adhesion Kinase in Cells

    KAUST Repository

    Kadaré, Gress

    2015-01-02

    Focal adhesion (FA) kinase (FAK) regulates cell survival and motility by transducing signals from membrane receptors. The C-terminal FA targeting (FAT) domain of FAK fulfils multiple functions, including recruitment to FAs through paxillin binding. Phosphorylation of FAT on Tyr925 facilitates FA disassembly and connects to the MAPK pathway through Grb2 association, but requires dissociation of the first helix (H1) of the four-helix bundle of FAT. We investigated the importance of H1 opening in cells by comparing the properties of FAK molecules containing wild-type or mutated FAT with impaired or facilitated H1 openings. These mutations did not alter the activation of FAK, but selectively affected its cellular functions, including self-association, Tyr925 phosphorylation, paxillin binding, and FA targeting and turnover. Phosphorylation of Tyr861, located between the kinase and FAT domains, was also enhanced by the mutation that opened the FAT bundle. Similarly phosphorylation of Ser910 by ERK in response to bombesin was increased by FAT opening. Although FAK molecules with the mutation favoring FAT opening were poorly recruited at FAs, they efficiently restored FA turnover and cell shape in FAK-deficient cells. In contrast, the mutation preventing H1 opening markedly impaired FAK function. Our data support the biological importance of conformational dynamics of the FAT domain and its functional interactions with other parts of the molecule.

  3. Focal Adhesion Kinase: Insight into Molecular Roles and Functions in Hepatocellular Carcinoma

    Directory of Open Access Journals (Sweden)

    Nadia Panera

    2017-01-01

    Full Text Available Hepatocellular carcinoma (HCC is the third leading cause of cancer-related death worldwide. Due to the high incidence of post-operative recurrence after current treatments, the identification of new and more effective drugs is required. In previous years, new targetable genes/pathways involved in HCC pathogenesis have been discovered through the help of high-throughput sequencing technologies. Mutations in TP53 and β-catenin genes are the most frequent aberrations in HCC. However, approaches able to reverse the effect of these mutations might be unpredictable. In fact, if the reactivation of proteins, such as p53 in tumours, holds great promise as anticancer therapy, there are studies arguing that chronic activation of these types of molecules may be deleterious. Thus, recently the efforts on potential targets have focused on actionable mutations, such as those occurring in the gene encoding for focal adhesion kinase (FAK. This tyrosine kinase, localized to cellular focal contacts, is over-expressed in a variety of human tumours, including HCC. Moreover, several lines of evidence demonstrated that FAK depletion or inhibition impair in vitro and in vivo HCC growth and metastasis. Here, we provide an overview of FAK expression and activity in the context of tumour biology, discussing the current evidence of its connection with HCC development and progression.

  4. In vitro phosphorylation of the focal adhesion targeting domain of focal adhesion kinase by Src kinase.

    Science.gov (United States)

    Cable, Jennifer; Prutzman, Kirk; Gunawardena, Harsha P; Schaller, Michael D; Chen, Xian; Campbell, Sharon L

    2012-03-20

    Focal adhesion kinase (FAK), a key regulator of cell adhesion and migration, is overexpressed in many types of cancer. The C-terminal focal adhesion targeting (FAT) domain of FAK is necessary for proper localization of FAK to focal adhesions and subsequent activation. Phosphorylation of Y926 in the FAT domain by the tyrosine kinase Src has been shown to promote metastasis and invasion in vivo by linking the FAT domain to the MAPK pathway via its interaction with growth factor receptor-bound protein 2. Several groups have reported that inherent conformational dynamics in the FAT domain likely regulate phosphorylation of Y926; however, what regulates these dynamics is unknown. In this paper, we demonstrate that there are two sites of in vitro Src-mediated phosphorylation in the FAT domain: Y926, which has been shown to affect FAK function in vivo, and Y1008, which has no known biological role. The phosphorylation of these two tyrosine residues is pH-dependent, but this does not reflect the pH dependence of Src kinase activity. Circular dichroism and nuclear magnetic resonance data indicate that the stability and conformational dynamics of the FAT domain are sensitive to changes in pH over a physiological pH range. In particular, regions of the FAT domain previously shown to regulate phosphorylation of Y926 as well as regions near Y1008 show pH-dependent dynamics on the microsecond to millisecond time scale.

  5. The extracellular matrix and focal adhesion kinase signaling regulate cancer stem cell function in pancreatic ductal adenocarcinoma.

    Directory of Open Access Journals (Sweden)

    Asma Begum

    Full Text Available Cancer stem cells (CSCs play an important role in the clonogenic growth and metastasis of pancreatic ductal adenocarcinoma (PDAC. A hallmark of PDAC is the desmoplastic reaction, but the impact of the tumor microenvironment (TME on CSCs is unknown. In order to better understand the mechanisms, we examined the impact of extracellular matrix (ECM proteins on PDAC CSCs. We quantified the effect of ECM proteins, β1-integrin, and focal adhesion kinase (FAK on clonogenic PDAC growth and migration in vitro and tumor initiation, growth, and metastasis in vivo in nude mice using shRNA and overexpression constructs as well as small molecule FAK inhibitors. Type I collagen increased PDAC tumor initiating potential, self-renewal, and the frequency of CSCs through the activation of FAK. FAK overexpression increased tumor initiation, whereas a dominant negative FAK mutant or FAK kinase inhibitors reduced clonogenic PDAC growth in vitro and in vivo. Moreover, the FAK inhibitor VS-4718 extended the anti-tumor response to gemcitabine and nab-paclitaxel in patient-derived PDAC xenografts, and the loss of FAK expression limited metastatic dissemination of orthotopic xenografts. Type I collagen enhances PDAC CSCs, and both kinase-dependent and independent activities of FAK impact PDAC tumor initiation, self-renewal, and metastasis. The anti-tumor impact of FAK inhibitors in combination with standard chemotherapy support the clinical testing of this combination.

  6. Focal adhesion kinase maintains, but not increases the adhesion of dental pulp cells.

    Science.gov (United States)

    Qian, Yuyan; Shao, Meiying; Zou, Wenlin; Wang, Linyan; Cheng, Ran; Hu, Tao

    2017-04-01

    Focal adhesion kinase (FAK) functions as a key enzyme in the integrin-mediated adhesion-signalling pathway. Here, we aimed to investigate the effects of FAK on adhesion of human dental pulp (HDP) cells. We transfected lentiviral vectors to silence or overexpress FAK in HDP cells ex vivo. Early cell adhesion, cell survival and focal contacts (FCs)-related proteins (FAK and paxillin) were examined. By using immunofluorescence, the formation of FCs and cytoskeleton was detected, respectively. We found that both adhesion and survival of HDP cells were suppressed by FAK inhibition. However, FAK overexpression slightly inhibited cell adhesion and exhibited no change in cell survival compared with the control. A thick rim of cytoskeleton accumulated and smaller dot-shaped FCs appeared in FAK knockdown cells. Phosphorylation of paxillin (p-paxillin) was inhibited in FAK knockdown cells, verifying that the adhesion was inhibited. Less cytoskeleton and elongated FCs were observed in FAK-overexpressed cells. However, p-paxillin had no significant difference compared with the control. In conclusion, the data suggest that FAK maintains cell adhesion, survival and cytoskeleton formation, but excessive FAK has no positive effects on these aspects.

  7. How to awaken your nanomachines: Site-specific activation of focal adhesion kinases through ligand interactions

    KAUST Repository

    Walkiewicz, Katarzyna Wiktoria

    2015-06-17

    The focal adhesion kinase (FAK) and the related protein-tyrosine kinase 2-beta (Pyk2) are highly versatile multidomain scaffolds central to cell adhesion, migration, and survival. Due to their key role in cancer metastasis, understanding and inhibiting their functions are important for the development of targeted therapy. Because FAK and Pyk2 are involved in many different cellular functions, designing drugs with partial and function-specific inhibitory effects would be desirable. Here, we summarise recent progress in understanding the structural mechanism of how the tug-of-war between intramolecular and intermolecular interactions allows these protein ‘nanomachines’ to become activated in a site-specific manner.

  8. Quantitative measurement of changes in adhesion force involving focal adhesion kinase during cell attachment, spread, and migration

    International Nuclear Information System (INIS)

    Wu, C.-C.; Su, H.-W.; Lee, C.-C.; Tang, M.-J.; Su, F.-C.

    2005-01-01

    Focal adhesion kinase (FAK) is a critical protein for the regulation of integrin-mediated cellular functions and it can enhance cell motility in Madin-Darby canine kidney (MDCK) cells by hepatocyte growth factor (HGF) induction. We utilized optical trapping and cytodetachment techniques to measure the adhesion force between pico-Newton and nano-Newton (nN) for quantitatively investigating the effects of FAK on adhesion force during initial binding (5 s), beginning of spreading (30 min), spreadout (12 h), and migration (induced by HGF) in MDCK cells with overexpressed FAK (FAK-WT), FAK-related non-kinase (FRNK), as well as normal control cells. Optical tweezers was used to measure the initial binding force between a trapped cell and glass coverslide or between a trapped bead and a seeded cell. In cytodetachment, the commercial atomic force microscope probe with an appropriate spring constant was used as a cyto-detacher to evaluate the change of adhesion force between different FAK expression levels of cells in spreading, spreadout, and migrating status. The results demonstrated that FAK-WT significantly increased the adhesion forces as compared to FRNK cells throughout all the different stages of cell adhesion. For cells in HGF-induced migration, the adhesion force decreased to almost the same level (∼600 nN) regardless of FAK levels indicating that FAK facilitates cells to undergo migration by reducing the adhesion force. Our results suggest FAK plays a role of enhancing cell adhesive ability in the binding and spreading, but an appropriate level of adhesion force is required for HGF-induced cell migration

  9. Protein kinase C involvement in focal adhesion formation

    DEFF Research Database (Denmark)

    Woods, A; Couchman, J R

    1992-01-01

    Matrix molecules such as fibronectin can promote cell attachment, spreading and focal adhesion formation. Although some interactions of fibronectin with cell surface receptors have now been identified, the consequent activation of intracellular messenger systems by cell/matrix interactions have...... still to be elucidated. We show here that the kinase inhibitors H7 and HA1004 reduce focal adhesion and stress fiber formation in response to fibronectin in a dose-dependent manner, and that activators of protein kinase C can promote their formation under conditions where they do not normally form....... Fibroblasts spread within 1h on substrata composed of fibronectin and formed focal adhesions by 3h, as monitored by interference reflection microscopy (IRM) and by labeling for talin, vinculin and integrin beta 1 subunits. In addition, stress fibers were visible. When cells were allowed to spread for 1h...

  10. Phosphotyrosine enrichment identifies focal adhesion kinase and other tyrosine kinases for targeting in canine hemangiosarcoma.

    Science.gov (United States)

    Marley, K; Maier, C S; Helfand, S C

    2012-09-01

    Canine hemangiosarcoma (HSA) is an endothelial cell malignancy driven, in part, by activating mutations in receptor and non-receptor tyrosine kinases. Proteomics, Western blots and a tyrosine kinase inhibitor were used to elucidate activating mechanisms in HSA cell lines. Phosphotyrosine peptides from focal adhesion kinase (FAK) STAT3, Lyn, Fyn and other signal transduction kinases were identified by mass spectrometry. FAK was constitutively activated at tyrosine 397, the autophosphorylation site, and this was reversible with high concentrations of a FAK inhibitor. FAK inhibitor-14 suppressed migration and phosphorylation of FAK tyrosine 397 and tyrosines 576/577 and was cytotoxic to HSA cells suggesting FAK signalling may be an important contributor to canine HSA survival. © 2012 Blackwell Publishing Ltd.

  11. Protein kinase C, focal adhesions and the regulation of cell migration

    DEFF Research Database (Denmark)

    Fogh, Betina S; Multhaupt, Hinke A B; Couchman, John Robert

    2014-01-01

    in their intracellular compartment. Among these are tyrosine kinases, which have received a great deal of attention, whereas the serine/threonine kinase protein kinase C has received much less. Here the status of protein kinase C in focal adhesions and cell migration is reviewed, together with discussion of its roles...... and adhesion turnover. Focal adhesions, or focal contacts, are widespread organelles at the cell-matrix interface. They arise as a result of receptor interactions with matrix ligands, together with clustering. Recent analysis shows that focal adhesions contain a very large number of protein components...

  12. Focal adhesion kinase and paxillin promote migration and adhesion to fibronectin by swine skeletal muscle satellite cells

    Science.gov (United States)

    Wang, Dan; Gao, Chun-qi; Chen, Rong-qiang; Jin, Cheng-long; Li, Hai-chang; Yan, Hui-chao; Wang, Xiu-qi

    2016-01-01

    The focal adhesion kinase (FAK) signaling pathway contributes to the cell migration and adhesion that is critical for wound healing and regeneration of damaged muscle, but its function in skeletal muscle satellite cells (SCs) is less clear. We compared the migration and adhesion of SCs derived from two species of pig (Lantang and Landrace) in vitro, and explored how FAK signaling modulates the two processes. The results showed that Lantang SCs had greater ability to migrate and adhere to fibronection (P < 0.05) than Landrace SCs. Compared to Landrace SCs, Lantang SCs expressed many more focal adhesion (FA) sites, which were indicated by the presence of p-paxillin (Tyr118), and exhibited less F-actin reorganization 24 h after seeding onto fibronectin. Levels of p-FAK (Tyr397) and p-paxillin (Tyr118) were greater (P < 0.05) in Lantang SCs than Landrace SCs after migration for 24 h. Similarly, Lantang SCs showed much higher levels of p-FAK (Tyr397), p-paxillin (Tyr118) and p-Akt (Ser473) than Landrace SCs 2 h after adhesion. Treatment with the FAK inhibitor PF-573228 (5 or 10 μmol/L) inhibited Lantang SC migration and adhesion to fibronectin (P < 0.05), decreased levels of p-paxillin (Tyr118) and p-Akt (Ser473) (P < 0.05), and suppressed the formation of FA sites on migrating SCs. Thus FAK appears to play a key role in the regulation of SC migration and adhesion necessary for muscle regeneration. PMID:27127174

  13. Increased Expression of Focal Adhesion Kinase in Thyroid Cancer: Immunohistochemical Study

    OpenAIRE

    Kim, Sang Jin; Park, Jin Woo; Yoon, Ji Sung; Mok, Ji O; Kim, Yeo Joo; Park, Hyeong Kyu; Kim, Chul Hee; Byun, Dong Won; Lee, Yong Jin; Jin, So Young; Suh, Kyo Il; Yoo, Myung Hi

    2004-01-01

    Focal adhesion kinase (FAK) is a tyrosine kinase that is found in cellular structures called focal adhesions. FAK appears to be a key element in signal transduction pathways involved in cell adhesion and locomotion. FAK is overexpressed in various tumors, including tumors derived from regions of the head and neck, colon, breast, prostate, and liver. In this study, we investigated immunohistochemically whether FAK expression was increased in thyroid cancers. FAK staining was not seen in any of...

  14. Role of Cbl-associated protein/ponsin in receptor tyrosine kinase signaling and cell adhesion

    Directory of Open Access Journals (Sweden)

    Ritva Tikkanen

    2012-10-01

    Full Text Available The Cbl-associated protein/ponsin (CAP is an adaptor protein that contains a so-called Sorbin homology (SoHo domain and three Src homology 3 (SH3 domains which are engaged in diverse protein-protein interactions. CAP has been shown to function in the regulation of the actin cytoskeleton and cell adhesion and to be involved in the differentiation of muscle cells and adipocytes. In addition, it participates in signaling pathways through several receptor tyrosine kinases such as insulin and neurotrophin receptors. In the last couple of years, several studies have shed light on the details of these processes and identified novel interaction partners of CAP. In this review, we summarize these recent findings and provide an overview on the function of CAP especially in cell adhesion and membrane receptor signaling.

  15. Mitogen-Activated Protein Kinase (MAPK) Pathway Regulates Branching by Remodeling Epithelial Cell Adhesion

    Science.gov (United States)

    Ihermann-Hella, Anneliis; Lume, Maria; Miinalainen, Ilkka J.; Pirttiniemi, Anniina; Gui, Yujuan; Peränen, Johan; Charron, Jean; Saarma, Mart; Costantini, Frank; Kuure, Satu

    2014-01-01

    Although the growth factor (GF) signaling guiding renal branching is well characterized, the intracellular cascades mediating GF functions are poorly understood. We studied mitogen-activated protein kinase (MAPK) pathway specifically in the branching epithelia of developing kidney by genetically abrogating the pathway activity in mice lacking simultaneously dual-specificity protein kinases Mek1 and Mek2. Our data show that MAPK pathway is heterogeneously activated in the subset of G1- and S-phase epithelial cells, and its tissue-specific deletion results in severe renal hypodysplasia. Consequently to the deletion of Mek1/2, the activation of ERK1/2 in the epithelium is lost and normal branching pattern in mutant kidneys is substituted with elongation-only phenotype, in which the epithelium is largely unable to form novel branches and complex three-dimensional patterns, but able to grow without primary defects in mitosis. Cellular characterization of double mutant epithelium showed increased E-cadherin at the cell surfaces with its particular accumulation at baso-lateral locations. This indicates changes in cellular adhesion, which were revealed by electron microscopic analysis demonstrating intercellular gaps and increased extracellular space in double mutant epithelium. When challenged to form monolayer cultures, the mutant epithelial cells were impaired in spreading and displayed strong focal adhesions in addition to spiky E-cadherin. Inhibition of MAPK activity reduced paxillin phosphorylation on serine 83 while remnants of phospho-paxillin, together with another focal adhesion (FA) protein vinculin, were augmented at cell surface contacts. We show that MAPK activity is required for branching morphogenesis, and propose that it promotes cell cycle progression and higher cellular motility through remodeling of cellular adhesions. PMID:24603431

  16. Mitogen-activated protein kinase (MAPK pathway regulates branching by remodeling epithelial cell adhesion.

    Directory of Open Access Journals (Sweden)

    Anneliis Ihermann-Hella

    2014-03-01

    Full Text Available Although the growth factor (GF signaling guiding renal branching is well characterized, the intracellular cascades mediating GF functions are poorly understood. We studied mitogen-activated protein kinase (MAPK pathway specifically in the branching epithelia of developing kidney by genetically abrogating the pathway activity in mice lacking simultaneously dual-specificity protein kinases Mek1 and Mek2. Our data show that MAPK pathway is heterogeneously activated in the subset of G1- and S-phase epithelial cells, and its tissue-specific deletion results in severe renal hypodysplasia. Consequently to the deletion of Mek1/2, the activation of ERK1/2 in the epithelium is lost and normal branching pattern in mutant kidneys is substituted with elongation-only phenotype, in which the epithelium is largely unable to form novel branches and complex three-dimensional patterns, but able to grow without primary defects in mitosis. Cellular characterization of double mutant epithelium showed increased E-cadherin at the cell surfaces with its particular accumulation at baso-lateral locations. This indicates changes in cellular adhesion, which were revealed by electron microscopic analysis demonstrating intercellular gaps and increased extracellular space in double mutant epithelium. When challenged to form monolayer cultures, the mutant epithelial cells were impaired in spreading and displayed strong focal adhesions in addition to spiky E-cadherin. Inhibition of MAPK activity reduced paxillin phosphorylation on serine 83 while remnants of phospho-paxillin, together with another focal adhesion (FA protein vinculin, were augmented at cell surface contacts. We show that MAPK activity is required for branching morphogenesis, and propose that it promotes cell cycle progression and higher cellular motility through remodeling of cellular adhesions.

  17. Phosphatidylinositol 4,5-bisphosphate triggers activation of focal adhesion kinase by inducing clustering and conformational changes

    DEFF Research Database (Denmark)

    Goñi, Guillermina M; Epifano, Carolina; Boskovic, Jasminka

    2014-01-01

    Focal adhesion kinase (FAK) is a nonreceptor tyrosine kinase (NRTK) with key roles in integrating growth and cell matrix adhesion signals, and FAK is a major driver of invasion and metastasis in cancer. Cell adhesion via integrin receptors is well known to trigger FAK signaling, and many of the p...

  18. Mycosporine-Like Amino Acids Promote Wound Healing through Focal Adhesion Kinase (FAK) and Mitogen-Activated Protein Kinases (MAP Kinases) Signaling Pathway in Keratinocytes

    Science.gov (United States)

    Choi, Yun-Hee; Yang, Dong Joo; Kulkarni, Atul; Moh, Sang Hyun; Kim, Ki Woo

    2015-01-01

    Mycosporine-like amino acids (MAAs) are secondary metabolites found in diverse marine, freshwater, and terrestrial organisms. Evidence suggests that MAAs have several beneficial effects on skin homeostasis such as protection against UV radiation and reactive oxygen species (ROS). In addition, MAAs are also involved in the modulation of skin fibroblasts proliferation. However, the regulatory function of MAAs on wound repair in human skin is not yet clearly elucidated. To investigate the roles of MAAs on the wound healing process in human keratinocytes, three MAAs, Shinorine (SH), Mycosporine-glycine (M-Gly), and Porphyra (P334) were purified from Chlamydomonas hedlyei and Porphyra yezoensis. We found that SH, M-Gly, and P334 have significant effects on the wound healing process in human keratinocytes and these effects were mediated by activation of focal adhesion kinases (FAK), extracellular signal-regulated kinases (ERK), and c-Jun N-terminal kinases (JNK). These results suggest that MAAs accelerate wound repair by activating the FAK-MAPK signaling pathways. This study also indicates that MAAs can act as a new wound healing agent and further suggests that MAAs might be a novel biomaterial for wound healing therapies. PMID:26703626

  19. Mycosporine-Like Amino Acids Promote Wound Healing through Focal Adhesion Kinase (FAK and Mitogen-Activated Protein Kinases (MAP Kinases Signaling Pathway in Keratinocytes

    Directory of Open Access Journals (Sweden)

    Yun-Hee Choi

    2015-11-01

    Full Text Available Mycosporine-like amino acids (MAAs are secondary metabolites found in diverse marine, freshwater, and terrestrial organisms. Evidence suggests that MAAs have several beneficial effects on skin homeostasis such as protection against UV radiation and reactive oxygen species (ROS. In addition, MAAs are also involved in the modulation of skin fibroblasts proliferation. However, the regulatory function of MAAs on wound repair in human skin is not yet clearly elucidated. To investigate the roles of MAAs on the wound healing process in human keratinocytes, three MAAs, Shinorine (SH, Mycosporine-glycine (M-Gly, and Porphyra (P334 were purified from Chlamydomonas hedlyei and Porphyra yezoensis. We found that SH, M-Gly, and P334 have significant effects on the wound healing process in human keratinocytes and these effects were mediated by activation of focal adhesion kinases (FAK, extracellular signal-regulated kinases (ERK, and c-Jun N-terminal kinases (JNK. These results suggest that MAAs accelerate wound repair by activating the FAK-MAPK signaling pathways. This study also indicates that MAAs can act as a new wound healing agent and further suggests that MAAs might be a novel biomaterial for wound healing therapies.

  20. Focal adhesion kinase-dependent focal adhesion recruitment of SH2 domains directs SRC into focal adhesions to regulate cell adhesion and migration

    Science.gov (United States)

    Wu, Jui-Chung; Chen, Yu-Chen; Kuo, Chih-Ting; Wenshin Yu, Helen; Chen, Yin-Quan; Chiou, Arthur; Kuo, Jean-Cheng

    2015-01-01

    Directed cell migration requires dynamical control of the protein complex within focal adhesions (FAs) and this control is regulated by signaling events involving tyrosine phosphorylation. We screened the SH2 domains present in tyrosine-specific kinases and phosphatases found within FAs, including SRC, SHP1 and SHP2, and examined whether these enzymes transiently target FAs via their SH2 domains. We found that the SRC_SH2 domain and the SHP2_N-SH2 domain are associated with FAs, but only the SRC_SH2 domain is able to be regulated by focal adhesion kinase (FAK). The FAK-dependent association of the SRC_SH2 domain is necessary and sufficient for SRC FA targeting. When the targeting of SRC into FAs is inhibited, there is significant suppression of SRC-mediated phosphorylation of paxillin and FAK; this results in an inhibition of FA formation and maturation and a reduction in cell migration. This study reveals an association between FAs and the SRC_SH2 domain as well as between FAs and the SHP2_N-SH2 domains. This supports the hypothesis that the FAK-regulated SRC_SH2 domain plays an important role in directing SRC into FAs and that this SRC-mediated FA signaling drives cell migration. PMID:26681405

  1. The role of focal adhesion kinase in the regulation of cellular mechanical properties

    International Nuclear Information System (INIS)

    Mierke, Claudia Tanja

    2013-01-01

    The regulation of mechanical properties is necessary for cell invasion into connective tissue or intra- and extravasation through the endothelium of blood or lymph vessels. Cell invasion is important for the regulation of many healthy processes such as immune response reactions and wound healing. In addition, cell invasion plays a role in disease-related processes such as tumor metastasis and autoimmune responses. Until now the role of focal adhesion kinase (FAK) in regulating mechanical properties of cells and its impact on cell invasion efficiency is still not well known. Thus, this review focuses on mechanical properties regulated by FAK in comparison to the mechano-regulating protein vinculin. Moreover, it points out the connection between cancer cell invasion and metastasis and FAK by showing that FAK regulates cellular mechanical properties required for cellular motility. Furthermore, it sheds light on the indirect interaction of FAK with vinculin by binding to paxillin, which then impairs the binding of paxillin to vinculin. In addition, this review emphasizes whether FAK fulfills regulatory functions similar to vinculin. In particular, it discusses the differences and the similarities between FAK and vinculin in regulating the biomechanical properties of cells. Finally, this paper highlights that both focal adhesion proteins, vinculin and FAK, synergize their functions to regulate the mechanical properties of cells such as stiffness and contractile forces. Subsequently, these mechanical properties determine cellular invasiveness into tissues and provide a source sink for future drug developments to inhibit excessive cell invasion and hence, metastases formation. (paper)

  2. Focal adhesion kinase a potential therapeutic target for pancreatic cancer and malignant pleural mesothelioma.

    Science.gov (United States)

    Kanteti, Rajani; Mirzapoiazova, Tamara; Riehm, Jacob J; Dhanasingh, Immanuel; Mambetsariev, Bolot; Wang, Jiale; Kulkarni, Prakash; Kaushik, Garima; Seshacharyulu, Parthasarathy; Ponnusamy, Moorthy P; Kindler, Hedy L; Nasser, Mohd W; Batra, Surinder K; Salgia, Ravi

    2018-04-03

    The non-receptor cytoplasmic tyrosine kinase, Focal Adhesion Kinase (FAK) is known to play a key role in a variety of normal and cancer cellular functions such as survival, proliferation, migration and invasion. It is highly active and overexpressed in various cancers including Pancreatic Ductal Adenocarcinoma (PDAC) and Malignant Pleural Mesothelioma (MPM). Here, initially, we demonstrate that FAK is overexpressed in both PDAC and MPM cell lines. Then we analyze effects of two small molecule inhibitors PF-573228, and PF-431396, which are dual specificity inhibitors of FAK and proline rich tyrosine kinase 2 (PYK2), as well as VS-6063, another small molecule inhibitor that specifically inhibits FAK but not PYK2 for cell growth, motility and invasion of PDAC and MPM cell lines. Treatment with PF-573228, PF-431396 and VS-6063 cells resulted in a dose-dependent inhibition of growth and anchorage-independent colony formation in both cancer cell lines. Furthermore, these compounds suppressed the phosphorylation of FAK at its active site, Y397, and functionally induced significant apoptosis and cell cycle arrest in both cell lines. Using the ECIS (Electric cell-substrate impedance sensing) system, we found that treatment of both PF compounds suppressed adherence and migration of PDAC cells on fibronectin. Interestingly, 3D-tumor organoids derived from autochthonous KC (Kras;PdxCre) mice treated with PF-573228 revealed a significant decrease in tumor organoid size and increase in organoid cell death. Taken together, our results show that FAK is an important target for mesothelioma and pancreatic cancer therapy that merit further translational studies.

  3. Rsu1 contributes to cell adhesion and spreading in MCF10A cells via effects on P38 map kinase signaling.

    Science.gov (United States)

    Kim, Yong-Chul; Gonzalez-Nieves, Reyda; Cutler, Mary L

    2015-01-01

    The ILK, PINCH, Parvin (IPP) complex regulates adhesion and migration via binding of ILK to β1 integrin and α-parvin thus linking focal adhesions to actin cytoskeleton. ILK also binds the adaptor protein PINCH which connects signaling proteins including Rsu1 to the complex. A recent study of Rsu1 and PINCH1 in non-transformed MCF10A human mammary epithelial cells revealed that the siRNA-mediated depletion of either Rsu1 or PINCH1 decreased the number of focal adhesions (FAs) and altered the distribution and localization of FA proteins. This correlated with reduced adhesion, failure to spread or migrate in response to EGF and a loss of actin stress fibers and caveolae. The depletion of Rsu1 caused significant reduction in PINCH1 implying that Rsu1 may function in part by regulating levels of PINCH1. However, Rsu1, but not PINCH1, was required for EGF-induced activation of p38 Map kinase and ATF2 phosphorylation, suggesting a Rsu1 function independent from the IPP complex. Reconstitution of Rsu1-depleted cells with a Rsu1 mutant (N92D) that does not bind to PINCH1 failed to restore FAs or migration but did promote IPP-independent spreading and constitutive as well as EGF-induced p38 activation. In this commentary we discuss p38 activity in adhesion and how Rsu1 expression may be linked to Map kinase kinase (MKK) activation and detachment-induced stress kinase signaling.

  4. Functional Peptides from Laminin-1 Improve the Cell Adhesion Capacity of Recombinant Mussel Adhesive Protein.

    Science.gov (United States)

    Wang, Kai; Ji, Lina; Hua, Zichun

    2017-01-01

    Since cell adhesion is important for cell processes such as migration and proliferation, it is a crucial consideration in biomaterial design and development. Based on the fusion of mussel adhesive protein fp151 with laminin-1-originated functional peptides we designed fusion proteins (fLA4, fLG6 and fAG73) and explored their cell adhesion properties. In our study, cell adhesion analysis showed that protein fLG6 and fLA4 had a significantly higher cell adhesion property for A549 than fp151. Moreover, protein fAG73 also displayed a strong adhesion capacity for Hela cells. In conclusion, the incorporation of functional peptides with integrin and heparin/heparan sulphate binding capacity into mussel adhesive protein will promote the application of mussel adhesive protein as cell adhesion biomaterial. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

  5. Focal Adhesion Kinase regulates cell-cell contact formation in epithelial cells via modulation of Rho

    International Nuclear Information System (INIS)

    Playford, Martin P.; Vadali, Kavita; Cai Xinming; Burridge, Keith; Schaller, Michael D.

    2008-01-01

    Focal Adhesion Kinase (FAK) is a non-receptor tyrosine kinase that plays a key role in cellular processes such as cell adhesion, migration, proliferation and survival. Recent studies have also implicated FAK in the regulation of cell-cell adhesion. Here, evidence is presented showing that siRNA-mediated suppression of FAK levels in NBT-II cells and expression of dominant negative mutants of FAK caused loss of epithelial cell morphology and inhibited the formation of cell-cell adhesions. Rac and Rho have been implicated in the regulation of cell-cell adhesions and can be regulated by FAK signaling. Expression of active Rac or Rho in NBT-II cells disrupted formation of cell-cell contacts, thus promoting a phenotype similar to FAK-depleted cells. The loss of intercellular contacts in FAK-depleted cells is prevented upon expression of a dominant negative Rho mutant, but not a dominant negative Rac mutant. Inhibition of FAK decreased tyrosine phosphorylation of p190RhoGAP and elevated the level of GTP-bound Rho. This suggests that FAK regulates cell-cell contact formation by regulation of Rho

  6. Focal Adhesion Kinase as a Potential Target in AML and MDS.

    Science.gov (United States)

    Carter, Bing Z; Mak, Po Yee; Wang, Xiangmeng; Yang, Hui; Garcia-Manero, Guillermo; Mak, Duncan H; Mu, Hong; Ruvolo, Vivian R; Qiu, Yihua; Coombes, Kevin; Zhang, Nianxiang; Ragon, Brittany; Weaver, David T; Pachter, Jonathan A; Kornblau, Steven; Andreeff, Michael

    2017-06-01

    Although overexpression/activation of focal adhesion kinase (FAK) is widely known in solid tumors to control cell growth, survival, invasion, metastasis, gene expression, and stem cell self-renewal, its expression and function in myeloid leukemia are not well investigated. Using reverse-phase protein arrays in large cohorts of newly diagnosed acute myeloid leukemia (AML) and myeloid dysplastic syndrome (MDS) samples, we found that high FAK expression was associated with unfavorable cytogenetics ( P = 2 × 10 -4 ) and relapse ( P = 0.02) in AML. FAK expression was significantly lower in patients with FLT3 -ITD ( P = 0.0024) or RAS ( P = 0.05) mutations and strongly correlated with p-SRC and integrinβ3 levels. FAK protein levels were significantly higher in CD34 + ( P = 5.42 × 10 -20 ) and CD34 + CD38 - MDS ( P = 7.62 × 10 -9 ) cells compared with normal CD34 + cells. MDS patients with higher FAK in CD34 + cells tended to have better overall survival ( P = 0.05). FAK expression was significantly higher in MDS patients who later transformed to compared with those who did not transform to AML and in AML patients who transformed from MDS compared with those with de novo AML. Coculture with mesenchymal stromal cells (MSC) increased FAK expression in AML cells. Inhibition of FAK decreased MSC-mediated adhesion/migration and viability of AML cells and prolonged survival in an AML xenograft murine model. Our results suggest that FAK regulates leukemia-stromal interactions and supports leukemia cell survival; hence, FAK is a potential therapeutic target in myeloid leukemia. Mol Cancer Ther; 16(6); 1133-44. ©2017 AACR . ©2017 American Association for Cancer Research.

  7. Activation of AMP-activated protein kinase attenuates hepatocellular carcinoma cell adhesion stimulated by adipokine resistin

    International Nuclear Information System (INIS)

    Yang, Chen-Chieh; Chang, Shun-Fu; Chao, Jian-Kang; Lai, Yi-Liang; Chang, Wei-En; Hsu, Wen-Hsiu; Kuo, Wu-Hsien

    2014-01-01

    Resistin, adipocyte-secreting adipokine, may play critical role in modulating cancer pathogenesis. The aim of this study was to investigate the effects of resistin on HCC adhesion to the endothelium, and the mechanism underlying these resistin effects. Human SK-Hep1 cells were used to study the effect of resistin on intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1) expressions as well as NF-κB activation, and hence cell adhesion to human umbilical vein endothelial cells (HUVECs). 5-Aminoimidazole-4-carboxamide 1-β-D-ribofuranoside (AICAR), an AMP-activated protein kinase (AMPK) activator, was used to determine the regulatory role of AMPK on HCC adhesion to the endothelium in regard to the resistin effects. Treatment with resistin increased the adhesion of SK-Hep1 cells to HUVECs and concomitantly induced NF-κB activation, as well as ICAM-1 and VCAM-1 expressions in SK-Hep1 cells. Using specific blocking antibodies and siRNAs, we found that resistin-induced SK-Hep1 cell adhesion to HUVECs was through NF-κB-regulated ICAM-1 and VCAM-1 expressions. Moreover, treatment with AICAR demonstrated that AMPK activation in SK-Hep1 cells significantly attenuates the resistin effect on SK-Hep1 cell adhesion to HUVECs. These results clarify the role of resistin in inducing HCC adhesion to the endothelium and demonstrate the inhibitory effect of AMPK activation under the resistin stimulation. Our findings provide a notion that resistin play an important role to promote HCC metastasis and implicate AMPK may be a therapeutic target to against HCC metastasis

  8. Copper Deficiency Induced Emphysema Is Associated with Focal Adhesion Kinase Inactivation

    OpenAIRE

    Mizuno, Shiro; Yasuo, Masanori; Bogaard, Harm J.; Kraskauskas, Donatas; Alhussaini, Aysar; Gomez-Arroyo, Jose; Farkas, Daniela; Farkas, Laszlo; Voelkel, Norbert F.

    2012-01-01

    Background Copper is an important regulator of hypoxia inducible factor 1 alpha (HIF-1?) dependent vascular endothelial growth factor (VEGF) expression, and is also required for the activity of lysyl oxidase (LOX) to effect matrix protein cross-linking. Cell detachment from the extracellular matrix can induce apoptosis (anoikis) via inactivation of focal adhesion kinase (FAK). Methodology To examine the molecular mechanisms whereby copper depletion causes the destruction of the normal alveola...

  9. The Src homology 2 protein Shb promotes cell cycle progression in murine hematopoietic stem cells by regulation of focal adhesion kinase activity

    International Nuclear Information System (INIS)

    Gustafsson, Karin; Heffner, Garrett; Wenzel, Pamela L.; Curran, Matthew; Grawé, Jan; McKinney-Freeman, Shannon L.; Daley, George Q.; Welsh, Michael

    2013-01-01

    The widely expressed adaptor protein Shb has previously been reported to contribute to T cell function due to its association with the T cell receptor and furthermore, several of Shb's known interaction partners are established regulators of blood cell development and function. In addition, Shb deficient embryonic stem cells displayed reduced blood cell colony formation upon differentiation in vitro. The aim of the current study was therefore to explore hematopoietic stem and progenitor cell function in the Shb knockout mouse. Shb deficient bone marrow contained reduced relative numbers of long-term hematopoietic stem cells (LT-HSCs) that exhibited lower proliferation rates. Despite this, Shb knockout LT-HSCs responded promptly by entering the cell cycle in response to genotoxic stress by 5-fluorouracil treatment. In competitive LT-HSC transplantations, Shb null cells initially engrafted as well as the wild-type cells but provided less myeloid expansion over time. Moreover, Shb knockout bone marrow cells exhibited elevated basal activities of focal adhesion kinase/Rac1/p21-activated kinase signaling and reduced responsiveness to Stem Cell Factor stimulation. Consequently, treatment with a focal adhesion kinase inhibitor increased Shb knockout LT-HSC proliferation. The altered signaling characteristics thus provide a plausible mechanistic explanation for the changes in LT-HSC proliferation since these signaling intermediates have all been shown to participate in LT-HSC cell cycle control. In summary, the loss of Shb dependent signaling in bone marrow cells, resulting in elevated focal adhesion kinase activity and reduced proliferative responses in LT-HSCs under steady state hematopoiesis, confers a disadvantage to the maintenance of LT-HSCs over time. -- Highlights: • Shb is an adaptor protein operating downstream of tyrosine kinase receptors. • Shb deficiency reduces hematopoietic stem cell proliferation. • The proliferative effect of Shb occurs via increased

  10. The Src homology 2 protein Shb promotes cell cycle progression in murine hematopoietic stem cells by regulation of focal adhesion kinase activity

    Energy Technology Data Exchange (ETDEWEB)

    Gustafsson, Karin [Department of Medical Cell Biology, Uppsala University, Uppsala 751 23 (Sweden); Heffner, Garrett; Wenzel, Pamela L.; Curran, Matthew [HHMI, Children' s Hospital Boston, Harvard Medical School, Boston, 02115 MA (United States); Grawé, Jan [Department of Genetics and Pathology, Uppsala University, Uppsala 75185 (Sweden); McKinney-Freeman, Shannon L. [Department of Hematology, St. Jude Children' s Research Hospital, Memphis, TN 38105 (United States); Daley, George Q. [HHMI, Children' s Hospital Boston, Harvard Medical School, Boston, 02115 MA (United States); Welsh, Michael, E-mail: michael.welsh@mcb.uu.se [Department of Medical Cell Biology, Uppsala University, Uppsala 751 23 (Sweden)

    2013-07-15

    The widely expressed adaptor protein Shb has previously been reported to contribute to T cell function due to its association with the T cell receptor and furthermore, several of Shb's known interaction partners are established regulators of blood cell development and function. In addition, Shb deficient embryonic stem cells displayed reduced blood cell colony formation upon differentiation in vitro. The aim of the current study was therefore to explore hematopoietic stem and progenitor cell function in the Shb knockout mouse. Shb deficient bone marrow contained reduced relative numbers of long-term hematopoietic stem cells (LT-HSCs) that exhibited lower proliferation rates. Despite this, Shb knockout LT-HSCs responded promptly by entering the cell cycle in response to genotoxic stress by 5-fluorouracil treatment. In competitive LT-HSC transplantations, Shb null cells initially engrafted as well as the wild-type cells but provided less myeloid expansion over time. Moreover, Shb knockout bone marrow cells exhibited elevated basal activities of focal adhesion kinase/Rac1/p21-activated kinase signaling and reduced responsiveness to Stem Cell Factor stimulation. Consequently, treatment with a focal adhesion kinase inhibitor increased Shb knockout LT-HSC proliferation. The altered signaling characteristics thus provide a plausible mechanistic explanation for the changes in LT-HSC proliferation since these signaling intermediates have all been shown to participate in LT-HSC cell cycle control. In summary, the loss of Shb dependent signaling in bone marrow cells, resulting in elevated focal adhesion kinase activity and reduced proliferative responses in LT-HSCs under steady state hematopoiesis, confers a disadvantage to the maintenance of LT-HSCs over time. -- Highlights: • Shb is an adaptor protein operating downstream of tyrosine kinase receptors. • Shb deficiency reduces hematopoietic stem cell proliferation. • The proliferative effect of Shb occurs via

  11. Expression of MLN64 influences cellular matrix adhesion of breast cancer cells, the role for focal adhesion kinase.

    Science.gov (United States)

    Cai, Wei; Ye, Lin; Sun, Jiabang; Mansel, Robert E; Jiang, Wen G

    2010-04-01

    The metastatic lymph node 64 (MLN64) gene was initially identified as highly expressed in the metastatic lymph node from breast cancer. It is localized in q12-q21 of the human chromosome 17 and is often co-amplified with erbB-2. However, the role played by MLN64 in breast cancer remains unclear. In the present study, the expression of MLN64 was examined in a breast cancer cohort using quantitative real-time PCR and immunohistochemical staining. It demonstrated that MLN64 was highly expressed in breast tumours compared to corresponding background tissues at both transcript level and protein level. The elevated level of MLN64 transcripts was correlated with the poor prognosis and overall survival of the patients. A panel of breast cancer cell sublines was subsequently developed by knockdown of MLN64 expression. Loss of MLN64 expression in MCF-7 cells resulted in a significant reduction of cell growth (absorbance for MCF-7DeltaMLN64 being 0.87+/-0.07, Padhesion assay, MDA-MB-231DeltaMLN64 cells showed a significant increase in adhesion (86+/-14), padhesion kinase (FAK) in MDA-MB-231DeltaMLN64 cells using Western blot analysis and immunofluorescent staining of FAK. Moreover, addition of FAK inhibitor to these cells diminished the effect of MLN64 on cell-matrix adhesion, suggesting that FAK contributed to the increased adhesion in MDA-MB-231DeltaMLN64 cells. In conclusion, MLN64 is overexpressed in breast cancer, and its level correlates with poor prognosis and patient survival. MLN64 contributes to the development and progression of breast cancer through the regulation of cell proliferation and adhesive capacity.

  12. The role of phosphatidylinositol 3-kinase in neural cell adhesion molecule-mediated neuronal differentiation and survival

    DEFF Research Database (Denmark)

    Ditlevsen, Dorte K; Køhler, Lene B; Pedersen, Martin V

    2003-01-01

    The neural cell adhesion molecule, NCAM, is known to stimulate neurite outgrowth from primary neurones and PC12 cells presumably through signalling pathways involving the fibroblast growth factor receptor (FGFR), protein kinase A (PKA), protein kinase C (PKC), the Ras-mitogen activated protein...... that phosphatidylinositol 3-kinase (PI3K) is required for NCAM-mediated neurite outgrowth from PC12-E2 cells and from cerebellar and dopaminergic neurones in primary culture, and that the thr/ser kinase Akt/protein kinase B (PKB) is phosphorylated downstream of PI3K after stimulation with C3. Moreover, we present data...

  13. Organization and post-transcriptional processing of focal adhesion kinase gene

    Directory of Open Access Journals (Sweden)

    Enslen Hervé

    2006-08-01

    Full Text Available Abstract Background Focal adhesion kinase (FAK is a non-receptor tyrosine kinase critical for processes ranging from embryo development to cancer progression. Although isoforms with specific molecular and functional properties have been characterized in rodents and chicken, the organization of FAK gene throughout phylogeny and its potential to generate multiple isoforms are not well understood. Here, we study the phylogeny of FAK, the organization of its gene, and its post-transcriptional processing in rodents and human. Results A single orthologue of FAK and the related PYK2 was found in non-vertebrate species. Gene duplication probably occurred in deuterostomes after the echinoderma embranchment, leading to the evolution of PYK2 with distinct properties. The amino acid sequence of FAK and PYK2 is conserved in their functional domains but not in their linker regions, with the absence of autophosphorylation site in C. elegans. Comparison of mouse and human FAK genes revealed the existence of multiple combinations of conserved and non-conserved 5'-untranslated exons in FAK transcripts suggesting a complex regulation of their expression. Four alternatively spliced coding exons (13, 14, 16, and 31, previously described in rodents, are highly conserved in vertebrates. Cis-regulatory elements known to regulate alternative splicing were found in conserved alternative exons of FAK or in the flanking introns. In contrast, other reported human variant exons were restricted to Homo sapiens, and, in some cases, other primates. Several of these non-conserved exons may correspond to transposable elements. The inclusion of conserved alternative exons was examined by RT-PCR in mouse and human brain during development. Inclusion of exons 14 and 16 peaked at the end of embryonic life, whereas inclusion of exon 13 increased steadily until adulthood. Study of various tissues showed that inclusion of these exons also occurred, independently from each other, in a

  14. Phosphatidylinositol 4-kinases: Function, structure, and inhibition

    International Nuclear Information System (INIS)

    Boura, Evzen; Nencka, Radim

    2015-01-01

    The phosphatidylinositol 4-kinases (PI4Ks) synthesize phosphatidylinositol 4-phosphate (PI4P), a key member of the phosphoinositide family. PI4P defines the membranes of Golgi and trans-Golgi network (TGN) and regulates trafficking to and from the Golgi. Humans have two type II PI4Ks (α and β) and two type III enzymes (α and β). Recently, the crystal structures were solved for both type II and type III kinase revealing atomic details of their function. Importantly, the type III PI4Ks are hijacked by +RNA viruses to create so-called membranous web, an extensively phosphorylated and modified membrane system dedicated to their replication. Therefore, selective and potent inhibitors of PI4Ks have been developed as potential antiviral agents. Here we focus on the structure and function of PI4Ks and their potential in human medicine

  15. Phosphatidylinositol 4-kinases: Function, structure, and inhibition

    Energy Technology Data Exchange (ETDEWEB)

    Boura, Evzen, E-mail: boura@uochb.cas.cz; Nencka, Radim, E-mail: nencka@uochb.cas.cz

    2015-10-01

    The phosphatidylinositol 4-kinases (PI4Ks) synthesize phosphatidylinositol 4-phosphate (PI4P), a key member of the phosphoinositide family. PI4P defines the membranes of Golgi and trans-Golgi network (TGN) and regulates trafficking to and from the Golgi. Humans have two type II PI4Ks (α and β) and two type III enzymes (α and β). Recently, the crystal structures were solved for both type II and type III kinase revealing atomic details of their function. Importantly, the type III PI4Ks are hijacked by +RNA viruses to create so-called membranous web, an extensively phosphorylated and modified membrane system dedicated to their replication. Therefore, selective and potent inhibitors of PI4Ks have been developed as potential antiviral agents. Here we focus on the structure and function of PI4Ks and their potential in human medicine.

  16. Impaired Expression of Focal Adhesion Kinase in Mesenchymal Stromal Cells from Low-Risk Myelodysplastic Syndrome Patients

    Directory of Open Access Journals (Sweden)

    Yuenv Wu

    2017-08-01

    Full Text Available The pathogenic role of mesenchymal stromal cells (MSCs in myelodysplastic syndromes (MDS development and progression has been investigated by numerous studies, yet, it remains controversial in some aspects (1, 2. In the present study, we found distinct features of MSCs from low-risk (LR-MDS stromal microenvironment as compared to those from healthy subjects. At the molecular level, focal adhesion kinase, a key tyrosine kinase in control of cell proliferation, survival, and adhesion process, was found profoundly suppressed in expression and activation in LR-MDS MSC. At a functional level, LR-MDS MSCs showed impaired growth and clonogenic capacity, which were independent of cellular senescence and apoptosis. The pro-adipogenic differentiation and attenuated osteogenic capacity along with reduced SDF-1 expression could be involved in creating an unfavorable microenvironment for hematopoiesis. In conclusion, our experiments support the theory that the stromal microenvironment is fundamentally altered in LR-MDS, and these preliminary data offer a new perspective on LR-MDS pathophysiology.

  17. Comparing the mechanical influence of vinculin, focal adhesion kinase and p53 in mouse embryonic fibroblasts

    International Nuclear Information System (INIS)

    Klemm, Anna H.; Diez, Gerold; Alonso, Jose-Luis; Goldmann, Wolfgang H.

    2009-01-01

    Cytoskeletal reorganization is an ongoing process when cells adhere, move or invade extracellular substrates. The cellular force generation and transmission are determined by the intactness of the actomyosin-(focal adhesion complex)-integrin connection. We investigated the intracellular course of action in mouse embryonic fibroblasts deficient in the focal adhesion proteins vinculin and focal adhesion kinase (FAK) and the nuclear matrix protein p53 using magnetic tweezer and nanoparticle tracking techniques. Results show that the lack of these proteins decrease cellular stiffness and affect cell rheological behavior. The decrease in cellular binding strength was higher in FAK- to vinculin-deficient cells, whilst p53-deficient cells showed no effect compared to wildtype cells. The intracellular cytoskeletal activity was lowest in wildtype cells, but increased in the following order when cells lacked FAK+p53 > p53 > vinculin. In summary, cell mechanical processes are differently affected by the focal adhesion proteins vinculin and FAK than by the nuclear matrix protein, p53.

  18. New Treatment Opportunities in Phosphatase and Tensin Homolog (PTEN-Deficient Tumors: Focus on PTEN/Focal Adhesion Kinase Pathway

    Directory of Open Access Journals (Sweden)

    Roberta Alfieri

    2017-08-01

    Full Text Available Deep genetic studies revealed that phosphatase and tensin homolog (PTEN mutations or loss of expression are not early events in cancer development but characterize tumor progression and invasion. Loss of PTEN function causes a full activation of the prosurvival phosphoinositide 3-kinase (PI3K/AKT/mTOR pathway, but the treatment with specific inhibitors of PI3K/AKT/mTOR did not produce the expected results. One of the alternative targets of PTEN is the focal adhesion kinase (FAK kinase, mainly involved in the control of cancer cell spread. The connection between PTEN and FAK has been demonstrated in different tumor types, with reduced PTEN activity often correlated with increased expression and phosphorylation of FAK. FAK inhibition may thus represent a promising strategy, and some clinical trials are testing FAK inhibitors alone or combined with other agents in a number of solid tumors. However, only few preclinical and clinical data described the effects of the combination of PI3K/AKT/mTOR and FAK inhibitors. Increasing knowledge on the PTEN/FAK connection could confirm PTEN as a good prognostic marker for a combination strategy based on concomitant inhibition of PI3K/AKT and FAK signaling, in advanced metastatic malignancies with altered or reduced PTEN expression.

  19. Non-Catalytic Functions of Pyk2 and Fyn Regulate Late Stage Adhesion in Human T Cells

    Science.gov (United States)

    Houtman, Jon C. D.

    2012-01-01

    T cell activation drives the protective immune response against pathogens, but is also critical for the development of pathological diseases in humans. Cytoskeletal changes are required for downstream functions in T cells, including proliferation, cytokine production, migration, spreading, and adhesion. Therefore, investigating the molecular mechanism of cytoskeletal changes is crucial for understanding the induction of T cell-driven immune responses and for developing therapies to treat immune disorders related to aberrant T cell activation. In this study, we used a plate-bound adhesion assay that incorporated near-infrared imaging technology to address how TCR signaling drives human T cell adhesion. Interestingly, we observed that T cells have weak adhesion early after TCR activation and that binding to the plate was significantly enhanced 30–60 minutes after receptor activation. This late stage of adhesion was mediated by actin polymerization but was surprisingly not dependent upon Src family kinase activity. By contrast, the non-catalytic functions of the kinases Fyn and Pyk2 were required for late stage human T cell adhesion. These data reveal a novel TCR-induced signaling pathway that controls cellular adhesion independent of the canonical TCR signaling cascade driven by tyrosine kinase activity. PMID:23300847

  20. Integrin-Associated Focal Adhesion Kinase Protects Human Embryonic Stem Cells from Apoptosis, Detachment, and Differentiation

    Directory of Open Access Journals (Sweden)

    Loriana Vitillo

    2016-08-01

    Full Text Available Human embryonic stem cells (hESCs can be maintained in a fully defined niche on extracellular matrix substrates, to which they attach through integrin receptors. However, the underlying integrin signaling mechanisms, and their contribution to hESC behavior, are largely unknown. Here, we show that focal adhesion kinase (FAK transduces integrin activation and supports hESC survival, substrate adhesion, and maintenance of the undifferentiated state. After inhibiting FAK kinase activity we show that hESCs undergo cell detachment-dependent apoptosis or differentiation. We also report deactivation of FAK downstream targets, AKT and MDM2, and upregulation of p53, all key players in hESC regulatory networks. Loss of integrin activity or FAK also induces cell aggregation, revealing a role in the cell-cell interactions of hESCs. This study provides insight into the integrin signaling cascade activated in hESCs and reveals in FAK a key player in the maintenance of hESC survival and undifferentiated state.

  1. RACK1 Targets the Extracellular Signal-Regulated Kinase/Mitogen-Activated Protein Kinase Pathway To Link Integrin Engagement with Focal Adhesion Disassembly and Cell Motility

    Czech Academy of Sciences Publication Activity Database

    Vomastek, Tomáš; Iwanicki, M. P.; Schaeffer, J.; J.; Tarcsafalvi, A.; Parsons, J. T.; Weber, M. J.

    2007-01-01

    Roč. 27, č. 23 (2007), s. 8296-8305 ISSN 0270-7306 R&D Projects: GA AV ČR IAA500200716 Institutional research plan: CEZ:AV0Z50200510 Keywords : protein kinase * adhesion * cell Subject RIV: EE - Microbiology, Virology Impact factor: 6.420, year: 2007

  2. Switchable adhesion by chemical functionality and topography

    NARCIS (Netherlands)

    Kamperman, M.M.G.; Synytska, A.

    2012-01-01

    Progress in adhesion technology over the last few decades has led to widespread replacement of mechanical fasteners with adhesive bonds. Despite the advances, it remains challenging to produce materials that are sticky on demand. In this feature article we highlight recent efforts to develop

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

    Directory of Open Access Journals (Sweden)

    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.

  4. The secret life of kinases: functions beyond catalysis.

    LENUS (Irish Health Repository)

    Rauch, Jens

    2011-10-28

    Abstract Protein phosphorylation participates in the regulation of all fundamental biological processes, and protein kinases have been intensively studied. However, while the focus was on catalytic activities, accumulating evidence suggests that non-catalytic properties of protein kinases are essential, and in some cases even sufficient for their functions. These non-catalytic functions include the scaffolding of protein complexes, the competition for protein interactions, allosteric effects on other enzymes, subcellular targeting, and DNA binding. This rich repertoire often is used to coordinate phosphorylation events and enhance the specificity of substrate phosphorylation, but also can adopt functions that do not rely on kinase activity. Here, we discuss such kinase independent functions of protein and lipid kinases focussing on kinases that play a role in the regulation of cell proliferation, differentiation, apoptosis, and motility.

  5. PROLACTIN-INDUCED TYROSINE PHOSPHORYLATION, ACTIVATION AND RECEPTOR ASSOCIATION OF FOCAL ADHESION KINASE (FAK) IN MAMMARY EPITHELIAL CELLS

    Science.gov (United States)

    Prolactin-Induced Tyrosine Phosphorylation, Activation and ReceptorAssociation of Focal Adhesion Kinase (FAK) in Mammary Epithelial Cells. Suzanne E. Fenton1 and Lewis G. Sheffield2. 1U.S. Environmental ProtectionAgency, MD-72, Research Triangle Park, NC 27711, and

  6. Focal adhesion kinase is required for intestinal regeneration and tumorigenesis downstream of Wnt/c-Myc signaling

    NARCIS (Netherlands)

    Ashton, Gabrielle H.; Morton, Jennifer P.; Myant, Kevin; Phesse, Toby J.; Ridgway, Rachel A.; Marsh, Victoria; Wilkins, Julie A.; Athineos, Dimitris; Muncan, Vanesa; Kemp, Richard; Neufeld, Kristi; Clevers, Hans; Brunton, Valerie; Winton, Douglas J.; Wang, Xiaoyan; Sears, Rosalie C.; Clarke, Alan R.; Frame, Margaret C.; Sansom, Owen J.

    2010-01-01

    The intestinal epithelium has a remarkable capacity to regenerate after injury and DNA damage. Here, we show that the integrin effector protein Focal Adhesion Kinase (FAK) is dispensable for normal intestinal homeostasis and DNA damage signaling, but is essential for intestinal regeneration

  7. Mammary gland-specific ablation of focal adhesion kinase reduces the incidence of p53-mediated mammary tumour formation.

    NARCIS (Netherlands)

    Miltenburg, van M.H.; Nimwegen, van M.J.; Tijdens, R.B.; Lalai, R.A.; Kuiper, R.; Klarenbeek, S.; Schouten, P.C.; Vries, de A.; Jonkers, J.M.M.; Water, van de B.

    2014-01-01

    BACKGROUND Elevated expression of focal adhesion kinase (FAK) occurs in numerous human cancers including colon-, cervix- and breast cancer. Although several studies have implicated FAK in mammary tumour formation induced by ectopic oncogene expression, evidence supporting a role for FAK in

  8. v-Src SH3-enhanced Interaction with Focal Adhesion Kinase at Beta1 Integrin-containing Invadopodia Promotes Cell Invasion

    OpenAIRE

    Hauck, Christof R.; Hsia, Datsun A.; Ilić, Du ko; Schlaepfer, David D.

    2002-01-01

    In viral Src (v-Src) transformed cells, focal adhesion kinase (FAK) associates in a stable signaling complex with v-Src that is mediated by combined v-Src SH2 and gain-of-function v-Src SH3 domain binding to FAK. Here, we assess the significance of the Arg-95 to Trp gain-of-function mutation in the v-Src SH3 domain through comparisons of Src-/- fibroblasts transformed with either Prague C v-Src or a point-mutant (v-Src-RT) containing a normal (Arg-95) SH3 domain. Both v-Src isoforms exhibited...

  9. Activation of erythropoietin-producing hepatocellular receptor A2 attenuates cell adhesion of human fallopian tube epithelial cells via focal adhesion kinase dephosphorylation.

    Science.gov (United States)

    Yang, Xiao-Yi; Zhu, Wei-Jie; Jiang, Huan

    2012-02-01

    Tyrosine kinase receptor erythropoietin-producing hepatocellular receptor A2 (EphA2) and its predominant ligand EphrinA1 have been studied extensively for their roles of mediating cell adhesion in epithelial cells. However, EphA2 signaling in human fallopian tube epithelial cells is poorly understood. In this study, primary cultured fallopian tube epithelial cells were used as a model treated with EphrinA1-Fc or IgG-Fc (control), to explore the role of EphA2 signal and its network involved in the regulation of cell adhesion of tubal epithelia cells. The activation of EphA2 and focal adhesion kinase (FAK) was evaluated by western blotting assay in the cultured fallopian tube epithelia cells, of which the cell adhesion activity was determined by MTT assay. A significantly negative correlation was found between phosphorylated-EphA2 (Pho-EphA2) and phosphorylated-FAK (Pho-FAK) after exposure to EphrinA1-Fc (P = 0.000; r = -0.848). EphrinA1-Fc increased Pho-EphA2 and reduced Pho-FAK in seconds, with the apex level of Pho-EphA2 and the nadir level of Pho-FAK detected at the same time (10 min). Cell adhesion of the cultured cells supplemented with EphrinA1-Fc appeared to be weaker than that of the controls at the later time points of the treatment (from 30 to 120 min) (P < 0.05). Taken together, the EphrinA1 addition directly induces an elevated Pho-EphA2 accompanied by a decreased Pho-FAK in human fallopian tube epithelia cells. Furthermore, activation of EphA2 participates in the regulation of fallopian tube cell adhesion via FAK dephosphorylation.

  10. Functionalization of CoCr surfaces with cell adhesive peptides to promote HUVECs adhesion and proliferation

    Energy Technology Data Exchange (ETDEWEB)

    Castellanos, Maria Isabel, E-mail: maria.isabel.castellanos@upc.edu [Biomaterials, Biomechanics and Tissue Engineering Group, Department of Materials Science and Metallurgical Engineering, Technical University of Catalonia (UPC), ETSEIB, 08028 Barcelona (Spain); Centre for Research in Nanoengineering (CRNE), UPC, 08028 Barcelona (Spain); Mas-Moruno, Carlos, E-mail: carles.mas.moruno@upc.edu [Biomaterials, Biomechanics and Tissue Engineering Group, Department of Materials Science and Metallurgical Engineering, Technical University of Catalonia (UPC), ETSEIB, 08028 Barcelona (Spain); Centre for Research in Nanoengineering (CRNE), UPC, 08028 Barcelona (Spain); Grau, Anna, E-mail: agraugar@gmail.com [Biomaterials, Biomechanics and Tissue Engineering Group, Department of Materials Science and Metallurgical Engineering, Technical University of Catalonia (UPC), ETSEIB, 08028 Barcelona (Spain); Centre for Research in Nanoengineering (CRNE), UPC, 08028 Barcelona (Spain); Serra-Picamal, Xavier, E-mail: xserrapicamal@gmail.com [Institute for Bioengineering of Catalonia (IBEC), 08028 Barcelona (Spain); University of Barcelona and CIBER-BBN, 08036 Barcelona (Spain); Institució Catalana de Recerca i Estudis Avançats (ICREA), 08010 Barcelona (Spain); Trepat, Xavier, E-mail: xtrepat@ub.edu [Institute for Bioengineering of Catalonia (IBEC), 08028 Barcelona (Spain); University of Barcelona and CIBER-BBN, 08036 Barcelona (Spain); Institució Catalana de Recerca i Estudis Avançats (ICREA), 08010 Barcelona (Spain); Albericio, Fernando, E-mail: fernando.albericio@irbbarcelona.org [Department of Chemistry, University of Barcelona, CIBER-BBN, 08028 Barcelona (Spain); Joner, Michael, E-mail: michaeljoner@me.com [Department of Cardiology, Deutsches Herzzentrum München, 80636 Munich (Germany); CVPath Institute, Gaithersburg, MD 20878 (United States); and others

    2017-01-30

    Highlights: • We immobilized peptides on CoCr alloy through physisorption and covalent bonding. • Surface activation is an essential step prior to silanization to enhance peptide attachment. • Biofunctionalized surface characteristics were discussed. • RGDS, YIGSR and combination peptides display an improved HUVECs adhesion and proliferation. - Abstract: Biomimetic surface modification with peptides that have specific cell-binding moieties is a promising approach to improve endothelialization of metal-based stents. In this study, we functionalized CoCr surfaces with RGDS, REDV, YIGSR peptides and their combinations to promote endothelial cells (ECs) adhesion and proliferation. An extensive characterization of the functionalized surfaces was performed by XPS analysis, surface charge and quartz crystal microbalance with dissipation monitoring (QCM-D), which demonstrated the successful immobilization of the peptides to the surface. Cell studies demonstrated that the covalent functionalization of CoCr surfaces with an equimolar combination of RGDS and YIGSR represents the most powerful strategy to enhance the early stages of ECs adhesion and proliferation, indicating a positive synergistic effect between the two peptide motifs. Although these peptide sequences slightly increased smooth muscle cells (SMCs) adhesion, these values were ten times lower than those observed for ECs. The combination of RGDS with the REDV sequence did not show synergistic effects in promoting the adhesion or proliferation of ECs. The strategy presented in this study holds great potential to overcome clinical limitations of current metal stents by enhancing their capacity to support surface endothelialization.

  11. Focal adhesion kinase, a downstream mediator of Raf-1 signaling, suppresses cellular adhesion, migration, and neuroendocrine markers in BON carcinoid cells.

    Science.gov (United States)

    Ning, Li; Chen, Herbert; Kunnimalaiyaan, Muthusamy

    2010-05-01

    We have recently reported that activation of the Raf-1/mitogen-activated protein kinase/extracellular signal-regulated kinase (ERK) kinase 1/2 (MEK1/2)/ERK1/2 signaling cascade in gastrointestinal carcinoid cell line (BON) alters cellular morphology and neuroendocrine phenotype. The mechanisms by which Raf-1 mediates these changes in carcinoid cells are unclear. Here, we report that activation of the Raf-1 signaling cascade in BON cells induced the expression of focal adhesion kinase (FAK) protein, suppressed the production of neuroendocrine markers, and resulted in significant decreases in cellular adhesion and migration. Importantly, inactivation of MEK1/2 by 1,4-diamino-2,3-dicyano-1,4-bis[2-aminophenylthio]butadiene or abolition of FAK induction in Raf-1-activated BON cells by targeted siRNA led to reversal of the Raf-1-mediated reduction in neuroendocrine markers and cellular adhesion and migration. Phosphorylation site-specific antibodies detected the phosphorylated FAK(Tyr407), but not FAK(Tyr397), in these Raf-1-activated cells, indicating that FAK(Tyr407) may be associated with changes in the neuroendocrine phenotype. Overexpression of constitutively active FAK plasmids (wild-type FAK or FAK(Tyr397) mutant) into BON cells reduced neuroendocrine markers, whereas the FAK(Tyr407) mutant plasmid did not show any decrease in the levels of neuroendocrine markers, indicating that phosphorylation of FAK at the Tyr(407) residue may be important for these effects. Our results showed for the first time that FAK is an essential downstream effector of the Raf-1/MEK1/2/ERK1/2 signaling cascade and negatively regulated the neuroendocrine and metastatic phenotype in BON cells. (c)2010 AACR.

  12. Laminin α2-mediated focal adhesion kinase activation triggers Alport glomerular pathogenesis.

    Directory of Open Access Journals (Sweden)

    Duane Delimont

    Full Text Available It has been known for some time that laminins containing α1 and α2 chains, which are normally restricted to the mesangial matrix, accumulate in the glomerular basement membranes (GBM of Alport mice, dogs, and humans. We show that laminins containing the α2 chain, but not those containing the α1 chain activates focal adhesion kinase (FAK on glomerular podocytes in vitro and in vivo. CD151-null mice, which have weakened podocyte adhesion to the GBM rendering these mice more susceptible to biomechanical strain in the glomerulus, also show progressive accumulation of α2 laminins in the GBM, and podocyte FAK activation. Analysis of glomerular mRNA from both models demonstrates significant induction of MMP-9, MMP-10, MMP-12, MMPs linked to GBM destruction in Alport disease models, as well as the pro-inflammatory cytokine IL-6. SiRNA knockdown of FAK in cultured podocytes significantly reduced expression of MMP-9, MMP-10 and IL-6, but not MMP-12. Treatment of Alport mice with TAE226, a small molecule inhibitor of FAK activation, ameliorated fibrosis and glomerulosclerosis, significantly reduced proteinuria and blood urea nitrogen levels, and partially restored GBM ultrastructure. Glomerular expression of MMP-9, MMP-10 and MMP-12 mRNAs was significantly reduced in TAE226 treated animals. Collectively, this work identifies laminin α2-mediated FAK activation in podocytes as an important early event in Alport glomerular pathogenesis and suggests that FAK inhibitors, if safe formulations can be developed, might be employed as a novel therapeutic approach for treating Alport renal disease in its early stages.

  13. Heparan sulfate chain valency controls syndecan-4 function in cell adhesion

    DEFF Research Database (Denmark)

    Gopal, Sandeep; Bober, Adam; Whiteford, James R

    2010-01-01

    , clustering of one-chain syndecan-4 forms with antibodies overcame the block, indicating that valency of interactions with ligands is a key component of syndecan-4 function. Measurements of focal contact/adhesion size and focal adhesion kinase phosphorylation correlated with syndecan-4 status and alpha...... of the core protein cytoplasmic domain, though not interactions with PDZ proteins. A second key requirement is multiple heparan sulfate chains. Mutant syndecan-4 with no chains, or only one chain, failed to restore the wild type phenotype, while those expressing two or three were competent. However......-smooth muscle actin organization, being reduced where syndecan-4 function was compromised by a lack of multiple heparan sulfate chains....

  14. Disruption of laminin-integrin-CD151-focal adhesion kinase axis sensitizes breast cancer cells to ErbB2 antagonists.

    Science.gov (United States)

    Yang, Xiuwei H; Flores, Ludmila M; Li, Qinglin; Zhou, Pengcheng; Xu, Fenghui; Krop, Ian E; Hemler, Martin E

    2010-03-15

    Resistance to anti-ErbB2 agents is a significant problem in the treatment of human ErbB2+ breast cancers. We show here that adhesion of human ErbB2+ breast cancer cells to basement membrane laminin-5 provides substantial resistance to trastuzumab and lapatinib, agents that respectively target the extracellular and kinase domains of ErbB2. Knockdown of laminin-binding integrins (alpha6beta4, alpha3beta1) or associated tetraspanin protein CD151 reversed laminin-5 resistance and sensitized ErbB2+ cells to trastuzumab and lapatinib. CD151 knockdown, together with trastuzumab treatment, inhibited ErbB2 activation and downstream signaling through Akt, Erk1/2, and focal adhesion kinase (FAK). Hence, ErbB2 function in mammary tumor cells is promoted by integrin-mediated adhesion to laminin-5, with strong support by CD151, leading to signaling through FAK. Consequently, removal or inhibition of any of these components (laminin-5, integrin, CD151, FAK) markedly sensitizes cells to anti-ErbB2 agents. These new insights should be useful when devising strategies for overcoming drug resistance in ErbB2+ cancers.

  15. Protein kinase substrate identification on functional protein arrays

    Directory of Open Access Journals (Sweden)

    Zhou Fang

    2008-02-01

    Full Text Available Abstract Background Over the last decade, kinases have emerged as attractive therapeutic targets for a number of different diseases, and numerous high throughput screening efforts in the pharmaceutical community are directed towards discovery of compounds that regulate kinase function. The emerging utility of systems biology approaches has necessitated the development of multiplex tools suitable for proteomic-scale experiments to replace lower throughput technologies such as mass spectroscopy for the study of protein phosphorylation. Recently, a new approach for identifying substrates of protein kinases has applied the miniaturized format of functional protein arrays to characterize phosphorylation for thousands of candidate protein substrates in a single experiment. This method involves the addition of protein kinases in solution to arrays of immobilized proteins to identify substrates using highly sensitive radioactive detection and hit identification algorithms. Results To date, the factors required for optimal performance of protein array-based kinase substrate identification have not been described. In the current study, we have carried out a detailed characterization of the protein array-based method for kinase substrate identification, including an examination of the effects of time, buffer compositions, and protein concentration on the results. The protein array approach was compared to standard solution-based assays for assessing substrate phosphorylation, and a correlation of greater than 80% was observed. The results presented here demonstrate how novel substrates for protein kinases can be quickly identified from arrays containing thousands of human proteins to provide new clues to protein kinase function. In addition, a pooling-deconvolution strategy was developed and applied that enhances characterization of specific kinase-substrate relationships and decreases reagent consumption. Conclusion Functional protein microarrays are an

  16. Multi-targeted molecular therapeutic approach in aggressive neuroblastoma: the effect of Focal Adhesion Kinase-Src-Paxillin system.

    Science.gov (United States)

    Kratimenos, Panagiotis; Koutroulis, Ioannis; Marconi, Dante; Syriopoulou, Vasiliki; Delivoria-Papadopoulos, Maria; Chrousos, George P; Theocharis, Stamatios

    2014-12-01

    Nonreceptor tyrosine kinases play key roles in the integrin system. Located at the focal adhesions, they consist of large protein complexes through which the cytoskeleton connects to the extracellular matrix. The focal adhesion kinase (FAK)-Src-paxillin complex, a major mediator of the integrin pathway, contributes to cell migration and motility. Its overexpression is increased in children with advanced neuroblastoma (NB), one of the most common malignancies of childhood, with poor survival. We review the most recent data on FAK-Src-paxillin and their implications in NB, the molecular structure and the regulatory mechanisms of each molecule and their interactions and up-to-date information on their use as the newest biomarkers and their potential use as therapeutic targets in NB. Based on the current literature, we hypothesize that combined and concurrent inhibition of the FAK-Src-Paxillin system may result in significant tumor suppression and prevention or delay of metastasis.

  17. Loss of keratinocyte focal adhesion kinase stimulates dermal proteolysis through upregulation of MMP9 in wound healing.

    Science.gov (United States)

    Wong, Victor W; Garg, Ravi K; Sorkin, Michael; Rustad, Kristine C; Akaishi, Satoshi; Levi, Kemal; Nelson, Emily R; Tran, Misha; Rennert, Robert; Liu, Wei; Longaker, Michael T; Dauskardt, Reinhold H; Gurtner, Geoffrey C

    2014-12-01

    To investigate how epithelial mechanotransduction pathways impact wound repair. Mechanical forces are increasingly recognized to influence tissue repair, but their role in chronic wound pathophysiology remains unknown. Studies have shown that chronic wounds exhibit high levels of matrix metalloproteinase 9 (MMP9), a key proteolytic enzyme that regulates wound remodeling. We hypothesized that epithelial mechanosensory pathways regulated by keratinocyte-specific focal adhesion kinase (FAK) control dermal remodeling via MMP9. A standard wound model was applied to keratinocyte-specific FAK knockout (KO) and control mice. Rates of wound healing were measured and tissue was obtained for histologic and molecular analyses. Transcriptional and immunoblot assays were used to assess the activation of FAK, intracellular kinases, and MMP9 in vitro. A cell suspension model was designed to validate the importance of FAK mechanosensing, p38, and MMP9 secretion in human cells. Biomechanical testing was utilized to evaluate matrix tensile properties in FAK KO and control wounds. Wound healing in FAK KO mice was significantly delayed compared with controls (closure at 15 days compared with 20 days, P = 0.0003). FAK KO wounds demonstrated decreased dermal thickness and collagen density. FAK KO keratinocytes exhibited overactive p38 and MMP9 signaling in vitro, findings recapitulated in human keratinocytes via the deactivation of FAK in the cell suspension model. Functionally, FAK KO wounds were significantly weaker and more brittle than control wounds, results consistent with the histologic and molecular analyses. Keratinocyte FAK is highly responsive to mechanical cues and may play a critical role in matrix remodeling via regulation of p38 and MMP9. These findings suggest that aberrant epithelial mechanosensory pathways may contribute to pathologic dermal proteolysis and wound chronicity.

  18. Inhibition of tumor necrosis factor alpha-stimulated monocyte adhesion to human aortic endothelial cells by AMP-activated protein kinase.

    Science.gov (United States)

    Ewart, Marie-Ann; Kohlhaas, Christine F; Salt, Ian P

    2008-12-01

    Proatherosclerotic adhesion of leukocytes to the endothelium is attenuated by NO. As AMP-activated protein kinase (AMPK) regulates endothelial NO synthesis, we investigated the modulation of adhesion to cultured human aortic endothelial cells (HAECs) by AMPK. HAECs incubated with the AMPK activator, AICAR, or expressing constitutively active AMPK demonstrated reduced TNFalpha-stimulated adhesion of promonocytic U-937 cells. Rapid inhibition of TNFalpha-stimulated U-937 cell adhesion by AICAR was NO-dependent, associated with unaltered cell surface adhesion molecule expression, and reduced MCP-1 secretion by HAECs. In contrast, inhibition of TNFalpha-stimulated U-937 cell adhesion by prolonged AMPK activation was NO-independent and associated with reduced cell surface adhesion molecule expression. AMPK activation in HAECs inhibits TNFalpha-stimulated leukocyte adhesion by a rapid NO-dependent mechanism associated with reduced MCP-1 secretion and a late NO-independent mechanism whereby adhesion molecule expression, in particular E-selectin, is suppressed.

  19. Focal adhesion kinase as a mechanotransducer during rapid brain growth of the chick embryo.

    Science.gov (United States)

    Desmond, Mary E; Knepper, Janice E; DiBenedetto, Angela J; Malaugh, Elizabeth; Callejo, Sagrario; Carretero, Raquel; Alonso, Maria-Isabel; Gato, Angel

    2014-01-01

    Expansion of the hollow fluid-filled embryonic brain occurs by an increase in intraluminal pressure created by accumulation of cerebrospinal fluid (CSF). Experiments have shown a direct correlation between cavity pressure and cell proliferation within the neuroepithelium. These findings lead us to ask how mechanistically this might come about. Are there perhaps molecules on the luminal surface of the embryonic neuroepithelium, such as focal adhesion kinases (FAKs) known to respond to tension in other epithelial cells? Immunodetection using antibodies to total FAK and p-FAK was performed with subsequent confocal analysis of the pattern of their activation under normal intraluminal pressure and induced chronic pressure. Western analysis was also done to look at the amount of FAK expression, as well as its activation under these same conditions. Using immunolocalization, we have shown that FAK is present and activated on both apical and basolateral surfaces and within the cytoplasm of the neuroepithelial cells. This pattern changed profoundly when the neuroepithelium was under pressure. By Western blot, we have shown that FAK was upregulated and activated in the neuroepithelium of the embryos just after the neural tube becomes a closed pressurized system, with phosphorylation detected on the luminal instead of the basal surface, along with an increase in cell proliferation. Chronic hyper-pressure does not induce an increase in phosphorylation of FAK. In conclusion, here we show that neuroepithelial cells respond to intraluminal pressure via FAK phosphorylation on the luminal surface.

  20. Copper deficiency induced emphysema is associated with focal adhesion kinase inactivation.

    Directory of Open Access Journals (Sweden)

    Shiro Mizuno

    Full Text Available Copper is an important regulator of hypoxia inducible factor 1 alpha (HIF-1α dependent vascular endothelial growth factor (VEGF expression, and is also required for the activity of lysyl oxidase (LOX to effect matrix protein cross-linking. Cell detachment from the extracellular matrix can induce apoptosis (anoikis via inactivation of focal adhesion kinase (FAK.To examine the molecular mechanisms whereby copper depletion causes the destruction of the normal alveolar architecture via anoikis, Male Sprague-Dawley rats were fed a copper deficient diet for 6 weeks while being treated with the copper chelator, tetrathiomolybdate. Other groups of rats were treated with the inhibitor of auto-phosphorylation of FAK, 1,2,4,5-benzenetetraamine tetrahydrochloride (1,2,4,5-BT or FAK small interfering RNA (siRNA.Copper depletion caused emphysematous changes, decreased HIF-1α activity, and downregulated VEGF expression in the rat lungs. Cleaved caspase-3, caspase-8 and Bcl-2 interacting mediator of cell death (Bim expression was increased, and the phosphorylation of FAK was decreased in copper depleted rat lungs. Administration of 1,2,4,5-BT and FAK siRNA caused emphysematous lung destruction associated with increased expression of cleaved capase-3, caspase-8 and Bim.These data indicate that copper-dependent mechanisms contribute to the pathogenesis of emphysema, which may be associated with decreased HIF-1α and FAK activity in the lung.

  1. Down-regulation of integrin β1 and focal adhesion kinase in renal glomeruli under various hemodynamic conditions.

    Directory of Open Access Journals (Sweden)

    Xiaoli Yuan

    Full Text Available Given that integrin β1 is an important component of the connection to maintain glomerular structural integrity, by binding with multiple extracellular matrix proteins and mediating intracellular signaling. Focal adhesion kinase (FAK is the most essential intracellular integrator in the integrin β1-FAK signalling pathway. Here, we investigated the changes of the two molecules and visualized the possible interaction between them under various hemodynamic conditions in podocytes. Mice kidney tissues were prepared using in vivo cryotechnique (IVCT and then were stained and observed using light microscopy, confocal laser scanning microscopy and immunoelectron microscopy. The expression of these molecules were examined by western blot. Under the normal condition, integrin β1 stained continually and evenly at the membrane, and FAK was located in the cytoplasm and nuclei of the podocytes. There were significant colocalized plaques of two molecules. But under acute hypertensive and cardiac arrest conditions, integrin β1 decreased and stained intermittently. Similarly, FAK decreased and appeared uneven. Additionally, FAK translocated to the nuclei of the podocytes. As a result, the colocalization of integrin β1 and FAK reduced obviously under these conditions. Western blot assay showed a consistent result with the immunostaining. Collectively, the abnormal redistribution and decreased expressions of integrin β1 and FAK are important molecular events in regulating the functions of podocytes under abnormal hemodynamic conditions. IVCT could offer considerable advantages for morphological analysis when researching renal diseases.

  2. Multiple myeloma is affected by multiple and heterogeneous somatic mutations in adhesion- and receptor tyrosine kinase signaling molecules

    International Nuclear Information System (INIS)

    Leich, E; Weißbach, S; Klein, H-U; Grieb, T; Pischimarov, J; Stühmer, T; Chatterjee, M; Steinbrunn, T; Langer, C; Eilers, M; Knop, S; Einsele, H; Bargou, R; Rosenwald, A

    2013-01-01

    Multiple myeloma (MM) is a largely incurable plasma cell malignancy with a poorly understood and heterogeneous clinical course. To identify potential, functionally relevant somatic mutations in MM, we performed whole-exome sequencing of five primary MM, corresponding germline DNA and six MM cell lines, and developed a bioinformatics strategy that also integrated published mutational data of 38 MM patients. Our analysis confirms that identical, recurrent mutations of single genes are infrequent in MM, but highlights that mutations cluster in important cellular pathways. Specifically, we show enrichment of mutations in adhesion molecules of MM cells, emphasizing the important role for the interaction of the MM cells with their microenvironment. We describe an increased rate of mutations in receptor tyrosine kinases (RTKs) and associated signaling effectors, for example, in EGFR, ERBB3, KRAS and MAP2K2, pointing to a role of aberrant RTK signaling in the development or progression of MM. The diversity of mutations affecting different nodes of a particular signaling network appears to be an intrinsic feature of individual MM samples, and the elucidation of intra- as well as interindividual redundancy in mutations that affect survival pathways will help to better tailor targeted therapeutic strategies to the specific needs of the MM patient

  3. Phosphatidylinositol 4-kinases: Function, structure, and inhibition

    Czech Academy of Sciences Publication Activity Database

    Bouřa, Evžen; Nencka, Radim

    2015-01-01

    Roč. 337, č. 2 (2015), s. 136-145 ISSN 0014-4827 R&D Projects: GA ČR GJ15-21030Y; GA MŠk LO1302; GA ČR GA15-09310S EU Projects: European Commission(XE) 333916 - STARPI4K Institutional support: RVO:61388963 Keywords : phosphatidylinositol 4-kinase * inhibitor * crystal structure * virus Subject RIV: CC - Organic Chemistry Impact factor: 3.378, year: 2015

  4. Reduced Mechanical Stretch Induces Enhanced Endothelin B Receptor-mediated Contractility via Activation of Focal Adhesion Kinase and Extra Cellular-regulated Kinase 1/2 in Cerebral Arteries from Rat

    DEFF Research Database (Denmark)

    Spray, Stine; Rasmussen, Marianne N P; Skovsted, Gry F

    2016-01-01

    ETB receptor agonist sarafotoxin 6c. The involvement of extracellular regulated kinase (ERK) 1/2 and focal adhesion kinase (FAK) were studied by their specific inhibitors U0126 and PF-228, respectively. Compared to their stretched counterparts, un-stretched MCA segments showed a significantly...

  5. Increased osteoblast adhesion on nanoparticulate crystalline hydroxyapatite functionalized with KRSR.

    Science.gov (United States)

    Nelson, Michael; Balasundaram, Ganesan; Webster, Thomas J

    2006-01-01

    The present in vitro study created nanometer crystalline hydroxyapatite (HA) and amorphous calcium phosphate for novel orthopedic applications. Specifically, nano-crystalline HA and amorphous calcium phosphate nanoparticles were synthesized by a wet chemical process followed by hydrothermal treatment for 2 hours at 200 degrees C and 70 degrees C, respectively. Resulting particles were then pressed into compacts. For the preparation of control conventional HA particles (or those currently used in orthopedics with micron diameters), the aforementioned calcium phosphate particles were pressed into compacts and sintered at 1100 degrees C for 2 hours. All calcium phosphate-based particles were fully characterized. Results showed that although there was an initial weight gain for all the compacts studied in this experiment, higher eventual degradation rates up to 3 weeks were observed for nano-amorphous calcium phosphate compared with nano-crystalline HA which was higher than conventional HA. Peptide functionalization (with the cell adhesive peptide lysine-arginine-serine-arginine [KRSR] and the non-cell-adhesive peptide lysine-serine-arginine-arginine [KSRR]) was accomplished by means of a three-step reaction procedure: silanization with 3-aminopropyltriethoxysilane (APTES), cross-linking with N-succinimidyl-3-maleimido propionate (SMP), and finally peptide immobilization. The peptide functionalization was fully characterized. Results demonstrated increased osteoblast (bone-forming cell) adhesion on non-functionalized and functionalized nano-crystalline HA compacts compared with nano amorphous calcium phosphate compacts; both increased osteoblast adhesion compared with conventional HA. To further exemplify the novel properties of nano crystalline HA, results also showed similar osteoblast adhesion between non-functionalized nano crystalline HA and KRSR functionalized conventional HA. Thus, results provided evidence that nanocrystalline HA should be further studied for

  6. Cell Adhesion Molecules and Ubiquitination—Functions and Significance

    Science.gov (United States)

    Homrich, Mirka; Gotthard, Ingo; Wobst, Hilke; Diestel, Simone

    2015-01-01

    Cell adhesion molecules of the immunoglobulin (Ig) superfamily represent the biggest group of cell adhesion molecules. They have been analyzed since approximately 40 years ago and most of them have been shown to play a role in tumor progression and in the nervous system. All members of the Ig superfamily are intensively posttranslationally modified. However, many aspects of their cellular functions are not yet known. Since a few years ago it is known that some of the Ig superfamily members are modified by ubiquitin. Ubiquitination has classically been described as a proteasomal degradation signal but during the last years it became obvious that it can regulate many other processes including internalization of cell surface molecules and lysosomal sorting. The purpose of this review is to summarize the current knowledge about the ubiquitination of cell adhesion molecules of the Ig superfamily and to discuss its potential physiological roles in tumorigenesis and in the nervous system. PMID:26703751

  7. Titanium dioxide nanoparticles induce human eosinophil adhesion onto endothelial EA.hy926 cells via activation of phosphoinositide 3-kinase/Akt cell signalling pathway.

    Science.gov (United States)

    Murphy-Marion, Maxime; Girard, Denis

    2018-02-01

    The use of nanoparticles (NPs) for developing new therapeutic strategies in a variety of diseases is gaining increasing attention. However, NPs could possess undesired effects, including pro-inflammatory activities. Despite the fact that several studies reported that NPs may induce or exacerbate eosinophilic inflammation in vivo in rodents, the information regarding the direct interaction between NPs and human eosinophils is lacking. In the present study, we test the possibility that NPs could alter the capacity of human eosinophils to adhere onto a cellular substratum. Using a panel of NPs, we found that several were able to increase the adhesion of human eosinophil onto endothelial EA.hy926 cells. Among them, TiO 2 NPs were the most potent and we therefore pursue this study with these NPs. TiO 2 NPs were found to increase the adhesion of eosinophils in a concentration dependent fashion. TiO 2 NPs did not alter the cell surface expression of a panel of cellular adhesion molecules, but CD29. Indeed, a weak to moderate, but significant, decrease of CD29 was observed after 30min but returned to normal levels after 90min. TiO 2 NPs were found to activate Akt, one important target of phosphoinositide 3-kinase (PI3K). However, despite the fact that cells were fully responsive to the cytokine GM-CSF activating both Akt and Erk-1/2, TiO 2 NPs did not activate Erk-1/2. Using a pharmacological approach with the PI3K/Akt inhibitor, wortmannin, the ability of TiO 2 NPs to activate Akt was drastically inhibited and, further, their capacity to increase adhesion of eosinophils was reversed. This study provides insights into the effects of NPs on the biology of human eosinophils indicating that as other agents, NPs, namely TiO 2 NPs, can induce intracellular events associated with a cellular function, adhesion. Copyright © 2017 Elsevier GmbH. All rights reserved.

  8. Indole-3-carbinol inhibits MDA-MB-231 breast cancer cell motility and induces stress fibers and focal adhesion formation by activation of Rho kinase activity.

    Science.gov (United States)

    Brew, Christine T; Aronchik, Ida; Kosco, Karena; McCammon, Jasmine; Bjeldanes, Leonard F; Firestone, Gary L

    2009-05-15

    Indole-3-carbinol (I3C), a phytochemical derived from cruciferous vegetables such as broccoli and Brussels sprouts, has potent antiproliferative effects in human breast cancer cells and has been shown to decrease metastatic spread of tumors in experimental animals. Using chemotaxis and fluorescent-bead cell motility assays, we demonstrated that I3C significantly decreased the in vitro migration of MDA-MB-231 cells, a highly invasive breast cancer cell line. Immunofluorescence staining of the actin cytoskeleton revealed that concurrent with the loss of cell motility, I3C treatment significantly increased stress fiber formation. Furthermore, I3C induced the localization of the focal adhesion component vinculin and tyrosine-phosphorylated proteins to the cell periphery, which implicates an indole-dependent enhancement of focal adhesions within the outer boundary of the cells. Coimmunoprecipitation analysis of focal adhesion kinase demonstrated that I3C stimulated the dynamic formation of the focal adhesion protein complex without altering the total level of individual focal adhesion proteins. The RhoA-Rho kinase pathway is involved in stress fiber and focal adhesion formation, and I3C treatment stimulated Rho kinase enzymatic activity and cofilin phosphorylation, which is a downstream target of Rho kinase signaling, but did not increase the level of active GTP-bound RhoA. Exposure of MDA-MB-231 cells to the Rho kinase inhibitor Y-27632, or expression of dominant negative RhoA ablated the I3C induced formation of stress fibers and of peripheral focal adhesions. Expression of constitutively active RhoA mimicked the I3C effects on both processes. Taken together, our data demonstrate that I3C induces stress fibers and peripheral focal adhesions in a Rho kinase-dependent manner that leads to an inhibition of motility in human breast cancer cells. (c) 2008 Wiley-Liss, Inc.

  9. Cortactin is a scaffolding platform for the E-cadherin adhesion complex and is regulated by protein kinase D1 phosphorylation.

    Science.gov (United States)

    Sroka, Robert; Van Lint, Johan; Katz, Sarah-Fee; Schneider, Marlon R; Kleger, Alexander; Paschke, Stephan; Seufferlein, Thomas; Eiseler, Tim

    2016-06-15

    Dynamic regulation of cell-cell adhesion by the coordinated formation and dissolution of E-cadherin-based adherens junctions is crucial for tissue homeostasis. The actin-binding protein cortactin interacts with E-cadherin and enables F-actin accumulation at adherens junctions. Here, we were interested to study the broader functional interactions of cortactin in adhesion complexes. In line with literature, we demonstrate that cortactin binds to E-cadherin, and that a posttranslational modification of cortactin, RhoA-induced phosphorylation by protein kinase D1 (PKD1; also known as PRKD1) at S298, impairs adherens junction assembly and supports their dissolution. Two new S298-phosphorylation-dependent interactions were also identified, namely, that phosphorylation of cortactin decreases its interaction with β-catenin and the actin-binding protein vinculin. In addition, binding of vinculin to β-catenin, as well as linkage of vinculin to F-actin, are also significantly compromised upon phosphorylation of cortactin. Accordingly, we found that regulation of cell-cell adhesion by phosphorylation of cortactin downstream of RhoA and PKD1 is vitally dependent on vinculin-mediated protein interactions. Thus, cortactin, unexpectedly, is an important integration node for the dynamic regulation of protein complexes during breakdown and formation of adherens junctions. © 2016. Published by The Company of Biologists Ltd.

  10. Focal Adhesion Kinase (FAK) Binds RET Kinase via Its FERM Domain, Priming a Direct and Reciprocal RET-FAK Transactivation Mechanism

    NARCIS (Netherlands)

    Plaza-Menacho, Ivan; Morandi, Andrea; Mologni, Luca; Boender, Piet; Gambacorti-Passerini, Carlo; Magee, Anthony I.; Hofstra, Robert M. W.; Knowles, Phillip; McDonald, Neil Q.; Isacke, Clare M.

    2011-01-01

    Whether RET is able to directly phosphorylate and activate downstream targets independently of the binding of proteins that contain Src homology 2 or phosphotyrosine binding domains and whether mechanisms in trans by cytoplasmic kinases can modulate RET function and signaling remain largely

  11. Understanding doublecortin-like kinase gene function through transgenesis

    NARCIS (Netherlands)

    Schenk, Geert J.

    2010-01-01

    Doublecortin (DCX) and DCX-domain containing Doublecortin-Like Kinase (DCLK) gene splice variants function during embryonic development, where they play a role in microtubule binding. Although a role for the DCLK gene during embryogenesis is clearly established, it encodes multiple, different

  12. Kinetic Mechanism and Rate-Limiting Steps of Focal Adhesion Kinase-1

    Energy Technology Data Exchange (ETDEWEB)

    Schneck, Jessica L.; Briand, Jacques; Chen, Stephanie; Lehr, Ruth; McDevitt, Patrick; Zhao, Baoguang; Smallwood, Angela; Concha, Nestor; Oza, Khyati; Kirkpatrick, Robert; Yan, Kang; Villa, James P.; Meek, Thomas D.; Thrall, Sara H. (Chemizon); (GSKPA)

    2010-12-07

    Steady-state kinetic analysis of focal adhesion kinase-1 (FAK1) was performed using radiometric measurement of phosphorylation of a synthetic peptide substrate (Ac-RRRRRRSETDDYAEIID-NH{sub 2}, FAK-tide) which corresponds to the sequence of an autophosphorylation site in FAK1. Initial velocity studies were consistent with a sequential kinetic mechanism, for which apparent kinetic values k{sub cat} (0.052 {+-} 0.001 s{sup -1}), K{sub MgATP} (1.2 {+-} 0.1 {micro}M), K{sub iMgATP} (1.3 {+-} 0.2 {micro}M), K{sub FAK-tide} (5.6 {+-} 0.4 {micro}M), and K{sub iFAK-tide} (6.1 {+-} 1.1 {micro}M) were obtained. Product and dead-end inhibition data indicated that enzymatic phosphorylation of FAK-tide by FAK1 was best described by a random bi bi kinetic mechanism, for which both E-MgADP-FAK-tide and E-MgATP-P-FAK-tide dead-end complexes form. FAK1 catalyzed the {beta}{gamma}-bridge:{beta}-nonbridge positional oxygen exchange of [{gamma}-{sup 18}O{sub 4}]ATP in the presence of 1 mM [{gamma}-{sup 18}O{sub 4}]ATP and 1.5 mM FAK-tide with a progressive time course which was commensurate with catalysis, resulting in a rate of exchange to catalysis of k{sub x}/k{sub cat} = 0.14 {+-} 0.01. These results indicate that phosphoryl transfer is reversible and that a slow kinetic step follows formation of the E-MgADP-P-FAK-tide complex. Further kinetic studies performed in the presence of the microscopic viscosogen sucrose revealed that solvent viscosity had no effect on k{sub cat}/K{sub FAK-tide}, while k{sub cat} and k{sub cat}/K{sub MgATP} were both decreased linearly at increasing solvent viscosity. Crystallographic characterization of inactive versus AMP-PNP-liganded structures of FAK1 showed that a large conformational motion of the activation loop upon ATP binding may be an essential step during catalysis and would explain the viscosity effect observed on k{sub cat}/K{sub m} for MgATP but not on k{sub cat}/K{sub m} for FAK-tide. From the positional isotope exchange, viscosity, and

  13. Electrostatic adhesion for added functionality of composite structures

    International Nuclear Information System (INIS)

    Heath, Callum J C; Bond, Ian P; Potter, Kevin D

    2016-01-01

    Electrostatic adhesion can be used as a means of reversible attachment. The incorporation of electrostatic adhesion into fibre reinforced polymer (FRP) composite structures could provide significant value added functionality. Imparting large potential differences (∼2 kV) across electrodes generates an attractive force, thus providing a means of attachment. This could be used as a reversible latching mechanism or as a means of controllable internal connectivity. Varying the connectivity for discrete elements of a substructure of a given design allows for control of internal load paths and moment of area of the cross section. This could facilitate variable stiffness (both in bending and torsion). Using a combination of existing fabrication techniques, functional electrodes have been integrated within a FRP. Copper polyimide thin film laminate material has been both co-cured with carbon fibre reinforced epoxy and bonded to PVC closed cell foam core material to provide a range of structural configurations with integrated electrodes. The ability of such integrated devices to confer variations in global bending stiffness of basic beam structures is investigated. Through the application of 4 kV across integrated electrostatic adhesive devices, a 112% increase in flexural stiffness has been demonstrated for a composite sandwich structure. (paper)

  14. Electrostatic adhesion for added functionality of composite structures

    Science.gov (United States)

    Heath, Callum J. C.; Bond, Ian P.; Potter, Kevin D.

    2016-02-01

    Electrostatic adhesion can be used as a means of reversible attachment. The incorporation of electrostatic adhesion into fibre reinforced polymer (FRP) composite structures could provide significant value added functionality. Imparting large potential differences (˜2 kV) across electrodes generates an attractive force, thus providing a means of attachment. This could be used as a reversible latching mechanism or as a means of controllable internal connectivity. Varying the connectivity for discrete elements of a substructure of a given design allows for control of internal load paths and moment of area of the cross section. This could facilitate variable stiffness (both in bending and torsion). Using a combination of existing fabrication techniques, functional electrodes have been integrated within a FRP. Copper polyimide thin film laminate material has been both co-cured with carbon fibre reinforced epoxy and bonded to PVC closed cell foam core material to provide a range of structural configurations with integrated electrodes. The ability of such integrated devices to confer variations in global bending stiffness of basic beam structures is investigated. Through the application of 4 kV across integrated electrostatic adhesive devices, a 112% increase in flexural stiffness has been demonstrated for a composite sandwich structure.

  15. Resensitization of breast cancer cells to anoikis by tropomyosin-1: role of Rho kinase-dependent cytoskeleton and adhesion.

    Science.gov (United States)

    Bharadwaj, Shantaram; Thanawala, Ruchi; Bon, Giulia; Falcioni, Rita; Prasad, G L

    2005-12-15

    Two most common properties of malignant cells are the presence of aberrant actin cytoskeleton and resistance to anoikis. Suppression of several key cytoskeletal proteins, including tropomyosin-1 (TM1), during neoplastic transformation is hypothesized to contribute to the altered cytoskeleton and neoplastic phenotype. Using TM1 as a paradigm, we have shown that cytoskeletal proteins induce anoikis in breast cancer (MCF-7 and MDA MB 231) cells. Here, we have tested the hypothesis that TM1-mediated cytoskeletal changes regulate integrin activity and the sensitivity to anoikis. TM1 expression in MDA MB 231 cells promotes the assembly of stress fibers, induces rapid anoikis via caspase-dependent pathways involving the release of cytochrome c. Further, TM1 inhibits binding of MDA MB 231 cells to collagen I, but promotes adhesion to laminin. Inhibition of Rho kinase disrupts TM1-mediated cytoskeletal reorganization and adhesion to the extracellular matrix components, whereas the parental cells attach to collagen I, spread and form extensive actin meshwork in the presence of Rho kinase inhibitor, underscoring the differences in parental and TM1-transduced breast cancer cells. Further, treatment with the cytoskeletal disrupting drugs rescues the cells from TM1-induced anoikis. These new findings demonstrate that the aberrant cytoskeleton contributes to neoplastic transformation by conferring resistance to anoikis. Restoration of stress fiber network through enhanced expression of key cytoskeletal proteins may modulate the activity of focal adhesions and sensitize the neoplastic cells to anoikis. Oncogene (2005) 24, 8291-8303. doi:10.1038/sj.onc.1208993; published online 19 September 2005.

  16. c-Src, Insulin-Like Growth Factor I Receptor, G-Protein-Coupled Receptor Kinases and Focal Adhesion Kinase are Enriched Into Prostate Cancer Cell Exosomes.

    Science.gov (United States)

    DeRita, Rachel M; Zerlanko, Brad; Singh, Amrita; Lu, Huimin; Iozzo, Renato V; Benovic, Jeffrey L; Languino, Lucia R

    2017-01-01

    It is well known that Src tyrosine kinase, insulin-like growth factor 1 receptor (IGF-IR), and focal adhesion kinase (FAK) play important roles in prostate cancer (PrCa) development and progression. Src, which signals through FAK in response to integrin activation, has been implicated in many aspects of tumor biology, such as cell proliferation, metastasis, and angiogenesis. Furthermore, Src signaling is known to crosstalk with IGF-IR, which also promotes angiogenesis. In this study, we demonstrate that c-Src, IGF-IR, and FAK are packaged into exosomes (Exo), c-Src in particular being highly enriched in Exo from the androgen receptor (AR)-positive cell line C4-2B and AR-negative cell lines PC3 and DU145. Furthermore, we show that the active phosphorylated form of Src (Src pY416 ) is co-expressed in Exo with phosphorylated FAK (FAK pY861 ), a known target site of Src, which enhances proliferation and migration. We further demonstrate for the first time exosomal enrichment of G-protein-coupled receptor kinase (GRK) 5 and GRK6, both of which regulate Src and IGF-IR signaling and have been implicated in cancer. Finally, Src pY416 and c-Src are both expressed in Exo isolated from the plasma of prostate tumor-bearing TRAMP mice, and those same mice have higher levels of exosomal c-Src than their wild-type counterparts. In summary, we provide new evidence that active signaling molecules relevant to PrCa are enriched in Exo, and this suggests that the Src signaling network may provide useful biomarkers detectable by liquid biopsy, and may contribute to PrCa progression via Exo. J. Cell. Biochem. 118: 66-73, 2017. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

  17. Two distinct functions for PI3-kinases in macropinocytosis.

    Science.gov (United States)

    Hoeller, Oliver; Bolourani, Parvin; Clark, Jonathan; Stephens, Len R; Hawkins, Phillip T; Weiner, Orion D; Weeks, Gerald; Kay, Robert R

    2013-09-15

    Class-1 PI3-kinases are major regulators of the actin cytoskeleton, whose precise contributions to chemotaxis, phagocytosis and macropinocytosis remain unresolved. We used systematic genetic ablation to examine this question in growing Dictyostelium cells. Mass spectroscopy shows that a quintuple mutant lacking the entire genomic complement of class-1 PI3-kinases retains only 10% of wild-type PtdIns(3,4,5)P3 levels. Chemotaxis to folate and phagocytosis of bacteria proceed normally in the quintuple mutant but macropinocytosis is abolished. In this context PI3-kinases show specialized functions, only one of which is directly linked to gross PtdIns(3,4,5)P3 levels: macropinosomes originate in patches of PtdIns(3,4,5)P3, with associated F-actin-rich ruffles, both of which depend on PI3-kinase 1/2 (PI3K1/2) but not PI3K4, whereas conversion of ruffles into vesicles requires PI3K4. A biosensor derived from the Ras-binding domain of PI3K1 suggests that Ras is activated throughout vesicle formation. Binding assays show that RasG and RasS interact most strongly with PI3K1/2 and PI3K4, and single mutants of either Ras have severe macropinocytosis defects. Thus, the fundamental function of PI3-kinases in growing Dictyostelium cells is in macropinocytosis where they have two distinct functions, supported by at least two separate Ras proteins.

  18. Adhesive Bonding and Corrosion Performance Investigated as a Function of Aluminum Oxide Chemistry and Adhesives

    NARCIS (Netherlands)

    Abrahami, S.T.; Hauffman, T.; de Kok, John M.M.; Terryn, H.A.; Mol, J.M.C.

    2017-01-01

    The long-term strength and durability of an adhesive bond is dependent on the stability of the oxide-adhesive interface. As such, changes in the chemistry of the oxide and/or the adhesive are expected to modify the interfacial properties and affect the joint performance in practice. The upcoming

  19. Junctional adhesion molecule-A: functional diversity through molecular promiscuity.

    Science.gov (United States)

    Steinbacher, Tim; Kummer, Daniel; Ebnet, Klaus

    2017-12-14

    Cell adhesion molecules (CAMs) of the immunoglobulin superfamily (IgSF) regulate important processes such as cell proliferation, differentiation and morphogenesis. This activity is primarily due to their ability to initiate intracellular signaling cascades at cell-cell contact sites. Junctional adhesion molecule-A (JAM-A) is an IgSF-CAM with a short cytoplasmic tail that has no catalytic activity. Nevertheless, JAM-A is involved in a variety of biological processes. The functional diversity of JAM-A resides to a large part in a C-terminal PDZ domain binding motif which directly interacts with nine different PDZ domain-containing proteins. The molecular promiscuity of its PDZ domain motif allows JAM-A to recruit protein scaffolds to specific sites of cell-cell adhesion and to assemble signaling complexes at those sites. Here, we review the molecular characteristics of JAM-A, including its dimerization, its interaction with scaffolding proteins, and the phosphorylation of its cytoplasmic domain, and we describe how these characteristics translate into diverse biological activities.

  20. Receptor tyrosine kinase structure and function in health and disease

    Directory of Open Access Journals (Sweden)

    Oleg A. Karpov

    2015-09-01

    Full Text Available Receptor tyrosine kinases (RTKs are membrane proteins that control the flow of information through signal transduction pathways, impacting on different aspects of cell function. RTKs are characterized by a ligand-binding ectodomain, a single transmembrane α-helix, a cytosolic region comprising juxtamembrane and kinase domains followed by a flexible C-terminal tail. Somatic and germline RTK mutations can induce aberrant signal transduction to give rise to cardiovascular, developmental and oncogenic abnormalities. RTK overexpression occurs in certain cancers, correlating signal strength and disease incidence. Diverse RTK activation and signal transduction mechanisms are employed by cells during commitment to health or disease. Small molecule inhibitors are one means to target RTK function in disease initiation and progression. This review considers RTK structure, activation, and signal transduction and evaluates biological relevance to therapeutics and clinical outcomes.

  1. A role for focal adhesion kinase in facilitating the contractile responses of murine gastric fundus smooth muscles.

    Science.gov (United States)

    Xie, Yeming; Han, Koon Hee; Grainger, Nathan; Li, Wen; Corrigan, Robert D; Perrino, Brian A

    2018-03-12

    Smooth muscle contraction involves regulating myosin light chain phosphorylation and dephosphorylation by myosin light chain kinase and myosin light chain phosphatase. CPI-17 and MYPT1 are crucial for regulating gastrointestinal smooth muscle contraction by inhibiting myosin light chain phosphatase. Integrin signalling involves the dynamic recruitment of several proteins, including FAK, to focal adhesions. FAK tyrosine kinase activation is involved in cell adhesion to the extracellular matrix via integrin signalling. FAK participates in linking the force generated by myofilament activation to the extracellular matrix and throughout the smooth muscle tissue. Here, we show that cholinergic stimulation activates FAK in gastric fundus smooth muscles. Electrical field stimulation in the presence of L-NAME and MRS2500 contracted gastric fundus smooth muscle strips and increased FAK Y397 phosphorylation (pY397). Atropine blocked the contractions and prevented the increase in pY397. The FAK inhibitor PF-431396 inhibited the contractions and the increase in pY397. PF-431396 also inhibited the EFS-induced increase in CPI-17 T38 phosphorylation, and reduced MYPT1 T696 and T853, and myosin light chain S19 phosphorylation. Ca 2+ influx was unaffected by PF-431396. Nicardipine inhibited the contractions but had no effect on the increase in pY397. PDBu or calyculin A contracted gastric fundus smooth muscle strips Ca 2+ independently and increased pY397. Our findings suggest that FAK is activated by mechanical forces during contraction, and reveal a novel role of FAK in the regulation of CPI-17 phosphorylation. This article is protected by copyright. All rights reserved. This article is protected by copyright. All rights reserved.

  2. Focal adhesion kinase-mediated activation of glycogen synthase kinase 3β regulates IL-33 receptor internalization and IL-33 signaling.

    Science.gov (United States)

    Zhao, Jing; Wei, Jianxin; Bowser, Rachel K; Traister, Russell S; Fan, Ming-Hui; Zhao, Yutong

    2015-01-15

    IL-33, a relatively new member of the IL-1 cytokine family, plays a crucial role in allergic inflammation and acute lung injury. Long form ST2 (ST2L), the receptor for IL-33, is expressed on immune effector cells and lung epithelia and plays a critical role in triggering inflammation. We have previously shown that ST2L stability is regulated by the ubiquitin-proteasome system; however, its upstream internalization has not been studied. In this study, we demonstrate that glycogen synthase kinase 3β (GSK3β) regulates ST2L internalization and IL-33 signaling. IL-33 treatment induced ST2L internalization, and an effect was attenuated by inhibition or downregulation of GSK3β. GSK3β was found to interact with ST2L on serine residue 446 in response to IL-33 treatment. GSK3β binding site mutant (ST2L(S446A)) and phosphorylation site mutant (ST2L(S442A)) are resistant to IL-33-induced ST2L internalization. We also found that IL-33 activated focal adhesion kinase (FAK). Inhibition of FAK impaired IL-33-induced GSK3β activation and ST2L internalization. Furthermore, inhibition of ST2L internalization enhanced IL-33-induced cytokine release in lung epithelial cells. These results suggest that modulation of the ST2L internalization by FAK/GSK3β might serve as a unique strategy to lessen pulmonary inflammation. Copyright © 2015 by The American Association of Immunologists, Inc.

  3. Identification and functional analysis of mitogen-activated protein kinase kinase kinase (MAPKKK) genes in canola (Brassica napus L.).

    Science.gov (United States)

    Sun, Yun; Wang, Chen; Yang, Bo; Wu, Feifei; Hao, Xueyu; Liang, Wanwan; Niu, Fangfang; Yan, Jingli; Zhang, Hanfeng; Wang, Boya; Deyholos, Michael K; Jiang, Yuan-Qing

    2014-05-01

    Mitogen-activated protein kinase (MAPK) signalling cascades, consisting of three types of reversibly phosphorylated kinases (MAPKKK, MAPKK, and MAPK), are involved in important processes including plant immunity and hormone responses. The MAPKKKs comprise the largest family in the MAPK cascades, yet only a few of these genes have been associated with physiological functions, even in the model plant Arabidopsis thaliana. Canola (Brassica napus L.) is one of the most important oilseed crops in China and worldwide. To explore MAPKKK functions in biotic and abiotic stress responses in canola, 66 MAPKKK genes were identified and 28 of them were cloned. Phylogenetic analysis of these canola MAPKKKs with homologous genes from representative species classified them into three groups (A-C), comprising four MAPKKKs, seven ZIKs, and 17 Raf genes. A further 15 interaction pairs between these MAPKKKs and the downstream BnaMKKs were identified through a yeast two-hybrid assay. The interactions were further validated through bimolecular fluorescence complementation (BiFC) analysis. In addition, by quantitative real-time reverse transcription-PCR, it was further observed that some of these BnaMAPKKK genes were regulated by different hormone stimuli, abiotic stresses, or fungal pathogen treatments. Interestingly, two novel BnaMAPKKK genes, BnaMAPKKK18 and BnaMAPKKK19, which could elicit hypersensitive response (HR)-like cell death when transiently expressed in Nicotiana benthamiana leaves, were successfully identified. Moreover, it was found that BnaMAPKKK19 probably mediated cell death through BnaMKK9. Overall, the present work has laid the foundation for further characterization of this important MAPKKK gene family in canola.

  4. Down-regulation of procaspase-8 expression by focal adhesion kinase protects HL-60 cells from TRAIL-induced apoptosis

    International Nuclear Information System (INIS)

    Tamagiku, Yuji; Sonoda, Yoshiko; Kunisawa, Mari; Ichikawa, Daiju; Murakami, Yayoi; Aizu-Yokota, Eriko; Kasahara, Tadashi

    2004-01-01

    We have demonstrated that focal adhesion kinase (FAK)-overexpressed (HL-60/FAK) cells have marked resistance against various apoptotic stimuli such as hydrogen peroxide, etoposide, and ionizing radiation compared with the vector-transfected (HL-60/Vect) cells. HL-60/FAK cells are highly resistant to TRAIL-induced apoptosis, while original HL-60 or HL-60/Vect cells were sensitive. TRAIL at 500 ng/ml induced significant DNA fragmentation, activation of caspase-8 and 3, the processing of a proapoptotic BID, and mitochondrial release of cytochrome c in HL-60/Vect cells, whereas no such events were observed in the HL-60/FAK cells. In particular, the expression of procaspase-8 gene and subsequent cleavage of caspase-8 were markedly reduced in HL-60/FAK cells, while expression of TRAIL-receptor 2 and 3, TRADD, and FADD was equivalent in both types of cells. In HL-60/FAK cells, the phosphoinositide 3 (PI3)-kinase/Akt survival pathway was constitutively activated, accompanied by significant induction of inhibitor-of-apoptosis proteins, XIAP, RIP, and Bcl-XL. The introduction of FAK siRNA in HL-60/FAK cells sensitized them against TRAIL-induced apoptosis, confirming that overexpressed FAK downregulates procaspase-8 expression, which subsequently inhibits downstream apoptosis pathway in the HL-60/FAK cells

  5. Homozygous mutation of focal adhesion kinase in embryonic stem cell derived neurons: normal electrophysiological and morphological properties in vitro

    Directory of Open Access Journals (Sweden)

    Komiyama NH

    2006-06-01

    Full Text Available Abstract Background Genetically manipulated embryonic stem (ES cell derived neurons (ESNs provide a powerful system with which to study the consequences of gene manipulation in mature, synaptically connected neurons in vitro. Here we report a study of focal adhesion kinase (FAK, which has been implicated in synapse formation and regulation of ion channels, using the ESN system to circumvent the embryonic lethality of homozygous FAK mutant mice. Results Mouse ES cells carrying homozygous null mutations (FAK-/- were generated and differentiated in vitro into neurons. FAK-/- ESNs extended axons and dendrites and formed morphologically and electrophysiologically intact synapses. A detailed study of NMDA receptor gated currents and voltage sensitive calcium currents revealed no difference in their magnitude, or modulation by tyrosine kinases. Conclusion FAK does not have an obligatory role in neuronal differentiation, synapse formation or the expression of NMDA receptor or voltage-gated calcium currents under the conditions used in this study. The use of genetically modified ESNs has great potential for rapidly and effectively examining the consequences of neuronal gene manipulation and is complementary to mouse studies.

  6. Focal adhesion kinase-promoted tumor glucose metabolism is associated with a shift of mitochondrial respiration to glycolysis.

    Science.gov (United States)

    Zhang, J; Gao, Q; Zhou, Y; Dier, U; Hempel, N; Hochwald, S N

    2016-04-14

    Cancer cells often gains a growth advantage by taking up glucose at a high rate and undergoing aerobic glycolysis through intrinsic cellular factors that reprogram glucose metabolism. Focal adhesion kinase (FAK), a key transmitter of growth factor and anchorage stimulation, is aberrantly overexpressed or activated in most solid tumors, including pancreatic ductal adenocarcinomas (PDACs). We determined whether FAK can act as an intrinsic driver to promote aerobic glycolysis and tumorigenesis. FAK inhibition decreases and overexpression increases intracellular glucose levels during unfavorable conditions, including growth factor deficiency and cell detachment. Amplex glucose assay, fluorescence and carbon-13 tracing studies demonstrate that FAK promotes glucose consumption and glucose-to-lactate conversion. Extracellular flux analysis indicates that FAK enhances glycolysis and decreases mitochondrial respiration. FAK increases key glycolytic proteins, including enolase, pyruvate kinase M2 (PKM2), lactate dehydrogenase and monocarboxylate transporter. Furthermore, active/tyrosine-phosphorylated FAK directly binds to PKM2 and promotes PKM2-mediated glycolysis. On the other hand, FAK-decreased levels of mitochondrial complex I can result in reduced oxidative phosphorylation (OXPHOS). Attenuation of FAK-enhanced glycolysis re-sensitizes cancer cells to growth factor withdrawal, decreases cell viability and reduces growth of tumor xenografts. These observations, for the first time, establish a vital role of FAK in cancer glucose metabolism through alterations in the OXPHOS-to-glycolysis balance. Broadly targeting the common phenotype of aerobic glycolysis and more specifically FAK-reprogrammed glucose metabolism will disrupt the bioenergetic and biosynthetic supply for uncontrolled growth of tumors, particularly glycolytic PDAC.

  7. Understanding the Structural Basis of Adhesion GPCR Functions.

    Science.gov (United States)

    Araç, Demet; Sträter, Norbert; Seiradake, Elena

    2016-01-01

    Unlike conventional G-protein-coupled receptors (GPCRs), adhesion GPCRs (aGPCRs) have large extracellular regions that are autoproteolytically cleaved from their membrane-embedded seven-pass transmembrane helices. Autoproteolysis occurs within the conserved GPCR-Autoproteolysis INducing (GAIN) domain that is juxtaposed to the transmembrane domain and cleaves the last beta strand of the GAIN domain. The other domains of the extracellular region are variable and specific to each aGPCR and are likely involved in adhering to various ligands. Emerging evidence suggest that extracellular regions may modulate receptor function and that ligand binding to the extracellular regions may induce receptor activation via multiple mechanisms. Here, we summarize current knowledge about the structural understanding for the extracellular regions of aGPCRs and discuss their possible functional roles that emerge from the available structural information.

  8. Protein kinase A-alpha directly phosphorylates FoxO1 in vascular endothelial cells to regulate expression of vascular cellular adhesion molecule-1 mRNA.

    Science.gov (United States)

    Lee, Ji-Won; Chen, Hui; Pullikotil, Philomena; Quon, Michael J

    2011-02-25

    FoxO1, a forkhead box O class transcription factor, is abundant in insulin-responsive tissues. Akt, downstream from phosphatidylinositol 3-kinase in insulin signaling, phosphorylates FoxO1 at Thr(24), Ser(256), and Ser(319), negatively regulating its function. We previously reported that dehydroepiandrosterone-stimulated phosphorylation of FoxO1 in endothelial cells requires cAMP-dependent protein kinase α (PKA-α). Therefore, we hypothesized that FoxO1 is a novel direct substrate for PKA-α. Using an immune complex kinase assay with [γ-(32)P]ATP, purified PKA-α directly phosphorylated wild-type FoxO1 but not FoxO1-AAA (mutant with alanine substitutions at known Akt phosphorylation sites). Phosphorylation of wild-type FoxO1 (but not FoxO1-AAA) was detectable using phospho-specific antibodies. Similar results were obtained using purified GST-FoxO1 protein as the substrate. Thus, FoxO1 is a direct substrate for PKA-α in vitro. In bovine aortic endothelial cells, interaction between endogenous PKA-α and endogenous FoxO1 was detected by co-immunoprecipitation. In human aortic endothelial cells (HAEC), pretreatment with H89 (PKA inhibitor) or siRNA knockdown of PKA-α decreased forskolin- or prostaglandin E(2)-stimulated phosphorylation of FoxO1. In HAEC transfected with a FoxO-promoter luciferase reporter, co-expression of the catalytic domain of PKA-α, catalytically inactive mutant PKA-α, or siRNA against PKA-α caused corresponding increases or decreases in transactivation of the FoxO promoter. Expression of vascular cellular adhesion molecule-1 mRNA, up-regulated by FoxO1 in endothelial cells, was enhanced by siRNA knockdown of PKA-α or treatment of HAEC with the PKA inhibitor H89. Adhesion of monocytes to endothelial cells was enhanced by H89 treatment or overexpression of FoxO1-AAA, similar to effects of TNF-α treatment. We conclude that FoxO1 is a novel physiological substrate for PKA-α in vascular endothelial cells.

  9. The new InsP3Kinase inhibitor BIP-4 is competitive to InsP3 and blocks proliferation and adhesion of lung cancer cells.

    Science.gov (United States)

    Schröder, Dominik; Tödter, Klaus; Gonzalez, Beatriz; Franco-Echevarría, Elsa; Rohaly, Gabor; Blecher, Christine; Lin, Hong-Ying; Mayr, Georg W; Windhorst, Sabine

    2015-07-15

    As ectopic expression of the neuronal inositol-1,4,5-trisphosphate-3-kinase A (InsP3Kinase) in tumor cells increases the metastatic potential, InsP3Kinase is an interesting target for tumor therapy. Recently, we have identified a membrane-permeable InsP3Kinase inhibitor (BAMB-4) exhibiting an IC50-value of 20 μM. Here we characterized a new InsP3Kinase inhibitor which shows a 130-fold lower IC50 value (157 ± 57 nM) as compared to BAMB-4. We demonstrate that this nitrophenolic compound, BIP-4, is non-competitive to ATP but competitive to InsP3, thus exhibits a high selectivity for inhibition of InsP3Kinase activity. Docking analysis suggested a putative binding mode of this molecule into the InsP3Kinase active site. Determination of cellular uptake in lung cancer cells (H1299) revealed that 6% of extracellular BIP-4 is internalized by non-endosomal uptake, showing that BIP-4 is not trapped inside endo/lysosomes but is available to inhibit cellular InsP3Kinase activity. Interestingly, we found that BIP-4 mediated inhibition of InsP3Kinase activity in the two lung cancer cell lines H1299 and LN4323 inhibited proliferation and adhesion at IC50 values of 3 μM or 2 μM, respectively. InsP3Kinase inhibition did not alter ATP-induced calcium signals but significantly reduced the level of Ins(1,3,4,5,6)P5. From these data we conclude that the inhibitory effect of BIP-4 on proliferation and adhesion of lung cancer cells does not result from alterations of calcium but from alterations of inositol phosphate signals. In summary, we reveal that inhibition of cellular InsP3Kinase by BIP-4 impairs proliferation and adhesion and therefore BIP-4 might be a promising compound to reduce the metastatic potential of lung carcinoma cells. Copyright © 2015 Elsevier Inc. All rights reserved.

  10. Protein kinase CK2 structure-function relationship

    DEFF Research Database (Denmark)

    Boldyreff, B; Meggio, F; Pinna, L A

    1994-01-01

    Protein kinase CK2 subunits alpha and beta were expressed either separately or together in a bacterial expression system (pT7-7/BL21(DE3)) and purified to homogeneity. After mixing the subunits, a CK2 holoenzyme (alpha 2 beta 2) was spontaneously reconstituted, which displays identical features...... inactivated through urea, protease, and heat treatment. In contrast, the holoenzyme, either reconstituted or native, is much more stable when similar negative insults prevail. The beta subunit has at least three functions: (a) it is necessary for maximum activity of the enzyme under physiological salt...

  11. Functional diversity of human protein kinase splice variants marks significant expansion of human kinome

    Directory of Open Access Journals (Sweden)

    Anamika Krishanpal

    2009-12-01

    Full Text Available Abstract Background Protein kinases are involved in diverse spectrum of cellular processes. Availability of draft version of the human genomic data in the year 2001 enabled recognition of repertoire of protein kinases. However, over the years the human genomic data is being refined and the current release of human genomic data has helped us to recognize a larger repertoire of over 900 human protein kinases represented mainly by splice variants. Results Many of these identified protein kinases are alternatively spliced products. Interestingly, some of the human kinase splice variants appear to be significantly diverged in terms of their functional properties as represented by incorporation or absence of one or more domains. Many sets of protein kinase splice variants have substantially different domain organization and in a few sets of splice variants kinase domains belong to different subfamilies of kinases suggesting potential participation in different signal transduction pathways. Conclusions Addition or deletion of a domain between splice variants of multi-domain kinases appears to be a means of generating differences in the functional features of otherwise similar kinases. It is intriguing that marked sequence diversity within the catalytic regions of some of the splice variant kinases result in kinases belonging to different subfamilies. These human kinase splice variants with different functions might contribute to diversity of eukaryotic cellular signaling.

  12. Progesterone receptor isoforms PRA and PRB differentially contribute to breast cancer cell migration through interaction with focal adhesion kinase complexes.

    Science.gov (United States)

    Bellance, Catherine; Khan, Junaid A; Meduri, Geri; Guiochon-Mantel, Anne; Lombès, Marc; Loosfelt, Hugues

    2013-05-01

    Progesterone receptor (PR) and progestins affect mammary tumorigenesis; however, the relative contributions of PR isoforms A and B (PRA and PRB, respectively) in cancer cell migration remains elusive. By using a bi-inducible MDA-MB-231 breast cancer cell line expressing PRA and/or PRB, we analyzed the effect of conditional PR isoform expression. Surprisingly, unliganded PRB but not PRA strongly enhanced cell migration as compared with PR(-) cells. 17,21-Dimethyl-19-norpregna-4,9-dien-3,20-dione (R5020) progestin limited this effect and was counteracted by the antagonist 11β-(4-dimethyl-amino)-phenyl-17β-hydroxy-17-(1-propynyl)-estra-4,9-dien-3-one (RU486). Of importance, PRA coexpression potentiated PRB-mediated migration, whereas PRA alone was ineffective. PR isoforms differentially regulated expressions of major players of cell migration, such as urokinase plasminogen activator (uPA), its inhibitor plasminogen activator inhibitor type 1, uPA receptor (uPAR), and β1-integrin, which affect focal adhesion kinase (FAK) signaling. Moreover, unliganded PRB but not PRA enhanced FAK Tyr397 phosphorylation and colocalized with activated FAK in cell protrusions. Because PRB, as well as PRA, coimmunoprecipitated with FAK, both isoforms can interact with FAK complexes, depending on their respective nucleocytoplasmic trafficking. In addition, FAK degradation was coupled to R5020-dependent turnovers of PRA and PRB. Such an effect of PRB/PRA expression on FAK signaling might thus affect adhesion/motility, underscoring the implication of PR isoforms in breast cancer invasiveness and metastatic evolution with underlying therapeutic outcomes.

  13. Activation of Focal Adhesion Kinase and Src Mediates Acquired Sorafenib Resistance in A549 Human Lung Adenocarcinoma Xenografts.

    Science.gov (United States)

    Zhou, Qingyu; Guo, Xiaofang; Choksi, Riya

    2017-12-01

    Despite encouraging clinical results with sorafenib monotherapy in patients with KRAS- mutant non-small-cell lung cancer (NSCLC), the overall survival benefit of this drug is limited by the inevitable development of acquired resistance. The exact mechanism underlying acquired sorafenib resistance in KRAS -mutant NSCLC is unclear. In this study, the mechanism of acquired sorafenib resistance was explored using a biologically relevant xenograft model, which was established by using the A549 human lung adenocarcinoma cell line and an in vivo-derived, sorafenib-resistant A549 subline (A549/SRFres). Results from the initial study demonstrated that sorafenib treatment significantly decreased E-cadherin ( P A549/SRFres tumors, whereas expression levels of phospho-protein kinase B (AKT), phospho-focal adhesion kinase (FAK), and phospho-Src were elevated in sorafenib-treated A549 and A549/SRFres tumors. We next examined whether concomitant dasatinib treatment could overcome acquired sorafenib resistance by blocking the FAK/Src escape route that mediates resistance. Despite the observed in vitro synergy between sorafenib and dasatinib, the in vivo antitumor effect of half-dose sorafenib-dasatinib combination therapy was inferior to that of the full-dose sorafenib treatment. Although the sorafenib-dasatinib combination effectively inhibited Src and AKT phosphorylation, it did not block the Y576/577-FAK phosphorylation, nor did it decrease vimentin protein expression; unexpectedly, it increased Y397-FAK phosphorylation and MMP9 protein expression in tumors. These results suggest that acquired sorafenib resistance in KRAS -mutant A549 xenografts involves the compensatory activation of FAK and Src, and Src inhibition alone is insufficient to diminish sorafenib-promoted epithelial-mesenchymal transition process and invasive potentials in tumors. Copyright © 2017 by The American Society for Pharmacology and Experimental Therapeutics.

  14. A kinome wide screen identifies novel kinases involved in regulation of monoamine transporter function

    DEFF Research Database (Denmark)

    Vuorenpää, Anne Elina; Ammendrup-Johnsen, Ina; Jorgensen, Trine N.

    2016-01-01

    cells (CAD) and rat chromocytoma (PC12) cells. Whereas SIK3 likely transcriptionally regulated expression of the three transfected transporters, depletion of PKA C-α was shown to decrease SERT function. Depletion of PrKX caused decreased surface expression and function of DAT without changing protein...... in regulation of monoamine transporter function and surface expression. A primary screen in HEK 293 cells stably expressing DAT or SERT with siRNAs against 573 human kinases revealed 93 kinases putatively regulating transporter function. All 93 hits, which also included kinases previously implicated...... in HEK 293 cells transiently expressing DAT, SERT or NET. Subsequently, three kinases; salt inducible kinase 3 (SIK3), cAMP-dependent protein kinase catalytic subunit alpha (PKA C-α) and protein kinase X-linked (PrKX); were selected for additional exploration in catecholaminergic CATH.a differentiated...

  15. Diacylglycerol kinases in T cell tolerance and effector function

    Directory of Open Access Journals (Sweden)

    Shelley S Chen

    2016-11-01

    Full Text Available Diacylglycerol kinases (DGKs are a family of enzymes that regulate the relative levels of diacylglycerol (DAG and phosphatidic acid (PA in cells by phosphorylating DAG to produce PA. Both DAG and PA are important second messengers cascading T cell receptor (TCR signal by recruiting multiple effector molecules such as RasGRP1, PKC, and mTOR. Studies have revealed important physiological functions of DGKs in the regulation of receptor signaling and the development and activation of immune cells. In this review, we will focus on recent progresses in our understanding of two DGK isoforms,  and , in CD8 T effector and memory cell differentiation, regulatory T cell development and function, and invariant NKT cell development and effector lineage differentiation.

  16. Surface functionalization of silicone rubber for permanent adhesion improvement.

    Science.gov (United States)

    Roth, Jan; Albrecht, Victoria; Nitschke, Mirko; Bellmann, Cornelia; Simon, Frank; Zschoche, Stefan; Michel, Stefan; Luhmann, Claudia; Grundke, Karina; Voit, Brigitte

    2008-11-04

    The surface properties of poly(dimethyl siloxane) (PDMS) layers screen printed onto silicon wafers were studied after oxygen and ammonia plasma treatments and subsequent grafting of poly(ethylene -alt-maleic anhydride) (PEMA) using X-ray photoelectron spectroscopy (XPS), roughness analysis, and contact angle and electrokinetic measurements. In the case of oxygen-plasma-treated PDMS, a hydrophilic, brittle, silica-like surface layer containing reactive silanol groups was obtained. These surfaces indicate a strong tendency for "hydrophobic recovery" due to the surface segregation of low-molecular-weight PDMS species. The ammonia plasma treatment of PDMS resulted in the generation of amino-functional surface groups and the formation of a weak boundary layer that could be washed off by polar liquids. To avoid the loss of the plasma modification effect and to achieve stabilization of the mechanically instable, functionalized PDMS top layer, PEMA was subsequently grafted directly or after using gamma-APS as a coupling agent on the plasma-activated PDMS surfaces. In this way, long-time stable surface functionalization of PDMS was obtained. The reactivity of the PEMA-coated PDMS surface caused by the availability of anhydride groups could be controlled by the number of amino functional surface groups of the PDMS surface necessary for the covalent binding of PEMA. The higher the number of amino functional surface groups available for the grafting-to procedure, the lower the hydrophilicity and hence the lower the reactivity of the PEMA-coated PDMS surface. Additionally, pull-off tests were applied to estimate the effect of surface modification on the adhesion between the silicone rubber and an epoxy resin.

  17. Focal Adhesion Kinase Directly Interacts with TSC2 Through Its FAT Domain and Regulates Cell Proliferation in Cashmere Goat Fetal Fibroblasts.

    Science.gov (United States)

    Zheng, Xu; Bao, Wenlei; Yang, Jiaofu; Zhang, Tao; Sun, Dongsheng; Liang, Yan; Li, Shuyu; Wang, Yanfeng; Feng, Xue; Hao, Huifang; Wang, Zhigang

    2016-09-01

    Focal adhesion kinase (FAK) is a cytoplasmic nonreceptor tyrosine kinase that senses a variety of extracellular signals, such as growth factors and integrins, to control the process of cell proliferation and metabolism. We cloned three goat FAK transcript variants (KM655805, KM658268, and KM658269) that encode 1052, 1006, and 962 amino-acid residue proteins. Bioinformatics analysis indicated that the putative FAK protein contains an FERM domain, a PTK domain, two Proline-rich regions, and a focal adhesion-targeting (FAT) domain. All the three transcript variants of FAK were detected in seven different goat tissues, and variant 1 had the most accumulation whereas variant 2 and variant 3 had lower accumulation. Treatment of goat fetal fibroblasts (GFbs) with a specific FAK inhibitor, TAE226, inhibited cell proliferation (p Cashmere goat tissues and its products participate in the mammalian target of rapamycin signaling pathway and cell proliferation through a direct interaction with TSC2 in GFBs.

  18. Heat shock protein 90β stabilizes focal adhesion kinase and enhances cell migration and invasion in breast cancer cells

    Energy Technology Data Exchange (ETDEWEB)

    Xiong, Xiangyang [Center for Experimental Medicine, The First Affiliated Hospital of Nanchang University, 17 Yongwai Street, Donghu District, Nanchang, Jiangxi 330006 (China); Department of Biochemistry and Molecular Biology, School of Basic Medical Sciences, Nanchang University, Nanchang, Jiangxi 330006 (China); State Key Laboratory of Food Science and Technology, Nanchang University, Nanchang, Jiangxi 330047 (China); Wang, Yao [Center for Experimental Medicine, The First Affiliated Hospital of Nanchang University, 17 Yongwai Street, Donghu District, Nanchang, Jiangxi 330006 (China); Liu, Chengmei [State Key Laboratory of Food Science and Technology, Nanchang University, Nanchang, Jiangxi 330047 (China); Lu, Quqin [Department of Biostatistics and Epidemiology, School of Public Health, Nanchang University, Nanchang, Jiangxi 330006 (China); Liu, Tao [Center for Experimental Medicine, The First Affiliated Hospital of Nanchang University, 17 Yongwai Street, Donghu District, Nanchang, Jiangxi 330006 (China); Chen, Guoan [Department of Hematology, The First Affiliated Hospital of Nanchang University, Nanchang, Jiangxi 330006 (China); Rao, Hai [Department of Molecular Medicine, University of Texas Health Science Center, San Antonio, TX 78229 (United States); Luo, Shiwen, E-mail: shiwenluo@ncu.edu.cn [Center for Experimental Medicine, The First Affiliated Hospital of Nanchang University, 17 Yongwai Street, Donghu District, Nanchang, Jiangxi 330006 (China)

    2014-08-01

    Focal adhesion kinase (FAK) acts as a regulator of cellular signaling and may promote cell spreading, motility, invasion and survival in malignancy. Elevated expression and activity of FAK frequently correlate with tumor cell metastasis and poor prognosis in breast cancer. However, the mechanisms by which the turnover of FAK is regulated remain elusive. Here we report that heat shock protein 90β (HSP90β) interacts with FAK and the middle domain (amino acids 233–620) of HSP90β is mainly responsible for this interaction. Furthermore, we found that HSP90β regulates FAK stability since HSP90β inhibitor 17-AAG triggers FAK ubiquitylation and subsequent proteasome-dependent degradation. Moreover, disrupted FAK-HSP90β interaction induced by 17-AAG contributes to attenuation of tumor cell growth, migration, and invasion. Together, our results reveal how HSP90β regulates FAK stability and identifies a potential therapeutic strategy to breast cancer. - Highlights: • HSP90β protects FAK from degradation by the ubiquitin-proteasome pathway. • Inhibition of HSP90β or FAK attenuates tumorigenesis of breast cancer cells. • Genetic repression of HSP90β or FAK inhibits tumor cell migration and proliferation. • Inhibition of HSP90β or FAK interferes cell invasion and cytoskeleton.

  19. Heat shock protein 90β stabilizes focal adhesion kinase and enhances cell migration and invasion in breast cancer cells

    International Nuclear Information System (INIS)

    Xiong, Xiangyang; Wang, Yao; Liu, Chengmei; Lu, Quqin; Liu, Tao; Chen, Guoan; Rao, Hai; Luo, Shiwen

    2014-01-01

    Focal adhesion kinase (FAK) acts as a regulator of cellular signaling and may promote cell spreading, motility, invasion and survival in malignancy. Elevated expression and activity of FAK frequently correlate with tumor cell metastasis and poor prognosis in breast cancer. However, the mechanisms by which the turnover of FAK is regulated remain elusive. Here we report that heat shock protein 90β (HSP90β) interacts with FAK and the middle domain (amino acids 233–620) of HSP90β is mainly responsible for this interaction. Furthermore, we found that HSP90β regulates FAK stability since HSP90β inhibitor 17-AAG triggers FAK ubiquitylation and subsequent proteasome-dependent degradation. Moreover, disrupted FAK-HSP90β interaction induced by 17-AAG contributes to attenuation of tumor cell growth, migration, and invasion. Together, our results reveal how HSP90β regulates FAK stability and identifies a potential therapeutic strategy to breast cancer. - Highlights: • HSP90β protects FAK from degradation by the ubiquitin-proteasome pathway. • Inhibition of HSP90β or FAK attenuates tumorigenesis of breast cancer cells. • Genetic repression of HSP90β or FAK inhibits tumor cell migration and proliferation. • Inhibition of HSP90β or FAK interferes cell invasion and cytoskeleton

  20. Patient-reported measures of quality of life and functional capacity in adhesive capsulitis

    OpenAIRE

    Fernandes, Marcos Rassi

    2017-01-01

    Summary Objective: To evaluate patient-reported measures of quality of life and functional capacity in adhesive capsulitis before and after suprascapular nerve block treatment; to analyze the influence of clinical and sociodemographic variables on both the outcome and correlation between the respective instruments. Method: A prospective clinical study was performed with adhesive capsulitis patients. Inclusion criteria were clinical diagnosis of adhesive capsulitis and a shoulder imaging tes...

  1. Dual functions of Bruton's tyrosine kinase and Tec kinase during Fcgamma receptor-induced signaling and phagocytosis.

    Science.gov (United States)

    Jongstra-Bilen, Jenny; Puig Cano, Adrianet; Hasija, Manvi; Xiao, Haiyan; Smith, C I Edvard; Cybulsky, Myron I

    2008-07-01

    Tec family nonreceptor tyrosine kinases are expressed by hematopoietic cells, activate phospholipase C (PLC)gamma, and regulate cytoskeletal rearrangement, yet their role in FcgammaR-induced signaling and phagocytosis remains unknown. We demonstrate in this study that Bruton's tyrosine kinase (Btk) and Tec, the only Tec kinases expressed by RAW 264.7 cells, are activated throughout phagocytosis. Activated Btk and Tec kinase accumulate at an early stage at the base of phagocytic cups and inhibition of their activity by the specific inhibitor LFM-A13 or expression by small interfering RNA significantly inhibited FcgammaR-induced phagocytosis. Similarly, a significant role for these kinases in phagocytosis was found in primary macrophages. FcgammaR-induced activation of Mac-1, which is required for optimal phagocytosis, was markedly inhibited and our findings suggest that the roles of kinases Btk and Tec in Mac-1 activation account for their functions in the early stages of phagocytosis. Initial activation of PLCgamma2, the predominant PLC isoform in RAW 264.7 cells, is dependent on Syk. In contrast, a late and prolonged activation of PLCgamma2 was dependent on Btk and Tec. We found accumulation of diacylglycerol (DAG), a PLCgamma product, in phagosome membranes, and activated Btk, but not Tec, colocalized with phagosomal DAG. Inhibition of Tec family kinase activity increased the level of DAG in phagosomes, suggesting a negative regulatory role for Btk. Tec, in contrast, clustered at sites near phagosome formation. In summary, we elucidated that Tec family kinases participate in at least two stages of FcgammaR-mediated phagocytosis: activation of Mac-1 during ingestion, and after phagosome formation, during which Btk and Tec potentially have distinct roles.

  2. Adhesion of oxide layer to metal-doped aluminum hydride surface: Density functional calculations

    Science.gov (United States)

    Takezawa, Tomoki; Itoi, Junichi; Kannan, Takashi

    2017-07-01

    The density functional theory (DFT) calculations were carried out to evaluate the adhesion energy of the oxide layer to the metal-doped surface of hydrogen storage material, aluminum hydride (alane, AlH3). The total energy calculations using slab model revealed that the surface doping of some metals to aluminum hydride weakens the adhesion strength of the oxide layer. The influence of titanium, iron, cobalt, and zirconium doping on adhesion strength were evaluated. Except for iron doping, the adhesion strength becomes weak by the doping.

  3. Dissecting signaling and functions of adhesion G protein-coupled receptors

    NARCIS (Netherlands)

    Araç, Demet; Aust, Gabriela; Calebiro, Davide; Engel, Felix B.; Formstone, Caroline; Goffinet, André; Hamann, Jörg; Kittel, Robert J.; Liebscher, Ines; Lin, Hsi-Hsien; Monk, Kelly R.; Petrenko, Alexander; Piao, Xianhua; Prömel, Simone; Schiöth, Helgi B.; Schwartz, Thue W.; Stacey, Martin; Ushkaryov, Yuri A.; Wobus, Manja; Wolfrum, Uwe; Xu, Lei; Langenhan, Tobias

    2012-01-01

    G protein-coupled receptors (GPCRs) comprise an expanded superfamily of receptors in the human genome. Adhesion class G protein-coupled receptors (adhesion-GPCRs) form the second largest class of GPCRs. Despite the abundance, size, molecular structure, and functions in facilitating cell and matrix

  4. Dissecting signaling and functions of adhesion G protein-coupled receptors

    DEFF Research Database (Denmark)

    Araç, Demet; Aust, Gabriela; Calebiro, Davide

    2012-01-01

    G protein-coupled receptors (GPCRs) comprise an expanded superfamily of receptors in the human genome. Adhesion class G protein-coupled receptors (adhesion-GPCRs) form the second largest class of GPCRs. Despite the abundance, size, molecular structure, and functions in facilitating cell and matrix...

  5. Crosstalk between focal adhesions and material mechanical properties governs cell mechanics and functions.

    Science.gov (United States)

    Fusco, Sabato; Panzetta, Valeria; Embrione, Valerio; Netti, Paolo A

    2015-09-01

    Mechanical properties of materials strongly influence cell fate and functions. Focal adhesions are involved in the extremely important processes of mechanosensing and mechanotransduction. To address the relationship between the mechanical properties of cell substrates, focal adhesion/cytoskeleton assembly and cell functions, we investigated the behavior of NIH/3T3 cells over a wide range of stiffness (3-1000kPa) using two of the most common synthetic polymers for cell cultures: polyacrylamide and polydimethylsiloxane. An overlapping stiffness region was created between them to compare focal adhesion characteristics and cell functions, taking into account their different time-dependent behavior. Indeed, from a rheological point of view, polyacrylamide behaves like a strong gel (elastically), whereas polydimethylsiloxane like a viscoelastic solid. First, focal adhesion characteristics and dynamics were addressed in terms of material stiffness, then cell spreading area, migration rate and cell mechanical properties were correlated with focal adhesion size and assembly. Focal adhesion size was found to increase in the whole range of stiffness and to be in agreement in the overlapping rigidity region for the investigated materials. Cell mechanics directly correlated with focal adhesion lengths, whereas migration rate followed an inverse correlation. Cell spreading correlated with the substrate stiffness on polyacrylamide hydrogel, while no specific trend was found on polydimethylsiloxane. Substrate mechanics can be considered as a key physical cue that regulates focal adhesion assembly, which in turn governs important cellular properties and functions. Copyright © 2015 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

  6. Focal Adhesion of Osteoblastic Cells on Titanium Surface with Amine Functionalities Formed by Plasma Polymerization

    Science.gov (United States)

    Song, Heesang; Jung, Sang Chul; Kim, Byung Hoon

    2012-08-01

    To enhance the focal adhesion of osteoblastic cells on a titanium surface, plasma polymerized allyl amine (AAm) thin films were deposited by plasma polymerization. This plasma polymer functionalization of titanium is advantageous for osteoblastic focal adhesion formation. Such Ti surfaces are useful for the fabrication of titanium-based dental implants for enhancement of osseointegration.

  7. Dissecting signaling and functions of adhesion G protein-coupled receptors.

    Science.gov (United States)

    Araç, Demet; Aust, Gabriela; Calebiro, Davide; Engel, Felix B; Formstone, Caroline; Goffinet, André; Hamann, Jörg; Kittel, Robert J; Liebscher, Ines; Lin, Hsi-Hsien; Monk, Kelly R; Petrenko, Alexander; Piao, Xianhua; Prömel, Simone; Schiöth, Helgi B; Schwartz, Thue W; Stacey, Martin; Ushkaryov, Yuri A; Wobus, Manja; Wolfrum, Uwe; Xu, Lei; Langenhan, Tobias

    2012-12-01

    G protein-coupled receptors (GPCRs) comprise an expanded superfamily of receptors in the human genome. Adhesion class G protein-coupled receptors (adhesion-GPCRs) form the second largest class of GPCRs. Despite the abundance, size, molecular structure, and functions in facilitating cell and matrix contacts in a variety of organ systems, adhesion-GPCRs are by far the most poorly understood GPCR class. Adhesion-GPCRs possess a unique molecular structure, with extended N-termini containing various adhesion domains. In addition, many adhesion-GPCRs are autoproteolytically cleaved into an N-terminal fragment (NTF, NT, α-subunit) and C-terminal fragment (CTF, CT, β-subunit) at a conserved GPCR autoproteolysis-inducing (GAIN) domain that contains a GPCR proteolysis site (GPS). These two features distinguish adhesion-GPCRs from other GPCR classes. Though active research on adhesion-GPCRs in diverse areas, such as immunity, neuroscience, and development and tumor biology has been intensified in the recent years, the general biological and pharmacological properties of adhesion-GPCRs are not well known, and they have not yet been used for biomedical purposes. The "6th International Adhesion-GPCR Workshop," held at the Institute of Physiology of the University of Würzburg on September 6-8, 2012, assembled a majority of the investigators currently actively pursuing research on adhesion-GPCRs, including scientists from laboratories in Europe, the United States, and Asia. The meeting featured the nascent mechanistic understanding of the molecular events driving the signal transduction of adhesion-GPCRs, novel models to evaluate their functions, and evidence for their involvement in human disease. © 2012 New York Academy of Sciences.

  8. Platelet adhesion enhances the glycoprotein VI-dependent procoagulant response: Involvement of p38 MAP kinase and calpain.

    Science.gov (United States)

    Siljander, P; Farndale, R W; Feijge, M A; Comfurius, P; Kos, S; Bevers, E M; Heemskerk, J W

    2001-04-01

    In the final stages of activation, platelets express coagulation-promoting activity by 2 simultaneous processes: exposure of aminophospholipids, eg, phosphatidylserine (PS), at the platelet surface, and formation of membrane blebs, which may be shed as microvesicles. Contact with collagen triggers both processes via platelet glycoprotein VI (GPVI). Here, we studied the capacity of 2 GPVI ligands, collagen-related peptide (CRP) and the snake venom protein convulxin (CVX), to elicit the procoagulant platelet response. In platelets in suspension, either ligand induced full aggregation and high Ca(2+) signals but little microvesiculation or PS exposure. However, most of the platelets adhering to immobilized CRP or CVX had exposed PS and formed membrane blebs after a prolonged increase in cytosolic [Ca(2+)](i). Platelets adhering to fibrinogen responded similarly but only when exposed to soluble CRP or CVX. By scanning electron microscopic analysis, the bleb-forming platelets were detected as either round, spongelike structures with associated microparticles or as arrays of vesicular cell fragments. The phosphorylation of p38 mitogen-activated protein kinase (MAPK) elicited by CRP and CVX was enhanced in fibrinogen-adherent platelets compared with that in platelets in suspension. The p38 inhibitor SB203580 and the calpain protease inhibitor calpeptin reduced only the procoagulant bleb formation, having no effect on PS exposure. Inhibition of p38 also downregulated calpain activity. We conclude that the procoagulant response evoked by GPVI stimulation is potentiated by platelet adhesion. The sequential activation of p38 MAPK and calpain appears to regulate procoagulant membrane blebbing but not PS exposure.

  9. Expression of focal adhesion kinase in uveal melanoma and the effects of Hsp90 inhibition by 17-AAG.

    Science.gov (United States)

    Faingold, Dana; Filho, Vasco Bravo; Fernandes, Bruno; Jagan, Lisa; de Barros, Alexandre M; Orellana, Maria Eugenia; Antecka, Emilia; Burnier, Miguel N

    2014-11-01

    Focal adhesion kinase (FAK) is implicated in tumor progression and metastatic cascade, and has been shown to be overexpressed in a variety of human cancers. However, the role of FAK in human uveal melanoma (UM) is not well defined. The purpose of this study was to evaluate the expression of FAK in UM tumors and normal eyes, and to determine the effect of Hsp90 inhibition on FAK expression in UM cells. FAK expression was assessed in 39 UM specimens, FAK[pY397] expression was assessed in 51 UM specimens, and both FAK and FAK[pY397] expression were assessed in 20 normal eyes. The expression of FAK and FAK[pY397] was detected by Western blot in five UM cell lines after treatment with 10 μmol/L of 17-AAG. FAK was positive in 87.2% and FAK[pY397] in 90% of UM specimens. Low FAK expression was detected in non-tumor structures and in normal eyes. The cell lines with the most proliferative, invasive phenotype (92.1, SP6.5 and MKT-BR) displayed high expression of FAK[pY397], and the levels of FAK and FAK[pY397] were decreased in the presence of 17-AAG starting with 24 h of exposure. FAK and FAK[pY397] were overexpressed in human UM tumors compared to normal ocular tissue and high levels of FAK[pY397] were seen in the most aggressive UM cell lines. Hsp90 inhibition led to downregulation of FAK expression. We propose a role for FAK in the pathogenesis of UM. Future studies are needed to explore the use of Hsp90 inhibitors as a feasible approach for modulating FAK in UM. Copyright © 2014 Elsevier GmbH. All rights reserved.

  10. Interleukin-2 induces beta2-integrin-dependent signal transduction involving the focal adhesion kinase-related protein B (fakB)

    DEFF Research Database (Denmark)

    Brockdorff, J; Kanner, S B; Nielsen, M

    1998-01-01

    beta2 integrin molecules are involved in a multitude of cellular events, including adhesion, migration, and cellular activation. Here, we studied the influence of beta2 integrins on interleukin-2 (IL-2)-mediated signal transduction in human CD4(+) T cell lines obtained from healthy donors...... and a leukocyte adhesion deficiency (LAD) patient. We show that IL-2 induces tyrosine phosphorylation of a 125-kDa protein and homotypic adhesion in beta2 integrin (CD18)-positive but not in beta2-integrin-negative T cells. EDTA, an inhibitor of integrin adhesion, blocks IL-2-induced tyrosine phosphorylation...... experiments indicate that the IL-2-induced 125-kDa phosphotyrosine protein is the focal adhesion kinase-related protein B (fakB). Thus, IL-2 induces strong tyrosine phosphorylation of fakB in beta2-integrin-positive but not in beta2-integrin-negative T cells, and CD18 mAb selectively blocks IL-2-induced fak...

  11. The PI3-kinase delta inhibitor idelalisib (GS-1101 targets integrin-mediated adhesion of chronic lymphocytic leukemia (CLL cell to endothelial and marrow stromal cells.

    Directory of Open Access Journals (Sweden)

    Stefania Fiorcari

    Full Text Available CLL cell trafficking between blood and tissue compartments is an integral part of the disease process. Idelalisib, a phosphoinositide 3-kinase delta (PI3Kδ inhibitor causes rapid lymph node shrinkage, along with an increase in lymphocytosis, prior to inducing objective responses in CLL patients. This characteristic activity presumably is due to CLL cell redistribution from tissues into the blood, but the underlying mechanisms are not fully understood. We therefore analyzed idelalisib effects on CLL cell adhesion to endothelial and bone marrow stromal cells (EC, BMSC. We found that idelalisib inhibited CLL cell adhesion to EC and BMSC under static and shear flow conditions. TNFα-induced VCAM-1 (CD106 expression in supporting layers increased CLL cell adhesion and accentuated the inhibitory effect of idelalisib. Co-culture with EC and BMSC also protected CLL from undergoing apoptosis, and this EC- and BMSC-mediated protection was antagonized by idelalisib. Furthermore, we demonstrate that CLL cell adhesion to EC and VLA-4 (CD49d resulted in the phosphorylation of Akt, which was sensitive to inhibition by idelalisib. These findings demonstrate that idelalisib interferes with integrin-mediated CLL cell adhesion to EC and BMSC, providing a novel mechanism to explain idelalisib-induced redistribution of CLL cells from tissues into the blood.

  12. The protein tyrosine kinase Tec regulates mast cell function.

    Science.gov (United States)

    Schmidt, Uwe; Abramova, Anastasia; Boucheron, Nicole; Eckelhart, Eva; Schebesta, Alexandra; Bilic, Ivan; Kneidinger, Michael; Unger, Bernd; Hammer, Martina; Sibilia, Maria; Valent, Peter; Ellmeier, Wilfried

    2009-11-01

    Mast cells play crucial roles in a variety of normal and pathophysiological processes and their activation has to be tightly controlled. Here, we demonstrate that the protein tyrosine kinase Tec is a crucial regulator of murine mast cell function. Tec was activated upon Fc epsilon RI stimulation of BM-derived mast cells (BMMC). The release of histamine in the absence of Tec was normal in vitro and in vivo; however, leukotriene C(4) levels were reduced in Tec(-) (/) (-) BMMC. Furthermore, the production of IL-4 was severely impaired, and GM-CSF, TNF-alpha and IL-13 levels were also diminished. Finally, a comparison of WT, Tec(-) (/) (-), Btk(-) (/) (-) and Tec(-) (/) (-)Btk(-) (/) (-) BMMC revealed a negative role for Btk in the regulation of IL-4 production, while for the efficient production of TNF-alpha, IL-13 and GM-CSF, both Tec and Btk were required. Our results demonstrate a crucial role for Tec in mast cells, which is partially different to the function of the well-characterized family member Btk.

  13. Investigation of adhesion of functional nanolayers to different substrates

    Science.gov (United States)

    Krzemiński, J.; Skalski, A.; Szałapak, J.; Jakubowska, M.

    2016-09-01

    The adhesion test are used in almost any industry. There are many different techniques and tools from scotch tape test to nanoscratch machine. The common test is pull-off test. Authors design the module for the testing machine and presented the researches about the adhesion of spray coated nanosilver layers to different substrates. In the article the main technique problems are discussed. Authors shows the typical graph result and described the separated sections. The experimental model was made and checked. The breaking forces of glass, Kapton and Aluminium samples are presented on the graph and discussed.

  14. Functional cine MR imaging for the detection and mapping of intraabdominal adhesions: method and surgical correlation

    Energy Technology Data Exchange (ETDEWEB)

    Buhmann-Kirchhoff, Sonja; Reiser, Maximilian; Lienemann, Andreas [University Hospital Grosshadern, Ludwig-Maximilians-University Munich, Department of Clinical Radiology, Munich (Germany); Lang, Reinhold; Steitz, Heinrich O.; Jauch, Karl W. [University Hospital Munich-Grosshadern, Department of Surgery, Munich (Germany); Kirchhoff, Chlodwig [University Hospital Munich-Innenstadt, Department of Surgery, Munich (Germany)

    2008-06-15

    The purpose of this study was to evaluate the presence and localization of intraabdominal adhesions using functional cine magnetic resonance imaging (MRI) and to correlate the MR findings with intraoperative results. In a retrospective study, patients who had undergone previous abdominal surgery with suspected intraabdominal adhesions were examined. A true fast imaging with steady state precession sequence in transverse/sagittal orientation was used for a section-by-section dynamic depiction of visceral slide on a 1.5-Tesla system. After MRI, all patients underwent anew surgery. A nine-segment abdominal map was used to document the location and type of the adhesions. The intraoperative results were taken as standard of reference. Ninety patients were enrolled. During surgery 71 adhesions were detected, MRI depicted 68 intraabdominal adhesions. The most common type of adhesion in MRI was found between the anterior abdominal wall and small bowel loops (n = 22, 32.5%) and between small bowel loops and pelvic organs (n = 14, 20.6%). Comparing MRI with the intraoperative findings, sensitivity varied between 31 and 75% with a varying specificity between 65 and 92% in the different segments leading to an overall MRI accuracy of 89%. Functional cine MRI proved to be a useful examination technique for the identification of intraabdominal adhesions in patients with acute or chronic pain and corresponding clinical findings providing accurate results. However, no differentiation for symptomatic versus asymptomatic adhesions is possible. (orig.)

  15. Adhesion GPCRs CD97 and GPR56: From structural regulation to cellular function

    NARCIS (Netherlands)

    Hsiao, C.-C.

    2015-01-01

    Cells correspond with their environment through receptors that translate extracellular signals into intracellular messages. Members of the large superfamily of G protein-coupled receptors (GPCRs) control various physiological functions and have been implicated in numerous diseases. Adhesion GPCRs

  16. Correlation between functional disability and quality of life in patients with adhesive capsulitis

    Science.gov (United States)

    Fernandes, Marcos Rassi

    2015-01-01

    OBJECTIVE: To determine the correlation between functional disability and quality of life of patients with adhesive capsulitis. METHODS: Two instruments (WHOQOL-BREF and DASH) were applied to evaluate the quality of life and functional capacity of patients with adhesive capsulitis. Inclusion criteria were age between 35 and 75 years old and achievement of shoulder imaging. Each domain of the WHOQOL-BREF was correlated with DASH. Pearson's correlation coefficient was used for parametric variables and Spearman's correlation coefficient was used when at least one variable had a non-normal distribution. The level of significance was p adhesive capsulitis. Level of Evidence IV, Prospective Study. PMID:27069405

  17. An Evolutionary-Conserved Function of Mammalian Notch Family Members as Cell Adhesion Molecules

    Science.gov (United States)

    Murata, Akihiko; Yoshino, Miya; Hikosaka, Mari; Okuyama, Kazuki; Zhou, Lan; Sakano, Seiji; Yagita, Hideo; Hayashi, Shin-Ichi

    2014-01-01

    Notch family members were first identified as cell adhesion molecules by cell aggregation assays in Drosophila studies. However, they are generally recognized as signaling molecules, and it was unclear if their adhesion function was restricted to Drosophila. We previously demonstrated that a mouse Notch ligand, Delta-like 1 (Dll1) functioned as a cell adhesion molecule. We here investigated whether this adhesion function was conserved in the diversified mammalian Notch ligands consisted of two families, Delta-like (Dll1, Dll3 and Dll4) and Jagged (Jag1 and Jag2). The forced expression of mouse Dll1, Dll4, Jag1, and Jag2, but not Dll3, on stromal cells induced the rapid and enhanced adhesion of cultured mast cells (MCs). This was attributed to the binding of Notch1 and Notch2 on MCs to each Notch ligand on the stromal cells themselves, and not the activation of Notch signaling. Notch receptor-ligand binding strongly supported the tethering of MCs to stromal cells, the first step of cell adhesion. However, the Jag2-mediated adhesion of MCs was weaker and unlike other ligands appeared to require additional factor(s) in addition to the receptor-ligand binding. Taken together, these results demonstrated that the function of cell adhesion was conserved in mammalian as well as Drosophila Notch family members. Since Notch receptor-ligand interaction plays important roles in a broad spectrum of biological processes ranging from embryogenesis to disorders, our finding will provide a new perspective on these issues from the aspect of cell adhesion. PMID:25255288

  18. FAK/src-family dependent activation of the Ste20-like kinase SLK is required for microtubule-dependent focal adhesion turnover and cell migration.

    Directory of Open Access Journals (Sweden)

    Simona Wagner

    2008-04-01

    Full Text Available Cell migration involves a multitude of signals that converge on cytoskeletal reorganization, essential for development, immune responses and tissue repair. Using knockdown and dominant negative approaches, we show that the microtubule-associated Ste20-like kinase SLK is required for focal adhesion turnover and cell migration downstream of the FAK/c-src complex. Our results show that SLK co-localizes with paxillin, Rac1 and the microtubules at the leading edge of migrating cells and is activated by scratch wounding. SLK activation is dependent on FAK/c-src/MAPK signaling, whereas SLK recruitment to the leading edge is src-dependent but FAK independent. Our results show that SLK represents a novel focal adhesion disassembly signal.

  19. Gene therapy targeting hepatocellular carcinoma by a dual-regulated oncolytic adenovirus harboring the focal adhesion kinase shRNA.

    Science.gov (United States)

    Gao, Yang; Zhu, Yayun; Huang, Xinyu; Ai, Kaixing; Zheng, Qi; Yuan, Zhou

    2015-08-01

    Cancer targeting gene-viro-therapy (CTGVT) approach has become a hotspot and a trend in the field of cancer biotherapy and oncolytic adenovirus is an ideal vector to carry the targeting genes. In this study, we used human telomerase reverse transcriptase (hTERT) promoter to control the adenovirus early region 1a (E1A) and the human α-fetoprotein (AFP) promoter integrated with hypoxia response element (HRE) to control the adenovirus early region 1b (E1B). Then the novel double-regulated adenovirus Ad-hTERT-HREAF (named SG505) was engineered. The short-hairpin RNA against focal adhesion kinase (FAK) was inserted into SG505 and thus forming Ad-hTERT-HREAF-shRNA (called SG505‑siFAK). Then various oncolytic adenoviruses were examined to verify whether they could suppress liver cancer cells selectively and efficiently both in vitro and in vivo. Both replicative and replication-defective adenoviruses carrying FAK-shRNA significantly inhibited the expression of FAK in Hep3B and SMMC-7721 cell lines and efficiently suppressed the growth of liver cancer cell lines with minor effect to normal cells. Furthermore, the recombined oncolytic adenoviruses, SG505-siFAK, SG505-EGFP and SG505 were able to selectively propagate in AFP-positive liver cancer cells in vitro and the SG505-siFAK efficiently suppressed the expression of FAK. SG505-siFAK showed the most potent tumor inhibition capability among the three recombined adenovirus with IC50 levels of 0.092±0.009 and 0.424±0.414 pfu/cell in the Hep3B and HepG2 cell line, respectively. Animal experiment further confirmed that SG505-siFAK achieved the most significant tumor inhibition of Hep3B liver cancer xenografted growth by intratumoral injection comparing to the intravenous injection among the three recombined viruses. Immunohistochemical results indicated that FAK expression was downregulated significantly in the tumors treated with SG505-siFAK. The dual-regulated oncolytic adenovirus SG505-siFAK was proven to inhibit the

  20. Neural cell adhesion molecule-stimulated neurite outgrowth depends on activation of protein kinase C and the Ras-mitogen-activated protein kinase pathway

    DEFF Research Database (Denmark)

    Kolkova, K; Novitskaya, V; Pedersen, N

    2000-01-01

    transfected with expression plasmids encoding constitutively active forms of Ras, Raf, MAP kinase kinases MEK1 and 2, dominant negative forms of Ras and Raf, and the FAK-related nonkinase. Alternatively, PC12-E2 cells were submitted to treatment with antibodies to the fibroblast growth factor (FGF) receptor......, inhibitors of the nonreceptor tyrosine kinase p59(fyn), PLC, PKC and MEK and an activator of PKC, phorbol-12-myristate-13-acetate (PMA). MEK2 transfection rescued cells treated with all inhibitors. The same was found for PMA treatment, except when cells concomitantly were treated with the MEK inhibitor....... Arachidonic acid rescued cells treated with antibodies to the FGF receptor or the PLC inhibitor, but not cells in which the activity of PKC, p59(fyn), FAK, Ras, or MEK was inhibited. Interaction of NCAM with a synthetic NCAM peptide ligand, known to induce neurite outgrowth, was shown to stimulate...

  1. Prediction of the adhesive behavior of bio-inspired functionally graded materials against rough surfaces

    Directory of Open Access Journals (Sweden)

    Chen Peijian

    2014-06-01

    Full Text Available Roughness effect and adhesion properties are important characteristics to be accessed in the development of functionally graded materials for biological and biomimetic applications, particularly for the hierarchical composition in biomimetic gecko robot. A multi-asperities adhesion model to predict the adhesive forces is presented in this work. The effect of surface roughness and graded material properties, which significantly alter the adhesive strength between contact bodies, can be simultaneously considered in the generalized model. It is found that proper interfacial strength can be controlled by adjusting surface roughness σ / R, graded exponent k and material parameter E*R / Δγ. The results should be helpful in the design of new biomimetic materials and useful in application of micro functional instruments.

  2. Glycogen Synthase Kinase 3 (GSK-3) influences epithelial barrier function by regulating Occludin, Claudin-1 and E-cadherin expression

    International Nuclear Information System (INIS)

    Severson, Eric A.; Kwon, Mike; Hilgarth, Roland S.; Parkos, Charles A.; Nusrat, Asma

    2010-01-01

    The Apical Junctional Complex (AJC) encompassing the tight junction (TJ) and adherens junction (AJ) plays a pivotal role in regulating epithelial barrier function and epithelial cell proliferative processes through signaling events that remain poorly characterized. A potential regulator of AJC protein expression is Glycogen Synthase Kinase-3 (GSK-3). GSK-3 is a constitutively active kinase that is repressed during epithelial-mesenchymal transition (EMT). In the present study, we report that GSK-3 activity regulates the structure and function of the AJC in polarized model intestinal (SK-CO15) and kidney (Madin-Darby Canine Kidney (MDCK)) epithelial cells. Reduction of GSK-3 activity, either by small molecule inhibitors or siRNA targeting GSK-3 alpha and beta mRNA, resulted in increased permeability to both ions and bulk solutes. Immunofluorescence labeling and immunoblot analyses revealed that the barrier defects correlated with decreased protein expression of AJC transmembrane proteins Occludin, Claudin-1 and E-cadherin without influencing other TJ proteins, Zonula Occludens-1 (ZO-1) and Junctional Adhesion Molecule A (JAM-A). The decrease in Occludin and E-cadherin protein expression correlated with downregulation of the corresponding mRNA levels for these respective proteins following GSK-3 inhibition. These observations implicate an important role of GSK-3 in the regulation of the structure and function of the AJC that is mediated by differential modulation of mRNA transcription of key AJC proteins, Occludin, Claudin-1 and E-cadherin.

  3. Using hydroxyapatite nanoparticles and decreased crystallinity to promote osteoblast adhesion similar to functionalizing with RGD.

    Science.gov (United States)

    Balasundaram, Ganesan; Sato, Michiko; Webster, Thomas J

    2006-05-01

    Better materials are needed to promote bone growth. For this reason, the present study created nanometer crystalline hydroxyapatite (HA) and amorphous calcium phosphate compacts functionalized with the arginine-glycine-aspartic acid (RGD) peptide sequence. Crystalline HA and amorphous calcium phosphate nanoparticles were synthesized by a wet chemical process followed by a hydrothermal treatment for 2 h at 200 degrees C and 70 degrees C, respectively. Resulting particles were then pressed into compacts. For the preparation of conventional HA particles (or those with micron diameters), the aforementioned pressed compacts were sintered at 1,100 degrees C for 2 h. Peptide functionalization was conducted by means of a three step reaction procedure: silanization with 3-aminopropyltriethoxysilane (APTES), cross-linking with N-succinimidyl-3-maleimido propionate (SMP), and finally peptide immobilization. The three step reaction procedure was characterized by a novel 3-(4-carboxybenzoyl)quinoline-2-carboxaldehyde (CBQCA) fluorescence technique. For all materials, results showed that the immobilization of the cell adhesive RGD sequence increased osteoblast (bone-forming cell) adhesion compared to those non-functionalized and those functionalized with the noncell adhesive control peptide (RGE) after 4 h. However, surprisingly, results also showed that the adhesion of osteoblasts on non-functionalized amorphous nanoparticulate calcium phosphate was similar to conventional HA functionalized with RGD. Osteoblast adhesion on nanocrystalline HA (unfunctionalized and functionalized with RGD) was below that of the respective functionalized amorphous calcium phosphate but above that of the respective functionalized conventional HA. In this manner, results of this study suggest that decreasing the particulate size into the nanometer regime and reducing crystallinity of calcium phosphate based materials may promote osteoblast adhesion to the same degree as the well

  4. Glycan-functionalized diamond nanoparticles as potent E. coli anti-adhesives

    Science.gov (United States)

    Barras, Alexandre; Martin, Fernando Ariel; Bande, Omprakash; Baumann, Jean-Sébastien; Ghigo, Jean-Marc; Boukherroub, Rabah; Beloin, Christophe; Siriwardena, Aloysius; Szunerits, Sabine

    2013-02-01

    Bacterial attachment and subsequent biofilm formation on biotic surfaces or medical devices is an increasing source of infections in clinical settings. A large proportion of these biofilm-related infections are caused by Escherichia coli, a major nosocomial pathogen, in which the major adhesion factor is the FimH adhesin located at the tip of type 1 fimbriae. Inhibition of FimH-mediated adhesion has been identified as an efficient antibiotic-alternative strategy to potentially reduce E. coli-related infections. In this article we demonstrate that nanodiamond particles, covently modified with mannose moieties by a ``click'' chemistry approach, are able to efficiently inhibit E. coli type 1 fimbriae-mediated adhesion to eukaryotic cells with relative inhibitory potency (RIP) of as high as 9259 (bladder cell adhesion assay), which is unprecedented when compared with RIP values previously reported for alternate multivalent mannose-functionalized nanostructures designed to inhibit E. coli adhesion. Also remarkable is that these novel mannose-modified NDs reduce E. coli biofilm formation, a property previously not observed for multivalent glyco-nanoparticles and rarely demonstrated for other multivalent or monovalent mannose glycans. This work sets the stage for the further evaluation of these novel NDs as an anti-adhesive therapeutic strategy against E. coli-derived infections.Bacterial attachment and subsequent biofilm formation on biotic surfaces or medical devices is an increasing source of infections in clinical settings. A large proportion of these biofilm-related infections are caused by Escherichia coli, a major nosocomial pathogen, in which the major adhesion factor is the FimH adhesin located at the tip of type 1 fimbriae. Inhibition of FimH-mediated adhesion has been identified as an efficient antibiotic-alternative strategy to potentially reduce E. coli-related infections. In this article we demonstrate that nanodiamond particles, covently modified with

  5. Exploring the function of protein kinases in schistosomes: perspectives from the laboratory and from comparative genomics

    Directory of Open Access Journals (Sweden)

    Anthony John Walker

    2014-07-01

    Full Text Available Eukaryotic protein kinases are well conserved through evolution. The genome of Schistosoma mansoni, which causes intestinal schistosomiasis, encodes over 250 putative protein kinases with all of the main eukaryotic groups represented. However, unraveling functional roles for these kinases is a considerable endeavour, particularly as protein kinases regulate multiple and sometimes overlapping cell and tissue functions in organisms. In this article, elucidating protein kinase signal transduction and function in schistosomes is considered from the perspective of the state-of-the-art methodologies used and comparative organismal biology, with a focus on current advances and future directions. Using the free-living nematode Caenorhabditis elegans as a comparator we predict roles for various schistosome protein kinases in processes vital for host invasion and successful parasitism such as sensory behaviour, growth and development. It is anticipated that the characterization of schistosome protein kinases in the context of parasite function will catalyze cutting edge research into host-parasite interactions and will reveal new targets for developing drug interventions against human schistosomiasis.

  6. Kinase Independent Functions of Cyclin D1 Which Contribute to its Oncogenic Potential In Vivo

    National Research Council Canada - National Science Library

    Landis, Mark

    2002-01-01

    .... Upon phosphorylation, pRb is inactivated, and cells pass from G1 into S phase. We and others have demonstrated that cyclin D1 has other functions, many of which are independent of kinase activity in vino...

  7. Non-small-cell lung cancer cells combat epidermal growth factor receptor tyrosine kinase inhibition through immediate adhesion-related responses

    Directory of Open Access Journals (Sweden)

    Wang HY

    2016-05-01

    Full Text Available Hsian-Yu Wang,1,2 Min-Kung Hsu,3,4 Kai-Hsuan Wang,1 Ching-Ping Tseng,2,4 Feng-Chi Chen,3,4 John T-A Hsu1,4 1Institute of Biotechnology and Pharmaceutical Research, National Health Research Institutes (NHRI, Zhunan, Miaoli County, 2Institute of Molecular Medicine and Bioengineering, National Chiao Tung University (NCTU, Hsinchu, 3Division of Biostatistics and Bioinformatics, Institute of Population Health Sciences, National Health Research Institutes (NHRI, Zhunan, Miaoli County, 4Department of Biological Science and Technology, National Chiao Tung University (NCTU, Hsinchu, Taiwan, Republic of China Background: Epidermal growth factor receptor (EGFR tyrosine kinase inhibitors (TKIs, such as gefitinib, erlotinib, and afatinib, have greatly improved treatment efficacy in non-small cell lung cancer (NSCLC patients with drug-sensitive EGFR mutations. However, in some TKI responders, the benefits of such targeted therapies are limited by the rapid development of resistance, and strategies to overcome this resistance are urgently needed. Studies of drug resistance in cancer cells typically involve long term in vitro induction to obtain stably acquired drug-resistant cells followed by elucidation of resistance mechanisms, but the immediate responses of cancer cells upon drug treatment have been ignored. The aim of this study was to investigate the immediate responses of NSCLC cells upon treatment with EGFR TKIs.Results: Both NSCLC cells, ie, PC9 and H1975, showed immediate enhanced adhesion-related responses as an apoptosis-countering mechanism upon first-time TKI treatment. By gene expression and pathway analysis, adhesion-related pathways were enriched in gefitinib-treated PC9 cells. Pathway inhibition by small-hairpin RNAs or small-molecule drugs revealed that within hours of EGFR TKI treatment, NSCLC cells used adhesion-related responses to combat the drugs. Importantly, we show here that the Src family inhibitor, dasatinib, dramatically inhibits

  8. A Molecular Mechanism for the Requirement of PAT-4 (Integrin-linked Kinase (ILK)) for the Localization of UNC-112 (Kindlin) to Integrin Adhesion Sites*

    Science.gov (United States)

    Qadota, Hiroshi; Moerman, Donald G.; Benian, Guy M.

    2012-01-01

    Caenorhabditis elegans muscle cells attach to basement membrane through adhesion plaques. PAT-3 (β-integrin), UNC-112 (kindlin), and PAT-4 (integrin-linked kinase) are associated with these structures. Genetic analysis indicated that PAT-4 is required for UNC-112 to be properly localized. We investigated the molecular basis of this requirement. We show that the cytoplasmic tail of PAT-3 binds to full-length UNC-112 and that the N- and C-terminal halves of UNC-112 bind to each other. We demonstrate competition between the UNC-112 C-terminal half and PAT-4 for binding to the UNC-112 N-terminal half. The D382V mutation results in lack of binding to PAT-4 and lack of localization to adhesion structures. T346A or E349K mutations, which abolish interaction of the N- and C-terminal halves, permit D382V UNC-112 to localize to adhesion structures. The following model is proposed. UNC-112 exists in closed inactive and open active conformations, and upon binding of PAT-4 to the UNC-112 N-terminal half, UNC-112 is converted into the open state, able to bind to PAT-3. PMID:22761445

  9. A molecular mechanism for the requirement of PAT-4 (integrin-linked kinase (ILK)) for the localization of UNC-112 (Kindlin) to integrin adhesion sites.

    Science.gov (United States)

    Qadota, Hiroshi; Moerman, Donald G; Benian, Guy M

    2012-08-17

    Caenorhabditis elegans muscle cells attach to basement membrane through adhesion plaques. PAT-3 (β-integrin), UNC-112 (kindlin), and PAT-4 (integrin-linked kinase) are associated with these structures. Genetic analysis indicated that PAT-4 is required for UNC-112 to be properly localized. We investigated the molecular basis of this requirement. We show that the cytoplasmic tail of PAT-3 binds to full-length UNC-112 and that the N- and C-terminal halves of UNC-112 bind to each other. We demonstrate competition between the UNC-112 C-terminal half and PAT-4 for binding to the UNC-112 N-terminal half. The D382V mutation results in lack of binding to PAT-4 and lack of localization to adhesion structures. T346A or E349K mutations, which abolish interaction of the N- and C-terminal halves, permit D382V UNC-112 to localize to adhesion structures. The following model is proposed. UNC-112 exists in closed inactive and open active conformations, and upon binding of PAT-4 to the UNC-112 N-terminal half, UNC-112 is converted into the open state, able to bind to PAT-3.

  10. Recent Progress on Liver Kinase B1 (LKB1: Expression, Regulation, Downstream Signaling and Cancer Suppressive Function

    Directory of Open Access Journals (Sweden)

    Ren-You Gan

    2014-09-01

    Full Text Available Liver kinase B1 (LKB1, known as a serine/threonine kinase, has been identified as a critical cancer suppressor in many cancer cells. It is a master upstream kinase of 13 AMP-activated protein kinase (AMPK-related protein kinases, and possesses versatile biological functions. LKB1 gene is mutated in many cancers, and its protein can form different protein complexes with different cellular localizations in various cell types. The expression of LKB1 can be regulated through epigenetic modification, transcriptional regulation and post-translational modification. LKB1 dowcnstream pathways mainly include AMPK, microtubule affinity regulating kinase (MARK, salt-inducible kinase (SIK, sucrose non-fermenting protein-related kinase (SNRK and brain selective kinase (BRSK signalings, etc. This review, therefore, mainly discusses recent studies about the expression, regulation, downstream signaling and cancer suppressive function of LKB1, which can be helpful for better understanding of this molecular and its significance in cancers.

  11. Sickle cell disease biochip: a functional red blood cell adhesion assay for monitoring sickle cell disease

    Science.gov (United States)

    ALAPAN, YUNUS; KIM, CEONNE; ADHIKARI, ANIMA; GRAY, KAYLA E.; GURKAN-CAVUSOGLU, EVREN; LITTLE, JANE A.; GURKAN, UMUT A.

    2016-01-01

    Sickle cell disease (SCD) afflicts millions of people worldwide and is associated with considerable morbidity and mortality. Chronic and acute vaso-occlusion are the clinical hallmarks of SCD and can result in pain crisis, widespread organ damage, and early movtality. Even though the molecular underpinnings of SCD were identified more than 60 years ago, there are no molecular or biophysical markers of disease severity that are feasibly measured in the clinic. Abnormal cellular adhesion to vascular endothelium is at the root of vaso-occlusion. However, cellular adhesion is not currently evaluated clinically. Here, we present a clinically applicable microfluidic device (SCD biochip) that allows serial quantitative evaluation of red blood cell (RBC) adhesion to endothelium-associated protein-immobilized microchannels, in a closed and preprocessing-free system. With the SCD biochip, we have analyzed blood samples from more than 100 subjects and have shown associations between the measured RBC adhesion to endothelium-associated proteins (fibronectin and laminin) and individual RBC characteristics, including hemoglobin content, fetal hemoglobin concentration, plasma lactate dehydrogenase level, and reticulocyte count. The SCD biochip is a functional adhesion assay, reflecting quantitative evaluation of RBC adhesion, which could be used at baseline, during crises, relative to various long-term complications, and before and after therapeutic interventions. PMID:27063958

  12. LEFTY2 Controls Migration of Human Endometrial Cancer Cells via Focal Adhesion Kinase Activity (FAK) and miRNA-200a.

    Science.gov (United States)

    Alowayed, Nour; Salker, Madhuri S; Zeng, Ni; Singh, Yogesh; Lang, Florian

    2016-01-01

    LEFTY2, a suppressor of cell proliferation, tumor growth, regulator of stemness and embryonic differentiation, is a negative regulator of cancer cell reprogramming. Malignant transformation may lead to migration requiring loss of adhesion and gain of migratory activity. Signaling involved in the orchestration of migration, proliferation and spreading of cells include focal adhesion kinase (FAK) and adhesion molecule E-cadherin. The present study explored whether LEFTY2 influences the proliferation marker MKi67, FAK activity, E-cadherin abundance and migration of Ishikawa human endometrial carcinoma cells. Moreover, the study explored the involvement of microRNA-200a (miR-200a), which is known to regulate cellular adhesion by targeting E-Cadherin. FAK activity was estimated from FAK phosphorylation quantified by Western blotting, migration utilizing a wound healing assay, miR-200a and MKi67 expression levels utilizing qRT-PCR, cell proliferation and apoptosis using BrdU and Annexin V staining, respectively, and E-Cadherin (E-Cad) abundance, using confocal microscopy. LEFTY2 (25 ng/ml, 48 hours) treatment was followed by decrease of MKi67 expression, FAK activity and migration. LEFTY2 upregulated miRNA-200a and E-Cad protein level in Ishikawa cells. The effect of LEFTY2 on migration was mimicked by FAK inhibitor PF 573228 (50 µM). Addition of LEFTY2 in the presence of PF-573228 did not result in a further significant decline of migration. In conclusion, LEFTY2 down-regulates MKi67 expression and FAK activity, up-regulates miR-200a and E-cadherin, and is thus a powerful negative regulator of endometrial cell proliferation and migration. © 2016 The Author(s) Published by S. Karger AG, Basel.

  13. Extracorporeal shockwave therapy improves short-term functional outcomes of shoulder adhesive capsulitis.

    Science.gov (United States)

    Chen, Chih-Yu; Hu, Chia-Chian; Weng, Pei-Wei; Huang, Yu-Ming; Chiang, Chang-Jung; Chen, Chia-Hsien; Tsuang, Yang-Hwei; Yang, Rong-Sen; Sun, Jui-Sheng; Cheng, Cheng-Kung

    2014-12-01

    The treatment of adhesive capsulitis is a dilemma for orthopaedic rehabilitation specialists. In this study, we assessed whether extracorporeal shockwave therapy (ESWT) improves the functional outcome of primary shoulder adhesive capsulitis. In this prospective, randomized, controlled, single-blind clinical trial, we enrolled 40 patients with primary adhesive capsulitis to assess whether ESWT can improve the functional outcome of primary adhesive capsulitis better than oral steroid therapy. Patients were allocated to the oral steroid group or ESWT group with randomization. Functional outcome evaluations were performed using the Constant Shoulder Score (CSS) and Oxford Shoulder Score. Both groups showed significant improvement in the Oxford Shoulder Score evaluation throughout the study period. In the ESWT group, the total CSS and range of motion (ROM) parameter of the CSS in the ESWT group showed significant improvement from the fourth week that was better than that in the steroid group; the activities–of–daily living (ADL) parameter of the CSS achieved significance and was better than that in the steroid group at the sixth week. For the steroid group, pain was significantly reduced from baseline to the fourth week of the study; ADL and ROM improved at the fourth to 12th week. For the ESWT group, ADL and ROM improvements were significant from baseline to the sixth week. Our results showed that ESWT can be an alternative treatment, at least in the short-term, for primary adhesive capsulitis of the shoulder. In addition, all of the side effects of ESWT were transient and tolerable.

  14. Multi-functional surfaces with controllable wettability and water adhesion

    Science.gov (United States)

    Anastasiadis, Spiros H.; Frysali, Melani A.; Kenanakis, George; Kaklamani, Georgia; Papoutsakis, Lampros

    The design of multifunctional surfaces based on biomimetic structures has gained the interest of the scientific community. Novel multifunctional surfaces have been developed, able to alter their wetting properties in response to temperature and pH as well as light illumination, by combining proper chemistry and surface micro/nano-structuring using ultrafast (femtosecond) laser irradiation. The combination of the hierarchical surface with a ZnO and/or a responsive polymer coating results in efficient photo-active properties as well as reversible superhydrophobic / superhydrophilic surfaces in response to external stimuli. These surfaces can be optimized to exhibit high or zero water adhesion and/or controllable directionality as well. Moreover, they can be seeded with human fibroblasts to examine the cellular response on both surface roughness and surface chemistry. Acknowledgements: This research has been co-financed by the General Secretariat for Research and Technology (''ARISTEIA II'' Action, SMART-SURF) and the European Union (NFFA Europe -Grant agreement No. 654360).

  15. Focal adhesion kinase-mediated activation of glycogen synthase kinase 3β regulates IL-33 receptor internalization and IL-33 signaling

    OpenAIRE

    Zhao, Jing; Wei, Jianxin; Bowser, Rachel K; Traister, Russell S; Fan, Ming-Hui; Zhao, Yutong

    2014-01-01

    IL-33, a relatively new member of the IL-1 cytokine family, plays a crucial role in allergic inflammation and acute lung injury. ST2L, the receptor for IL-33, is expressed on immune effector cells and lung epithelia, and plays a critical role in triggering inflammation. We have previously shown that ST2L stability is regulated by the ubiquitin-proteasome system, however its upstream internalization has not been studied. Here, we demonstrate that glycogen synthase kinase 3β (GSK3β) regulates S...

  16. Neural cell adhesion molecule-stimulated neurite outgrowth depends on activation of protein kinase C and the Ras-mitogen-activated protein kinase pathway

    DEFF Research Database (Denmark)

    Kolkova, K; Novitskaya, V; Pedersen, N

    2000-01-01

    , inhibitors of the nonreceptor tyrosine kinase p59(fyn), PLC, PKC and MEK and an activator of PKC, phorbol-12-myristate-13-acetate (PMA). MEK2 transfection rescued cells treated with all inhibitors. The same was found for PMA treatment, except when cells concomitantly were treated with the MEK inhibitor....... Arachidonic acid rescued cells treated with antibodies to the FGF receptor or the PLC inhibitor, but not cells in which the activity of PKC, p59(fyn), FAK, Ras, or MEK was inhibited. Interaction of NCAM with a synthetic NCAM peptide ligand, known to induce neurite outgrowth, was shown to stimulate...

  17. TrkB kinase activity maintains synaptic function and structural integrity at adult neuromuscular junctions

    Science.gov (United States)

    Stowe, Jessica M.; Sieck, Dylan C.; Ermilov, Leonid G.; Greising, Sarah M.; Zhang, Chao; Shokat, Kevan M.; Sieck, Gary C.

    2014-01-01

    Activation of the tropomyosin-related kinase receptor B (TrkB) by brain-derived neurotrophic factor acutely regulates synaptic transmission at adult neuromuscular junctions (NMJs). The role of TrkB kinase activity in the maintenance of NMJ function and structure at diaphragm muscle NMJs was explored using a chemical-genetic approach that permits reversible inactivation of TrkB kinase activity in TrkBF616A mice by 1NMPP1. Inhibiting TrkB kinase activity for 7 days resulted in significant, yet reversible, impairments in neuromuscular transmission at diaphragm NMJs. Neuromuscular transmission failure following 2 min of repetitive phrenic nerve stimulation increased from 42% in control to 59% in 1NMPP1-treated TrkBF616A mice (P = 0.010). Recovery of TrkB kinase activity following withdrawal of 1NMPP1 treatment improved neuromuscular transmission (P = 0.006). Electrophysiological measurements at individual diaphragm NMJs documented lack of differences in quantal content in control and 1NMPP1-treated mice (P = 0.845). Morphological changes at diaphragm NMJs were modest following inhibition and recovery of TrkB kinase activity. Three-dimensional reconstructions of diaphragm NMJs revealed no differences in volume at motor end plates (labeled by α-bungarotoxin; P = 0.982) or presynaptic terminals (labeled by synaptophysin; P = 0.515). Inhibition of TrkB kinase activity by 1NMPP1 resulted in more compact NMJs, with increased apposition of presynaptic terminals and motor end plates (P = 0.017) and reduced fragmentation of motor end plates (P = 0.005). Recovery of TrkB kinase activity following withdrawal of 1NMPP1 treatment resulted in postsynaptic remodeling likely reflecting increased gutter depth (P = 0.007), without significant presynaptic changes. These results support an essential role for TrkB kinase activity in maintaining synaptic function and structural integrity at NMJs in the adult mouse diaphragm muscle. PMID:25170066

  18. Distinct expression patterns of ICK/MAK/MOK protein kinases in the intestine implicate functional diversity.

    Directory of Open Access Journals (Sweden)

    Tufeng Chen

    Full Text Available ICK/MRK (intestinal cell kinase/MAK-related kinase, MAK (male germ cell-associated kinase, and MOK (MAPK/MAK/MRK-overlapping kinase are closely related serine/threonine protein kinases in the protein kinome. The biological functions and regulatory mechanisms of the ICK/MAK/MOK family are still largely elusive. Despite significant similarities in their catalytic domains, they diverge markedly in the sequence and structural organization of their C-terminal non-catalytic domains, raising the question as to whether they have distinct, overlapping, or redundant biological functions. In order to gain insights into their biological activities and lay a fundamental groundwork for functional studies, we investigated the spatio-temporal distribution patterns and the expression dynamics of ICK/MAK/MOK protein kinases in the intestine. We found that ICK/MAK/MOK proteins display divergent expression patterns along the duodenum-to-colon axis and during postnatal murine development. Furthermore, they are differentially partitioned between intestinal epithelium and mesenchyme. A significant increase in the protein level of ICK, but not MAK, was induced in human primary colon cancer specimens. ICK protein level was up-regulated whereas MOK protein level was down-regulated in mouse intestinal adenomas as compared with their adjacent normal intestinal mucosa. These data suggest distinct roles for ICK/MAK/MOK protein kinases in the regulation of intestinal neoplasia. Taken together, our findings demonstrate that the expressions of ICK/MAK/MOK proteins in the intestinal tract can be differentially and dynamically regulated, implicating a significant functional diversity within this group of protein kinases.

  19. Neural Cell Adhesion Molecules of the Immunoglobulin Superfamily Regulate Synapse Formation, Maintenance, and Function.

    Science.gov (United States)

    Sytnyk, Vladimir; Leshchyns'ka, Iryna; Schachner, Melitta

    2017-05-01

    Immunoglobulin superfamily adhesion molecules are among the most abundant proteins in vertebrate and invertebrate nervous systems. Prominent family members are the neural cell adhesion molecules NCAM and L1, which were the first to be shown to be essential not only in development but also in synaptic function and as key regulators of synapse formation, synaptic activity, plasticity, and synaptic vesicle recycling at distinct developmental and activity stages. In addition to interacting with each other, adhesion molecules interact with ion channels and cytokine and neurotransmitter receptors. Mutations in their genes are linked to neurological disorders associated with abnormal development and synaptic functioning. This review presents an overview of recent studies on these molecules and their crucial impact on neurological disorders. Copyright © 2017 Elsevier Ltd. All rights reserved.

  20. Lactobacillaceae and cell adhesion: genomic and functional screening.

    Directory of Open Access Journals (Sweden)

    Williams Turpin

    Full Text Available The analysis of collections of lactic acid bacteria (LAB from traditional fermented plant foods in tropical countries may enable the detection of LAB with interesting properties. Binding capacity is often the main criterion used to investigate the probiotic characteristics of bacteria. In this study, we focused on a collection of 163 Lactobacillaceace comprising 156 bacteria isolated from traditional amylaceous fermented foods and seven strains taken from a collection and used as controls. The collection had a series of analyses to assess binding potential for the selection of new probiotic candidates. The presence/absence of 14 genes involved in binding to the gastrointestinal tract was assessed. This enabled the detection of all the housekeeping genes (ef-Tu, eno, gap, groEl and srtA in the entire collection, of some of the other genes (apf, cnb, fpbA, mapA, mub in 86% to 100% of LAB, and of the other genes (cbsA, gtf, msa, slpA in 0% to 8% of LAB. Most of the bacteria isolated from traditional fermented foods exhibited a genetic profile favorable for their binding to the gastrointestinal tract. We selected 30 strains with different genetic profiles to test their binding ability to non-mucus (HT29 and mucus secreting (HT29-MTX cell lines as well as their ability to degrade mucus. Assays on both lines revealed high variability in binding properties among the LAB, depending on the cell model used. Finally, we investigated if their binding ability was linked to tighter cross-talk between bacteria and eukaryotic cells by measuring the expression of bacterial genes and of the eukaryotic MUC2 gene. Results showed that wild LAB from tropical amylaceous fermented food had a much higher binding capacity than the two LAB currently known to be probiotics. However their adhesion was not linked to any particular genetic equipment.

  1. A Site-Specific Phosphorylation of the Focal Adhesion Kinase Controls the Formation of Spheroid Cell Clusters

    DEFF Research Database (Denmark)

    Beck, Hans Christian; Gosau, Martin; Kristensen, Lars Peter

    2014-01-01

    approach revealed regulated phosphorylated proteins in SCCs, which were derived from DFCs after 24 and 48 h in SFM. These regulated proteins were categorized using the Kyoto encyclopedia of genes and genomes program. Here, cellular processes and signaling pathway were identified such as the focal adhesion...

  2. Interfacial adhesion of laser clad functionally graded materials

    NARCIS (Netherlands)

    Ocelik, V.; Pei, Y.T.; de Hosson, J.T.M.; Popoola, O; Dahotre, NB; Midea, SJ; Kopech, HM

    2003-01-01

    Two functionally graded coatings were prepared by different laser surface engineering techniques. Laser cladding of AlSi40 powder leads to the formation of functionally graded material (FGM) coating on AI-Si cast alloy substrate. Mapping of strain fields near the laser clad track using the digital

  3. Osthole Suppresses the Migratory Ability of Human Glioblastoma Multiforme Cells via Inhibition of Focal Adhesion Kinase-Mediated Matrix Metalloproteinase-13 Expression

    Directory of Open Access Journals (Sweden)

    Cheng-Fang Tsai

    2014-03-01

    Full Text Available Glioblastoma multiforme (GBM is the most common type of primary and malignant tumor occurring in the adult central nervous system. GBM often invades surrounding regions of the brain during its early stages, making successful treatment difficult. Osthole, an active constituent isolated from the dried C. monnieri fruit, has been shown to suppress tumor migration and invasion. However, the effects of osthole in human GBM are largely unknown. Focal adhesion kinase (FAK is important for the metastasis of cancer cells. Results from this study show that osthole can not only induce cell death but also inhibit phosphorylation of FAK in human GBM cells. Results from this study show that incubating GBM cells with osthole reduces matrix metalloproteinase (MMP-13 expression and cell motility, as assessed by cell transwell and wound healing assays. This study also provides evidence supporting the potential of osthole in reducing FAK activation, MMP-13 expression, and cell motility in human GBM cells.

  4. Amine functionalized nanodiamond promotes cellular adhesion, proliferation and neurite outgrowth

    International Nuclear Information System (INIS)

    Hopper, A P; Dugan, J M; Gill, A A; Haycock, J W; Claeyssens, F; Fox, O J L; May, P W

    2014-01-01

    In this study, we report the production of amine functionalized nanodiamond. The amine functionalized nanodiamond forms a conformal monolayer on a negatively charged surface produced via plasma polymerization of acrylic acid. Nanodiamond terminated surfaces were studied as substrates for neuronal cell culture. NG108-15 neuroblastoma-glyoma hybrid cells were successfully cultured upon amine functionalized nanodiamond coated surfaces for between 1 and 7 d. Additionally, primary dorsal root ganglion (DRG) neurons and Schwann cells isolated from Wistar rats were also successfully cultured over a period of 21 d illustrating the potential of the coating for applications in the treatment of peripheral nerve injury. (paper)

  5. Ligation of the adhesion-GPCR EMR2 regulates human neutrophil function

    NARCIS (Netherlands)

    Yona, Simon; Lin, Hsi-Hsien; Dri, Pietro; Davies, John Q.; Hayhoe, Richard P. G.; Lewis, Sion M.; Heinsbroek, Sigrid E. M.; Brown, K. Alun; Perretti, Mauro; Hamann, Jörg; Treacher, David F.; Gordon, Siamon; Stacey, Martin

    2008-01-01

    At present, approximately 150 different members of the adhesion-G protein-coupled receptor (GPCR) family have been identified in metazoans. Surprisingly, very little is known about their function, although they all possess large extracellular domains coupled to a seven-transmembrane domain,

  6. Biophysical characterization of functionalized titania nanoparticles and their application in dental adhesives.

    Science.gov (United States)

    Sun, Jirun; Petersen, Elijah J; Watson, Stephanie S; Sims, Christopher M; Kassman, Alexander; Frukhtbeyn, Stanislav; Skrtic, Drago; Ok, Meryem T; Jacobs, Debbie S; Reipa, Vytas; Ye, Qiang; Nelson, Bryant C

    2017-04-15

    It is demonstrated that carboxylic acid-functionalized titanium dioxide (TiO 2 ) NPs produce significantly higher levels of reactive oxygen species (ROS) after visible light irradiation (400-800nm, 1600mW/cm 2 ) in comparison to nonfunctionalized TiO 2 NPs. The level of ROS produced under these irradiation conditions was not capable of generating oxidatively induced DNA damage in a cell-free system for TiO 2 concentrations of 0.5mg/L or 5mg/L. In addition, specific incorporation of the acrylic acid-functionalized TiO 2 NPs into dental composites allowed us to utilize the generated ROS to enhance photopolymerization (curing and degree of vinyl conversion (DC)) of resin adhesives and create mechanically superior and biocompatible materials for dental applications. Incorporation of the TiO 2 NPs into selected dental composites increased the mean DC values by ≈7%. The modified TiO 2 materials and dental composite materials were extensively characterized using thermogravimetric analysis, electron microscopy, Fourier transform infrared spectroscopy, and electron paramagnetic resonance. Notably, dental adhesives incorporated with acrylic acid-functionalized TiO 2 NPs produced stronger bonds to human teeth following visible light curing in comparison to traditional dental adhesives not containing NPs with an increase in the shear bond strength of ≈29%. In addition, no leaching of the incorporated NPs was detectable from the dental adhesives after 2500 thermal cycles using inductively coupled plasma-optical emission spectroscopy, indicating that biocompatibility of the adhesives was not compromised after extensive aging. These findings suggest that NP-induced ROS may be useful to produce enhanced nanocomposite materials for selected applications in the medical device field. Titanium dioxide nanoparticles (TiO 2 NPs) have unique photocatalytic, antibacterial and UV-absorbing properties that make them beneficial additives in adhesives and composites. However, there is

  7. Cell Adhesion Selectivity of Stent Material to improve Bio-functionality by Ion Beam Modification

    Energy Technology Data Exchange (ETDEWEB)

    Lee, Jaesang; Park, JUngchan; Jung, Myunghwan; Kim, Yongki [Korea Atomic Energy Research Institute, Daejeon (Korea, Republic of); Park, Junkyu [Bio alpha., Co. Ltd., Gimhae (Korea, Republic of)

    2014-05-15

    In this study, ion implantation into collagen coated Co-Cr alloy, which is a cheaper material of the artificial stent product comparing with Ti alloy, has been studied to develop small diameter artificial stent by the cell adhesion control. The size of stent was 1.6mm of the diameter and 18mm of the length. The life-time of artificial stent depends on adhesion property of endothelial-cells. We successfully controlled cell adhesion selectivity between endothelial cell and muscle cell by using collagen coated and He{sup +} ion beam irradiated Co-Cr-alloy to apply to artificial stent. But, we did not achieve the inhibition of platelet adhesion, yet by using collagen coating and He{sup +} ion beam irradiation. Based on this study, we have plan to research about separation between collagen coating effect and ion beam effect. Also, we will have more detail analysis of the mechanism of cell attachment. In recent years, ion implantation has been applied to the surface modification of prosthesis to improve blood compatibility and tissue compatibility in field of biomedical application. As well known, bio compatibility was concerned with the cell adhesion selectivity for bio-functionality. The biomedical application of ion beam technology would be used more widely in the future such as catheter and artificial graft.

  8. Interfacial adhesion between graphene and silicon dioxide by density functional theory with van der Waals corrections

    International Nuclear Information System (INIS)

    Gao, Wei; Liechti, Kenneth M; Huang, Rui; Xiao, Penghao; Henkelman, Graeme

    2014-01-01

    Interfacial adhesion between graphene and a SiO 2 substrate is studied by density functional theory (DFT) with dispersion corrections. The results demonstrate the van der Waals (vdW) interaction as the predominant mechanism at the graphene/SiO 2 interface. It is found that the interaction strength is strongly influenced by changes of the SiO 2 surface structures due to surface reactions with water. The adhesion energy is reduced when the reconstructed SiO 2 surface is hydroxylated, and further reduced when covered by a monolayer of adsorbed water molecules. Moreover, it is noted that vdW forces are required to accurately model the graphene/SiO 2 interface with DFT and that the adhesion energy is underestimated by empirical force fields commonly used in atomistic simulations. (paper)

  9. Cognitive Functioning in Children with Pantothenate-Kinase-Associated Neurodegeneration Undergoing Deep Brain Stimulation

    Science.gov (United States)

    Mahoney, Rachel; Selway, Richard; Lin, Jean-Pierre

    2011-01-01

    Aim: To examine the cognitive functioning of young people with pantothenate-kinase-associated neurodegeneration (PKAN) after pallidal deep brain stimulation (DBS). PKAN is characterized by progressive generalized dystonia and has historically been associated with cognitive decline. With growing evidence that DBS can improve motor function in…

  10. Selective disruption of aurora C kinase reveals distinct functions from aurora B kinase during meiosis in mouse oocytes.

    Directory of Open Access Journals (Sweden)

    Ahmed Z Balboula

    2014-02-01

    Full Text Available Aurora B kinase (AURKB is the catalytic subunit of the chromosomal passenger complex (CPC, an essential regulator of chromosome segregation. In mitosis, the CPC is required to regulate kinetochore microtubule (K-MT attachments, the spindle assembly checkpoint, and cytokinesis. Germ cells express an AURKB homolog, AURKC, which can also function in the CPC. Separation of AURKB and AURKC function during meiosis in oocytes by conventional approaches has not been successful. Therefore, the meiotic function of AURKC is still not fully understood. Here, we describe an ATP-binding-pocket-AURKC mutant, that when expressed in mouse oocytes specifically perturbs AURKC-CPC and not AURKB-CPC function. Using this mutant we show for the first time that AURKC has functions that do not overlap with AURKB. These functions include regulating localized CPC activity and regulating chromosome alignment and K-MT attachments at metaphase of meiosis I (Met I. We find that AURKC-CPC is not the sole CPC complex that regulates the spindle assembly checkpoint in meiosis, and as a result most AURKC-perturbed oocytes arrest at Met I. A small subset of oocytes do proceed through cytokinesis normally, suggesting that AURKC-CPC is not the sole CPC complex during telophase I. But, the resulting eggs are aneuploid, indicating that AURKC is a critical regulator of meiotic chromosome segregation in female gametes. Taken together, these data suggest that mammalian oocytes contain AURKC to efficiently execute meiosis I and ensure high-quality eggs necessary for sexual reproduction.

  11. Constrained Adherable Area of Nanotopographic Surfaces Promotes Cell Migration through the Regulation of Focal Adhesion via Focal Adhesion Kinase/Rac1 Activation.

    Science.gov (United States)

    Lim, Jiwon; Choi, Andrew; Kim, Hyung Woo; Yoon, Hyungjun; Park, Sang Min; Tsai, Chia-Hung Dylan; Kaneko, Makoto; Kim, Dong Sung

    2018-05-02

    Cell migration is crucial in physiological and pathological processes such as embryonic development and wound healing; such migration is strongly guided by the surrounding nanostructured extracellular matrix. Previous studies have extensively studied the cell migration on anisotropic nanotopographic surfaces; however, only a few studies have reported cell migration on isotropic nanotopographic surfaces. We herein, for the first time, propose a novel concept of adherable area on cell migration using isotropic nanopore surfaces with sufficient nanopore depth by adopting a high aspect ratio. As the pore size of the nanopore surface was controlled to 200, 300, and 400 nm in a fixed center-to-center distance of 480 nm, it produced 86, 68, and 36% of adherable area, respectively, on the fabricated surface. A meticulous investigation of the cell migration in response to changes in the constrained adherable area of the nanotopographic surface showed 1.4-, 1.5-, and 1.6-fold increase in migration speeds and a 1.4-, 2-, and 2.5-fold decrease in the number of focal adhesions as the adherable area was decreased to 86, 68, and 36%, respectively. Furthermore, a strong activation of FAK/Rac1 signaling was observed to be involved in the promoted cell migration. These results suggest that the reduced adherable area promotes cell migration through decreasing the FA formation, which in turn upregulates FAK/Rac1 activation. The findings in this study can be utilized to control the cell migration behaviors, which is a powerful tool in the research fields involving cell migration such as promoting wound healing and tissue repair.

  12. The direct effect of Focal Adhesion Kinase (FAK, dominant-negative FAK, FAK-CD and FAK siRNA on gene expression and human MCF-7 breast cancer cell tumorigenesis

    Directory of Open Access Journals (Sweden)

    Zhang Li

    2009-08-01

    Full Text Available Abstract Background Focal adhesion kinase (FAK is a non-receptor tyrosine kinase that plays an important role in survival signaling. FAK has been shown to be overexpressed in breast cancer tumors at early stages of tumorigenesis. Methods To study the direct effect of FAK on breast tumorigenesis, we developed Tet-ON (tetracycline-inducible system of MCF-7 breast cancer cells stably transfected with FAK or dominant-negative, C-terminal domain of FAK (FAK-CD, and also FAKsiRNA with silenced FAK MCF-7 stable cell line. Increased expression of FAK in isogenic Tet-inducible MCF-7 cells caused increased cell growth, adhesion and soft agar colony formation in vitro, while expression of dominant-negative FAK inhibitor caused inhibition of these cellular processes. To study the role of induced FAK and FAK-CD in vivo, we inoculated these Tet-inducible cells in nude mice to generate tumors in the presence or absence of doxycycline in the drinking water. FAKsiRNA-MCF-7 cells were also injected into nude mice to generate xenograft tumors. Results Induction of FAK resulted in significant increased tumorigenesis, while induced FAK-CD resulted in decreased tumorigenesis. Taq Man Low Density Array assay demonstrated specific induction of FAKmRNA in MCF-7-Tet-ON-FAK cells. DMP1, encoding cyclin D binding myb-like protein 1 was one of the genes specifically affected by Tet-inducible FAK or FAK-CD in breast xenograft tumors. In addition, silencing of FAK in MCF-7 cells with FAK siRNA caused increased cell rounding, decreased cell viability in vitro and inhibited tumorigenesis in vivo. Importantly, Affymetrix microarray gene profiling analysis using Human Genome U133A GeneChips revealed >4300 genes, known to be involved in apoptosis, cell cycle, and adhesion that were significantly down- or up-regulated (p Conclusion Thus, these data for the first time demonstrate the direct effect of FAK expression and function on MCF-7 breast cancer tumorigenesis in vivo and reveal

  13. Abl family kinases regulate endothelial barrier function in vitro and in mice.

    Directory of Open Access Journals (Sweden)

    Elizabeth M Chislock

    Full Text Available The maintenance of endothelial barrier function is essential for normal physiology, and increased vascular permeability is a feature of a wide variety of pathological conditions, leading to complications including edema and tissue damage. Use of the pharmacological inhibitor imatinib, which targets the Abl family of non-receptor tyrosine kinases (Abl and Arg, as well as other tyrosine kinases including the platelet-derived growth factor receptor (PDGFR, Kit, colony stimulating factor 1 receptor (CSF1R, and discoidin domain receptors, has shown protective effects in animal models of inflammation, sepsis, and other pathologies characterized by enhanced vascular permeability. However, the imatinib targets involved in modulation of vascular permeability have not been well-characterized, as imatinib inhibits multiple tyrosine kinases not only in endothelial cells and pericytes but also immune cells important for disorders associated with pathological inflammation and abnormal vascular permeability. In this work we employ endothelial Abl knockout mice to show for the first time a direct role for Abl in the regulation of vascular permeability in vivo. Using both Abl/Arg-specific pharmacological inhibition and endothelial Abl knockout mice, we demonstrate a requirement for Abl kinase activity in the induction of endothelial permeability by vascular endothelial growth factor both in vitro and in vivo. Notably, Abl kinase inhibition also impaired endothelial permeability in response to the inflammatory mediators thrombin and histamine. Mechanistically, we show that loss of Abl kinase activity was accompanied by activation of the barrier-stabilizing GTPases Rac1 and Rap1, as well as inhibition of agonist-induced Ca(2+ mobilization and generation of acto-myosin contractility. In all, these findings suggest that pharmacological targeting of the Abl kinases may be capable of inhibiting endothelial permeability induced by a broad range of agonists and that use

  14. Cellular Adhesion and Adhesion Molecules

    OpenAIRE

    SELLER, Zerrin

    2014-01-01

    In recent years, cell adhesion and cell adhesion molecules have been shown to be important for many normal biological processes, including embryonic cell migration, immune system functions and wound healing. It has also been shown that they contribute to the pathogenesis of a large number of common human disorders, such as rheumatoid arthritis and tumor cell metastasis in cancer. In this review, the basic mechanisms of cellular adhesion and the structural and functional features of adhes...

  15. Multi-scale modeling of an upper respiratory airway: Effect of mucosal adhesion on Eustachian tube function in young children.

    Science.gov (United States)

    Malik, Jennifer; Ghadiali, Samir N

    2018-01-27

    The Eustachian tube is a collapsible upper respiratory airway that is periodically opened to maintain a healthy middle ear. Young children, function in young children. Multi-scale finite element models were used to simulate the muscle-assisted opening of the Eustachian tube in healthy adults and young children. Airflow during opening was quantified as a function of adhesion strength, muscle forces and tissue mechanics. Although Eustachian tube function was sensitive to increased mucosal adhesion in both adults and children, young children developed Eustachian tube dysfunction at significantly lower values of mucosal adhesion. Specifically, the critical adhesion value was 2 orders of magnitude lower in young children as compared to healthy adults. Although increased adhesion did not alter the sensitivity of Eustachian tube function to tensor and levator veli palatini muscles forces, increased adhesion in young children did reduced the sensitivity of Eustachian tube function to changes in cartilage and mucosal tissue stiffness. These results indicate that increased mucosal adhesion can significantly alter the biomechanical mechanisms of Eustachian tube function in young children and that clinical assessment of adhesion levels may be important in therapy selection. Copyright © 2018 Elsevier Ltd. All rights reserved.

  16. Enhanced Cellular Adhesion on Titanium by Silk Functionalized with titanium binding and RGD peptides

    Science.gov (United States)

    Vidal, Guillaume; Blanchi, Thomas; Mieszawska, Aneta J.; Calabrese, Rossella; Rossi, Claire; Vigneron, Pascale; Duval, Jean-Luc; Kaplan, David L.; Egles, Christophe

    2012-01-01

    Soft tissue adhesion on titanium represents a challenge for implantable materials. In order to improve adhesion at the cell/material interface we used a new approach based on the molecular recognition of titanium by specific peptides. Silk fibroin protein was chemically grafted with titanium binding peptide (TiBP) to increase adsorption of these chimeric proteins to the metal surface. Quartz Crystal Microbalance was used to quantify the specific adsorption of TiBP-functionalized silk and an increase in protein deposition by more than 35% was demonstrated due to the presence of the binding peptide. A silk protein grafted with TiBP and fibronectin-derived RGD peptide was then prepared. The adherence of fibroblasts on the titanium surface modified with the multifunctional silk coating demonstrated an increase in the number of adhering cells by 60%. The improved adhesion was demonstrated by Scanning Electron Microscopy and immunocytochemical staining of focal contact points. Chick embryo organotypic culture also revealed strong adhesion of endothelial cells expanding on the multifunctional silk-peptide coating. These results demonstrated that silk functionalized with TiBP and RGD represents a promising approach to modify cell-biomaterial interfaces, opening new perspectives for implantable medical devices, especially when reendothelialization is required. PMID:22975628

  17. Function-blocking antibodies to human vascular adhesion protein-1: a potential anti-inflammatory therapy.

    Science.gov (United States)

    Kirton, Christopher M; Laukkanen, Marja-Leena; Nieminen, Antti; Merinen, Marika; Stolen, Craig M; Armour, Kathryn; Smith, David J; Salmi, Marko; Jalkanen, Sirpa; Clark, Michael R

    2005-11-01

    Human vascular adhesion protein-1 (VAP-1) is a homodimeric 170-kDa sialoglycoprotein that is expressed on the surface of endothelial cells and functions as a semicarbazide-sensitive amine oxidase and as an adhesion molecule. Blockade of VAP-1 has been shown to reduce leukocyte adhesion and transmigration in in vivo and in vitro models, suggesting that VAP-1 is a potential target for anti-inflammatory therapy. In this study we have constructed mouse-human chimeric antibodies by genetic engineering in order to circumvent the potential problems involved in using murine antibodies in man. Our chimeric anti-VAP-1 antibodies, which were designed to lack Fc-dependent effector functions, bound specifically to cell surface-expressed recombinant human VAP-1 and recognized VAP-1 in different cell types in tonsil. Furthermore, the chimeric antibodies prevented leukocyte adhesion and transmigration in vitro and in vivo. Hence, these chimeric antibodies have the potential to be used as a new anti-inflammatory therapy.

  18. Bioactivity and properties of a dental adhesive functionalized with polyhedral oligomeric silsesquioxanes (POSS) and bioactive glass.

    Science.gov (United States)

    Rizk, Marta; Hohlfeld, Lisa; Thanh, Loan Tao; Biehl, Ralf; Lühmann, Nicole; Mohn, Dirk; Wiegand, Annette

    2017-09-01

    This study aimed to analyze the effect of infiltrating a commercial adhesive with nanosized bioactive glass (BG-Bi) particles or methacryl-functionalized polyhedral oligomeric silsesquioxanes (POSS) on material properties and bioactivity. An acetone-based dental adhesive (Solobond Plus adhesive, VOCO GmbH, Cuxhaven, Germany) was infiltrated with nanosized bioactive glass particles (0.1 or 1wt%), or with monofunctional or multifunctional POSS particles (10 or 20wt%). Unfilled adhesive served as control. Dispersion and hydrodynamic radius of the nanoparticles were studied by dynamic light scattering. Set specimens were immersed for 28days in artificial saliva at 37°C, and surfaces were mapped for the formation of calcium phospate (Ca/P) precipitates (scanning electron microscopy/energy-dispersive X-ray spectroscopy). Viscosity (rheometry) and the structural characteristic of the networks were studied, such as degree of conversion (FTIR spectroscopy), sol fraction and water sorption. POSS particles showed a good dispersion of the particles for both types of particles being smaller than 3nm, while the bioactive glass particles had a strong tendency to agglomerate. All nanoparticles induced the formation of Ca/P precipitates. The viscosity of the adhesive was not or only slightly increased by POSS particle addition but strongly increased by the bioactive glass particles. The degree of conversion, water sorption and sol fraction showed a maintained or improved network structure and properties when filled with BG-Bi and multifunctional POSS, however, less polymerization was found when loading a monofunctional POSS. Multifunctional POSS may be incorporated into dental adhesives to provide a bioactive potential without changing material properties adversely. Copyright © 2017 The Academy of Dental Materials. Published by Elsevier Ltd. All rights reserved.

  19. Silk-based biomaterials functionalized with fibronectin type II promotes cell adhesion.

    Science.gov (United States)

    Pereira, Ana Margarida; Machado, Raul; da Costa, André; Ribeiro, Artur; Collins, Tony; Gomes, Andreia C; Leonor, Isabel B; Kaplan, David L; Reis, Rui L; Casal, Margarida

    2017-01-01

    The objective of this work was to exploit the fibronectin type II (FNII) module from human matrix metalloproteinase-2 as a functional domain for the development of silk-based biopolymer blends that display enhanced cell adhesion properties. The DNA sequence of spider dragline silk protein (6mer) was genetically fused with the FNII coding sequence and expressed in Escherichia coli. The chimeric protein 6mer+FNII was purified by non-chromatographic methods. Films prepared from 6mer+FNII by solvent casting promoted only limited cell adhesion of human skin fibroblasts. However, the performance of the material in terms of cell adhesion was significantly improved when 6mer+FNII was combined with a silk-elastin-like protein in a concentration-dependent behavior. With this work we describe a novel class of biopolymer that promote cell adhesion and potentially useful as biomaterials for tissue engineering and regenerative medicine. This work reports the development of biocompatible silk-based composites with enhanced cell adhesion properties suitable for biomedical applications in regenerative medicine. The biocomposites were produced by combining a genetically engineered silk-elastin-like protein with a genetically engineered spider-silk-based polypeptide carrying the three domains of the fibronectin type II module from human metalloproteinase-2. These composites were processed into free-standing films by solvent casting and characterized for their biological behavior. To our knowledge this is the first report of the exploitation of all three FNII domains as a functional domain for the development of bioinspired materials with improved biological performance. The present study highlights the potential of using genetically engineered protein-based composites as a platform for the development of new bioinspired biomaterials. Copyright © 2016 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

  20. Patient-reported measures of quality of life and functional capacity in adhesive capsulitis

    Directory of Open Access Journals (Sweden)

    Marcos Rassi Fernandes

    Full Text Available Summary Objective: To evaluate patient-reported measures of quality of life and functional capacity in adhesive capsulitis before and after suprascapular nerve block treatment; to analyze the influence of clinical and sociodemographic variables on both the outcome and correlation between the respective instruments. Method: A prospective clinical study was performed with adhesive capsulitis patients. Inclusion criteria were clinical diagnosis of adhesive capsulitis and a shoulder imaging test. The WHOQOL-BREF and DASH instruments were administered before and after treatment. A Constant test score of 55 points was used as a cutoff point for discontinuation of treatment. Mean values were compared using paired t-test and Wilcoxon. The Pearson or Spearman coefficients were used for correlation analysis. Multiple linear regression analysis was carried out using variables with p<0.20 as predictors in univariate analysis and WHOQOL domains and DASH as outcomes. The significance level was 5%. Results: Forty-three patients were evaluated. The comparison between WHOQOL-BREF and DASH mean values before and after the blocks, p<0.05. DASH correlated negatively with the physical, psychological and environmental WHOQOL-BREF domains. Older patients and those with higher levels of education influenced the improvement in patients' quality of life and functional capacity. Conclusion: The improvement of adhesive capsulitis with treatment involves an improvement in quality of life and functional capacity. The greater functional capacity of the shoulder matches a better quality of life for patients. Age and education level are the variables that most influence improvement in quality of life and functional capacity.

  1. Patient-reported measures of quality of life and functional capacity in adhesive capsulitis.

    Science.gov (United States)

    Fernandes, Marcos Rassi

    2017-04-01

    To evaluate patient-reported measures of quality of life and functional capacity in adhesive capsulitis before and after suprascapular nerve block treatment; to analyze the influence of clinical and sociodemographic variables on both the outcome and correlation between the respective instruments. A prospective clinical study was performed with adhesive capsulitis patients. Inclusion criteria were clinical diagnosis of adhesive capsulitis and a shoulder imaging test. The WHOQOL-BREF and DASH instruments were administered before and after treatment. A Constant test score of 55 points was used as a cutoff point for discontinuation of treatment. Mean values were compared using paired t-test and Wilcoxon. The Pearson or Spearman coefficients were used for correlation analysis. Multiple linear regression analysis was carried out using variables with padhesive capsulitis with treatment involves an improvement in quality of life and functional capacity. The greater functional capacity of the shoulder matches a better quality of life for patients. Age and education level are the variables that most influence improvement in quality of life and functional capacity.

  2. Tuning adhesion forces between functionalized gold colloidal nanoparticles and silicon AFM tips: role of ligands and capillary forces.

    Science.gov (United States)

    Oras, Sven; Vlassov, Sergei; Berholts, Marta; Lõhmus, Rünno; Mougin, Karine

    2018-01-01

    Adhesion forces between functionalized gold colloidal nanoparticles (Au NPs) and scanning probe microscope silicon tips were experimentally investigated by atomic force microscopy (AFM) equipped with PeakForce QNM (Quantitative Nanoscale Mechanics) module. Au NPs were synthesized by a seed-mediated process and then functionalized with thiols containing different functional groups: amino, hydroxy, methoxy, carboxy, methyl, and thiol. Adhesion measurements showed strong differences between NPs and silicon tip depending on the nature of the tail functional group. The dependence of the adhesion on ligand density for different thiols with identical functional tail-group was also demonstrated. The calculated contribution of the van der Waals (vdW) forces between particles was in good agreement with experimentally measured adhesive values. In addition, the adhesion forces were evaluated between flat Au films functionalized with the same molecular components and silicon tips to exclude the effect of particle shape on the adhesion values. Although adhesion values on flat substrates were higher than on their nanoparticle counterparts, the dependance on functional groups remained the same.

  3. The regulation of SIRT2 function by cyclin-dependent kinases affects cell motility.

    NARCIS (Netherlands)

    Pandithage, R.; Lilischkis, R.; Harting, K.; Wolf, A.; Jedamzik, B.; Luscher-Firzlaff, J.; Vervoorts, J.; Lasonder, E.; Kremmer, E.; Knoll, B.; Luscher, B.

    2008-01-01

    Cyclin-dependent kinases (Cdks) fulfill key functions in many cellular processes, including cell cycle progression and cytoskeletal dynamics. A limited number of Cdk substrates have been identified with few demonstrated to be regulated by Cdk-dependent phosphorylation. We identify on protein

  4. Adhesion, invasion and evasion: the many functions of the surface proteins of Staphylococcus aureus

    Science.gov (United States)

    Foster, Timothy J.; Geoghegan, Joan A.; Ganesh, Vannakambadi K.; Höök, Magnus

    2014-01-01

    Staphylococcus aureus is an important opportunistic pathogen and persistently colonizes about 20% of the human population. Its surface is ‘decorated’ with proteins that are covalently anchored to the cell wall peptidoglycan. Structural and functional analysis has identified four distinct classes of surface proteins, of which microbial surface component recognizing adhesive matrix molecules (MSCRAMMs) are the largest class. These surface proteins have numerous functions, including adhesion to and invasion of host cells and tissues, evasion of immune responses and biofilm formation. Thus, cell wall-anchored proteins are essential virulence factors for the survival of S. aureus in the commensal state and during invasive infections, and targeting them with vaccines could combat S. aureus infections. PMID:24336184

  5. Surface functionalization of titanium substrates with chitosan-lauric acid conjugate to enhance osteoblasts functions and inhibit bacteria adhesion.

    Science.gov (United States)

    Zhao, Lu; Hu, Yan; Xu, Dawei; Cai, Kaiyong

    2014-07-01

    Orthopedic implants failures are generally related to poor osseointegration and/or bacterial infection in clinical application. Surface functionalization of an implant is one promising alternative for enhancing osseointegration and/or reducing bacterial infection, thus ensuring the long term survival of the implant. In this study, titanium (Ti) substrates were surface functionalized with a polydopamine (PDOP) film as an intermediate layer for post-immobilization of chitosan-lauric acid (Chi-LA) conjugate. Chi-LA conjugate was synthesized and characterized by Fourier transform infrared spectroscopy (FTIR) and hydrogen proton nuclear magnetic resonance (NMR) spectrometer, respectively. Lauric acid (LA), a natural saturated fatty acid, was used mainly due to its good antibacterial property. Scanning electron microscopy (SEM) and water contact angle measurements were employed to detect the morphology changes and surface wettability of Ti substrates. The results suggested that Chi-LA conjugate was successfully immobilized onto the surfaces of Ti substrates. In vitro tests confirmed that the cell adhesion, cell viability, intracellular alkaline phosphatase activity and mineralization capacity of osteoblasts were remarkably improved when cultured onto Chi-LA surface functionalized Ti substrates. Antibacterial assay against Staphylococcus aureus (S. aureus) and Pseudomonas aeruginosa (P. aeruginosa) showed that the Chi-LA modified Ti substrates efficiently inhibited the adhesion and growth of bacteria. Overall, this study developed a promising approach to fabricate functional Ti-based orthopedic implants, which could enhance the biological functions of osteoblasts and concurrently reduce bacteria adhesion. Copyright © 2014 Elsevier B.V. All rights reserved.

  6. The FGGY carbohydrate kinase family: insights into the evolution of functional specificities.

    Directory of Open Access Journals (Sweden)

    Ying Zhang

    2011-12-01

    Full Text Available Function diversification in large protein families is a major mechanism driving expansion of cellular networks, providing organisms with new metabolic capabilities and thus adding to their evolutionary success. However, our understanding of the evolutionary mechanisms of functional diversity in such families is very limited, which, among many other reasons, is due to the lack of functionally well-characterized sets of proteins. Here, using the FGGY carbohydrate kinase family as an example, we built a confidently annotated reference set (CARS of proteins by propagating experimentally verified functional assignments to a limited number of homologous proteins that are supported by their genomic and functional contexts. Then, we analyzed, on both the phylogenetic and the molecular levels, the evolution of different functional specificities in this family. The results show that the different functions (substrate specificities encoded by FGGY kinases have emerged only once in the evolutionary history following an apparently simple divergent evolutionary model. At the same time, on the molecular level, one isofunctional group (L-ribulokinase, AraB evolved at least two independent solutions that employed distinct specificity-determining residues for the recognition of a same substrate (L-ribulose. Our analysis provides a detailed model of the evolution of the FGGY kinase family. It also shows that only combined molecular and phylogenetic approaches can help reconstruct a full picture of functional diversifications in such diverse families.

  7. Inhibition of Src family kinases improves cognitive function after intraventricular hemorrhage or intraventricular thrombin.

    Science.gov (United States)

    Liu, Da Zhi; Waldau, Ben; Ander, Bradley P; Zhan, Xinhua; Stamova, Boryana; Jickling, Glen C; Lyeth, Bruce G; Sharp, Frank R

    2017-07-01

    Intraventricular hemorrhage causes spatial memory loss, but the mechanism remains unknown. Our recent studies demonstrated that traumatic brain injury activates Src family kinases, which cause spatial memory loss. To test whether the spatial memory loss was due to blood in the ventricles, which activated Src family kinases, we infused autologous whole blood or thrombin into the lateral ventricles of adult rats to model non-traumatic intraventricular hemorrhage. Hippocampal neuron loss was examined 1 day to 5 weeks later. Spatial memory function was assessed 29 to 33 days later using the Morris water maze. Five weeks after the ventricular injections of blood or thrombin, there was death of most hippocampal neurons and significant memory deficits compared with sham operated controls. These data show that intraventricular thrombin is sufficient to kill hippocampal neurons and produce spatial memory loss. In addition, systemic administration of the non-specific Src family kinase inhibitor PP2 or intraventricular injection of siRNA-Fyn, a Src family kinase family member, prevented hippocampal neuronal loss and spatial memory deficits following intraventricular hemorrhage. The data support the conclusions that thrombin mediates the hippocampal neuronal cell death and spatial memory deficits produced by intraventricular blood and that these can be blocked by non-specific inhibition of Src family kinases or by inhibiting Fyn.

  8. Structural Basis of Wee Kinases Functionality and Inactivation by Diverse Small Molecule Inhibitors.

    Science.gov (United States)

    Zhu, Jin-Yi; Cuellar, Rebecca A; Berndt, Norbert; Lee, Hee Eun; Olesen, Sanne H; Martin, Mathew P; Jensen, Jeffrey T; Georg, Gunda I; Schönbrunn, Ernst

    2017-09-28

    Members of the Wee family of kinases negatively regulate the cell cycle via phosphorylation of CDK1 and are considered potential drug targets. Herein, we investigated the structure-function relationship of human Wee1, Wee2, and Myt1 (PKMYT1). Purified recombinant full-length proteins and kinase domain constructs differed substantially in phosphorylation states and catalytic competency, suggesting complex mechanisms of activation. A series of crystal structures reveal unique features that distinguish Wee1 and Wee2 from Myt1 and establish the structural basis of differential inhibition by the widely used Wee1 inhibitor MK-1775. Kinome profiling and cellular studies demonstrate that, in addition to Wee1 and Wee2, MK-1775 is an equally potent inhibitor of the polo-like kinase PLK1. Several previously unrecognized inhibitors of Wee kinases were discovered and characterized. Combined, the data provide a comprehensive view on the catalytic and structural properties of Wee kinases and a framework for the rational design of novel inhibitors thereof.

  9. Role of focal adhesion tyrosine kinases in GPVI-dependent platelet activation and reactive oxygen species formation.

    Directory of Open Access Journals (Sweden)

    Naadiya Carrim

    Full Text Available We have previously shown the presence of a TRAF4/p47phox/Hic5/Pyk2 complex associated with the platelet collagen receptor, GPVI, consistent with a potential role of this complex in GPVI-dependent ROS formation. In other cell systems, NOX-dependent ROS formation is facilitated by Pyk2, which along with its closely related homologue FAK are known to be activated and phosphorylated downstream of ligand binding to GPVI.To evaluate the relative roles of Pyk2 and FAK in GPVI-dependent ROS formation and to determine their location within the GPVI signaling pathway.Human and mouse washed platelets (from WT or Pyk2 KO mice were pre-treated with pharmacological inhibitors targeting FAK or Pyk2 (PF-228 and Tyrphostin A9, respectively and stimulated with the GPVI-specific agonist, CRP. FAK, but not Pyk2, was found to be essential for GPVI-dependent ROS production and aggregation. Subsequent human platelet studies with PF-228 confirmed FAK is essential for GPVI-mediated phosphatidylserine exposure, α-granule secretion (P-selectin (CD62P surface expression and integrin αIIbβ3 activation. To determine the precise location of FAK within the GPVI pathway, we analyzed the effect of PF-228 inhibition in CRP-stimulated platelets in conjunction with immunoprecipitation and pulldown analysis to show that FAK is downstream of Lyn, Spleen tyrosine kinase (Syk, PI3-K and Bruton's tyrosine kinase (Btk and upstream of Rac1, PLCγ2, Ca2+ release, PKC, Hic-5, NOX1 and αIIbβ3 activation.Overall, these data suggest a novel role for FAK in GPVI-dependent ROS formation and platelet activation and elucidate a proximal signaling role for FAK within the GPVI pathway.

  10. Identification of interphase functions for the NIMA kinase involving microtubules and the ESCRT pathway.

    Directory of Open Access Journals (Sweden)

    Meera Govindaraghavan

    2014-03-01

    Full Text Available The Never in Mitosis A (NIMA kinase (the founding member of the Nek family of kinases has been considered a mitotic specific kinase with nuclear restricted roles in the model fungus Aspergillus nidulans. By extending to A. nidulans the results of a synthetic lethal screen performed in Saccharomyces cerevisiae using the NIMA ortholog KIN3, we identified a conserved genetic interaction between nimA and genes encoding proteins of the Endosomal Sorting Complex Required for Transport (ESCRT pathway. Absence of ESCRT pathway functions in combination with partial NIMA function causes enhanced cell growth defects, including an inability to maintain a single polarized dominant cell tip. These genetic insights suggest NIMA potentially has interphase functions in addition to its established mitotic functions at nuclei. We therefore generated endogenously GFP-tagged NIMA (NIMA-GFP which was fully functional to follow its interphase locations using live cell spinning disc 4D confocal microscopy. During interphase some NIMA-GFP locates to the tips of rapidly growing cells and, when expressed ectopically, also locates to the tips of cytoplasmic microtubules, suggestive of non-nuclear interphase functions. In support of this, perturbation of NIMA function either by ectopic overexpression or through partial inactivation results in marked cell tip growth defects with excess NIMA-GFP promoting multiple growing cell tips. Ectopic NIMA-GFP was found to locate to the plus ends of microtubules in an EB1 dependent manner, while impairing NIMA function altered the dynamic localization of EB1 and the cytoplasmic microtubule network. Together, our genetic and cell biological analyses reveal novel non-nuclear interphase functions for NIMA involving microtubules and the ESCRT pathway for normal polarized fungal cell tip growth. These insights extend the roles of NIMA both spatially and temporally and indicate that this conserved protein kinase could help integrate cell

  11. The Fanconi anemia proteins functionally interact with the protein kinase regulated by RNA (PKR).

    Science.gov (United States)

    Zhang, Xiaoling; Li, June; Sejas, Daniel P; Rathbun, Keaney R; Bagby, Grover C; Pang, Qishen

    2004-10-15

    Protein kinase regulated by RNA (PKR) plays critical roles in cell growth and apoptosis and is implicated as a potential pathogenic factor of Alzheimer's, Parkinson's, and Huntington's diseases. Here we report that this proapoptotic kinase is also involved in Fanconi anemia (FA), a disease characterized by bone marrow (BM) failure and leukemia. We have used a BM extract to show that three FA proteins, FANCA, FANCC, and FANCG, functionally interact with the PKR kinase, which in turn regulates translational control. By using a combined immunoprecipitation and reconstituted kinase assay, in which an active PKR kinase complex was captured from a normal cell extract, we demonstrated functional interactions between the FA proteins and the PKR kinase. In primary human BM cells, mutations in the FANCA, FANCC, and FANCG genes markedly increase the amount of PKR bound to FANCC, and this PKR accumulation is correlated with elevated PKR activation and hypersensitivity of BM progenitor cells to growth repression mediated by the inhibitory cytokines interferon-gamma and tumor necrosis factor-alpha. Specific inhibition of PKR by 2-aminopurine in these FA BM cells attenuates PKR activation and apoptosis induction. In lymphoblasts derived from an FA-C patient, overexpression of a dominant negative mutant PKR (PKRK296R) suppressed PKR activation and apoptosis induced by interferon-gamma and tumor necrosis factor-alpha. Furthermore, by using genetically matched wild-type and PKR-null cells, we demonstrated that forced expression of a patient-derived FA-C mutant (FANCCL554P) augmented double-stranded RNA-induced PKR activation and cell death. Thus, inappropriate activation of PKR as a consequence of certain FA mutations might play a role in bone marrow failure that frequently occurred in FA.

  12. Focal Adhesion Kinase functions as an Akt downstream target in migration of colorectal cancer cells

    Czech Academy of Sciences Publication Activity Database

    Turečková, Jolana; Vojtěchová, Martina; Krausová, Michaela; Šloncová, Eva; Kořínek, Vladimír

    2009-01-01

    Roč. 2, č. 4 (2009), s. 281-290 ISSN 1936-5233 R&D Projects: GA ČR GA204/06/1658; GA MŠk 2B06077 Institutional research plan: CEZ:AV0Z50520514 Keywords : FAK * migration * invasion Subject RIV: EB - Genetics ; Molecular Biology

  13. Adhesive interfacial characteristics and the related bonding performance of four self-etching adhesives with different functional monomers applied to dentin.

    Science.gov (United States)

    Wang, Ruirui; Shi, Yilin; Li, Tingting; Pan, Yueping; Cui, Yu; Xia, Wenwei

    2017-07-01

    To examine the interfacial chemical and morphological characteristics of four self-etching adhesives bonded to dentin with different functional monomers. Further, to evaluate the effects of this interaction between functional monomers and dentin on short-term in vitro bonding performance of the four adhesives. Clearfil SE Bond (CSE) and Scotchbond Universal (SU) containing 10-methacryloxydecyl dihydrogen phosphate (10-MDP), Optibond XTR (OX) containing glycero-phosphate dimethacrylate (GPDM), and Adper Easy One (AEO) containing 6-methacryloyloxyhexyl dihydrogen phosphate (6-MHP) were applied to the dentin surface according to the instructions supplied with each. Interaction between the functional monomers and dentin was characterized using thin-film X-ray diffraction (TF-XRD) and scanning electron microscopy (SEM). The hydrophilicity of each acidic monomer was also assessed by chemical structure drawing software. Micro-tensile bond strength (μTBS) and nanoleakage were used to evaluate the bonding effectiveness of the adhesives, either immediately or after thermo-cycling (5°C-55°C) for 5000 cycles. TF-XRD showed that both CSE and SU exhibited 10-MDP-Ca nano-layering at the adhesive interface, but with different intensity when reacted with dentin. OX, that contains GPDM, demineralized the dentin surface more severely, forming long resin tags into the dentinal tubules, and gained the highest μTBS at the immediate time-point. Thermo-cycling adversely affected the μTBS and nanoleakage of AEO and OX, but had no significant influence on CSE and SU which contain 10-MDP. Self-etching adhesives containing different structures/concentrations of functional monomers produced adhesive interfaces with obviously different chemical and morphological characteristics, which may have a direct impact on bonding effectiveness. Our findings support the concept that the stable chemical bonding produced by 10-MDP to the Ca of hydroxyapatite is advantageous for durability of adhesive

  14. A MAM7 peptide-based inhibitor of Staphylococcus aureus adhesion does not interfere with in vitro host cell function.

    Directory of Open Access Journals (Sweden)

    Catherine Alice Hawley

    Full Text Available Adhesion inhibitors that block the attachment of pathogens to host tissues may be used synergistically with or as an alternative to antibiotics. The wide-spread bacterial adhesin Multivalent Adhesion Molecule (MAM 7 has recently emerged as a candidate molecule for a broad-spectrum adhesion inhibitor which may be used to prevent bacterial colonization of wounds. Here we have tested if the antibacterial properties of a MAM-based inhibitor could be used to competitively inhibit adhesion of methicillin-resistant Staphylococcus aureus (MRSA to host cells. Additionally, we analyzed its effect on host cellular functions linked to the host receptor fibronectin, such as migration, adhesion and matrix formation in vitro, to evaluate potential side effects prior to advancing our studies to in vivo infection models. As controls, we used inhibitors based on well-characterized bacterial adhesin-derived peptides from F1 and FnBPA, which are known to affect host cellular functions. Inhibitors based on F1 or FnBPA blocked MRSA attachment but at the same time abrogated important cellular functions. A MAM7-based inhibitor did not interfere with host cell function while showing good efficacy against MRSA adhesion in a tissue culture model. These observations provide a possible candidate for a bacterial adhesion inhibitor that does not cause adverse effects on host cells while preventing bacterial infection.

  15. A conserved cysteine motif is critical for rice ceramide kinase activity and function.

    Directory of Open Access Journals (Sweden)

    Fang-Cheng Bi

    Full Text Available Ceramide kinase (CERK is a key regulator of cell survival in dicotyledonous plants and animals. Much less is known about the roles of CERK and ceramides in mediating cellular processes in monocot plants. Here, we report the characterization of a ceramide kinase, OsCERK, from rice (Oryza sativa spp. Japonica cv. Nipponbare and investigate the effects of ceramides on rice cell viability.OsCERK can complement the Arabidopsis CERK mutant acd5. Recombinant OsCERK has ceramide kinase activity with Michaelis-Menten kinetics and optimal activity at 7.0 pH and 40°C. Mg2+ activates OsCERK in a concentration-dependent manner. Importantly, a CXXXCXXC motif, conserved in all ceramide kinases and important for the activity of the human enzyme, is critical for OsCERK enzyme activity and in planta function. In a rice protoplast system, inhibition of CERK leads to cell death and the ratio of added ceramide and ceramide-1-phosphate, CERK's substrate and product, respectively, influences cell survival. Ceramide-induced rice cell death has apoptotic features and is an active process that requires both de novo protein synthesis and phosphorylation, respectively. Finally, mitochondria membrane potential loss previously associated with ceramide-induced cell death in Arabidopsis was also found in rice, but it occurred with different timing.OsCERK is a bona fide ceramide kinase with a functionally and evolutionarily conserved Cys-rich motif that plays an important role in modulating cell fate in plants. The vital function of the conserved motif in both human and rice CERKs suggests that the biochemical mechanism of CERKs is similar in animals and plants. Furthermore, ceramides induce cell death with similar features in monocot and dicot plants.

  16. The Interaction between Cancer Stem Cell Marker CD133 and Src Protein Promotes Focal Adhesion Kinase (FAK) Phosphorylation and Cell Migration.

    Science.gov (United States)

    Liu, Chanjuan; Li, Yinan; Xing, Yang; Cao, Benjin; Yang, Fan; Yang, Tianxiao; Ai, Zhilong; Wei, Yuanyan; Jiang, Jianhai

    2016-07-22

    CD133, a widely known cancer stem cell marker, has been proved to promote tumor metastasis. However, the mechanism by which CD133 regulates metastasis remains largely unknown. Here, we report that CD133 knockdown inhibits cancer cell migration, and CD133 overexpression promotes cell migration. CD133 expression is beneficial to activate the Src-focal adhesion kinase (FAK) signaling pathway. Further studies show that CD133 could interact with Src, and the region between amino acids 845 and 857 in the CD133 C-terminal domain is indispensable for its interaction with Src. The interaction activates Src to phosphorylate its substrate FAK and to promote cell migration. Likewise, a Src binding-deficient CD133 mutant loses the abilities to increase Src and FAK phosphorylation and to promote cell migration. Inhibition of Src activity by PP2, a known Src activity inhibitor, could block the activation of FAK phosphorylation and cell migration induced by CD133. In summary, our data suggest that activation of FAK by the interaction between CD133 and Src promotes cell migration, providing clues to understand the migratory mechanism of CD133(+) tumor cells. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

  17. Loss of Sphingosine Kinase Alters Life History Traits and Locomotor Function inCaenorhabditis elegans.

    Science.gov (United States)

    Chan, Jason P; Brown, Jaylene; Hark, Brandon; Nolan, Abby; Servello, Dustin; Hrobuchak, Hannah; Staab, Trisha A

    2017-01-01

    Sphingolipid metabolism is important to balance the abundance of bioactive lipid molecules involved in cell signaling, neuronal function, and survival. Specifically, the sphingolipid sphingosine mediates cell death signaling, whereas its phosphorylated form, sphingosine-1-phosphate (S1P), mediates cell survival signaling. The enzyme sphingosine kinase produces S1P, and the activity of sphingosine kinase impacts the ability of cells to survive under stress and challenges. To examine the influence of sphingolipid metabolism, particularly enzymes regulating sphingosine and S1P, in mediating aging, neuronal function and stress response, we examined life history traits, locomotor capacities and heat stress responses of young and old animals using the model organism Caenorhabditis elegans . We found that C. elegans sphk-1 mutants, which lack sphingosine kinase, had shorter lifespans, reduced brood sizes, and smaller body sizes compared to wild type animals. By analyzing a panel of young and old animals with genetic mutations in the sphingolipid signaling pathway, we showed that aged sphk-1 mutants exhibited a greater decline in neuromuscular function and locomotor behavior. In addition, aged animals lacking sphk-1 were more susceptible to death induced by acute and prolonged heat exposure. On the other hand, older animals with loss of function mutations in ceramide synthase ( hyl-1 ), which converts sphingosine to ceramide, showed improved neuromuscular function and stress response with age. This phenotype was dependent on sphk-1 . Together, our data show that loss of sphingosine kinase contributes to poor animal health span, suggesting that sphingolipid signaling may be important for healthy neuronal function and animal stress response during aging.

  18. Loss of Sphingosine Kinase Alters Life History Traits and Locomotor Function in Caenorhabditis elegans

    Directory of Open Access Journals (Sweden)

    Jason P. Chan

    2017-09-01

    Full Text Available Sphingolipid metabolism is important to balance the abundance of bioactive lipid molecules involved in cell signaling, neuronal function, and survival. Specifically, the sphingolipid sphingosine mediates cell death signaling, whereas its phosphorylated form, sphingosine-1-phosphate (S1P, mediates cell survival signaling. The enzyme sphingosine kinase produces S1P, and the activity of sphingosine kinase impacts the ability of cells to survive under stress and challenges. To examine the influence of sphingolipid metabolism, particularly enzymes regulating sphingosine and S1P, in mediating aging, neuronal function and stress response, we examined life history traits, locomotor capacities and heat stress responses of young and old animals using the model organism Caenorhabditis elegans. We found that C. elegans sphk-1 mutants, which lack sphingosine kinase, had shorter lifespans, reduced brood sizes, and smaller body sizes compared to wild type animals. By analyzing a panel of young and old animals with genetic mutations in the sphingolipid signaling pathway, we showed that aged sphk-1 mutants exhibited a greater decline in neuromuscular function and locomotor behavior. In addition, aged animals lacking sphk-1 were more susceptible to death induced by acute and prolonged heat exposure. On the other hand, older animals with loss of function mutations in ceramide synthase (hyl-1, which converts sphingosine to ceramide, showed improved neuromuscular function and stress response with age. This phenotype was dependent on sphk-1. Together, our data show that loss of sphingosine kinase contributes to poor animal health span, suggesting that sphingolipid signaling may be important for healthy neuronal function and animal stress response during aging.

  19. Tec kinases regulate T-lymphocyte development and function: new insights into the roles of Itk and Rlk/Txk.

    Science.gov (United States)

    Readinger, Julie A; Mueller, Kristen L; Venegas, Ana M; Horai, Reiko; Schwartzberg, Pamela L

    2009-03-01

    The Tec (tyrosine kinase expressed in hepatocellular carcinoma) family of non-receptor tyrosine kinases consists of five members: Tec, Bruton's tyrosine kinase (Btk), inducible T-cell kinase (Itk), resting lymphocyte kinase (Rlk/Txk), and bone marrow-expressed kinase (Bmx/Etk). Although their functions are probably best understood in antigen receptor signaling, where they participate in the phosphorylation and regulation of phospholipase C-gamma (PLC-gamma), it is now appreciated that these kinases contribute to signaling from many receptors and that they participate in multiple downstream pathways, including regulation of the actin cytoskeleton. In T cells, three Tec kinases are expressed, Itk, Rlk/Txk, and Tec. Itk is expressed at highest amounts and plays the major role in regulating signaling from the T-cell receptor. Recent studies provide evidence that these kinases contribute to multiple aspects of T-cell biology and have unique roles in T-cell development that have revealed new insight into the regulation of conventional and innate T-cell development. We review new findings on the Tec kinases with a focus on their roles in T-cell development and mature T-cell differentiation.

  20. Titanium Surface Coating with a Laminin-Derived Functional Peptide Promotes Bone Cell Adhesion

    Directory of Open Access Journals (Sweden)

    Seung-Ki Min

    2013-01-01

    Full Text Available Laminin-derived peptide coatings can enhance epithelial cell adhesion to implants, and the positive effect of these peptides on bone cell adhesion has been anticipated. The purpose of this study was to evaluate the improvement in bone cell attachment to and activity on titanium (Ti scaffolds coated with a laminin-derived functional peptide, Ln2-P3 (the DLTIDDSYWYRI motif. Four Ti disc surfaces were prepared, and a human osteosarcoma (HOS cell attachment test was performed to select two candidate surfaces for peptide coating. These two candidates were then coated with Ln2-P3 peptide, a scrambled peptide, or left uncoated to measure cell attachment to each surface, following which one surface was chosen to assess alkaline phosphatase (ALP activity and osteogenic marker gene expression with quantitative real-time PCR. On the commercially pure Ti surface, the Ln2-P3 coating significantly increased cellular ALP activity and the expression levels of ALP and bone sialoprotein mRNA as compared with the scrambled peptide-coated and uncoated surfaces. In conclusion, although further in vivo studies are needed, the findings of this in vitro study indicate that the Ln2-P3-coated implant surface promotes bone cell adhesion, which has clinical implications for reducing the overall treatment time of dental implant therapy.

  1. Chemical functionalization of ceramic tile surfaces by silane coupling agents: polymer modified mortar adhesion mechanism implications

    Directory of Open Access Journals (Sweden)

    Alexandra Ancelmo Piscitelli Mansur

    2008-09-01

    Full Text Available Adhesion between tiles and mortars are crucial to the stability of ceramic tile systems. From the chemical point of view, weak forces such as van der Waals forces and hydrophilic interactions are expected to be developed preferably at the tiles and polymer modified Portland cement mortar interface. The main goal of this paper was to use organosilanes as primers to modify ceramic tile hydrophilic properties to improve adhesion between ceramic tiles and polymer modified mortars. Glass tile surfaces were treated with several silane derivatives bearing specific functionalities. Contact angle measurements and Fourier Transform Infrared Spectroscopy (FTIR were used for evaluating the chemical changes on the tile surface. In addition, pull-off tests were conducted to assess the effect on adhesion properties between tile and poly(ethylene-co-vinyl acetate, EVA, modified mortar. The bond strength results have clearly shown the improvement of adherence at the tile-polymer modified mortar interface, reflecting the overall balance of silane, cement and polymer interactions.

  2. Silencing HIF-1α reduces the adhesion and secretion functions of acute leukemia hBMSCs

    Energy Technology Data Exchange (ETDEWEB)

    Zeng, Dong-Feng [Department of Hematology, XinQiao Hospital, Third Military Medical University, ChongQing (China); Liu, Ting [Department of Ophthalmology, DaPing Hospital, Third Military Medical University, ChongQing (China); Chang, Cheng; Zhang, Xi; Liang, Xue; Chen, Xing-Hua; Kong, Pei-Yan [Department of Hematology, XinQiao Hospital, Third Military Medical University, ChongQing (China)

    2012-06-29

    Hypoxia inducible factor-1α (HIF-1α) is an important transcription factor, which plays a critical role in the formation of solid tumor and its microenvironment. The objective of the present study was to evaluate the expression and function of HIF-1α in human leukemia bone marrow stromal cells (BMSCs) and to identify the downstream targets of HIF-1α. HIF-1α expression was detected at both the RNA and protein levels using real-time PCR and immunohistochemistry, respectively. Vascular endothelial growth factor (VEGF) and stromal cell-derived factor-1α (SDF-1α) were detected in stromal cells by enzyme-linked immunosorbent assay. HIF-1α was blocked by constructing the lentiviral RNAi vector system and infecting the BMSCs. The Jurkat cell/BMSC co-cultured system was constructed by putting the two cells into the same suitable cultured media and conditions. Cell adhesion and secretion functions of stromal cells were evaluated after transfection with the lentiviral RNAi vector of HIF-1α. Increased HIF-1α mRNA and protein was detected in the nucleus of the acute myeloblastic and acute lymphoblastic leukemia compared with normal BMSCs. The lentiviral RANi vector for HIF-1α was successfully constructed and was applied to block the expression of HIF-1α. When HIF-1α of BMSCs was blocked, the expression of VEGF and SDF-1α secreted by stromal cells was decreased. When HIF-1α was blocked, the co-cultured Jurkat cell's adhesion and migration functions were also decreased. Taken together, these results suggest that HIF-1α acts as an important transcription factor and can significantly affect the secretion and adhesion functions of leukemia BMSCs.

  3. Silencing HIF-1α reduces the adhesion and secretion functions of acute leukemia hBMSCs

    Directory of Open Access Journals (Sweden)

    Zeng Dong-Feng

    2012-10-01

    Full Text Available Hypoxia inducible factor-1α (HIF-1α is an important transcription factor, which plays a critical role in the formation of solid tumor and its microenviroment. The objective of the present study was to evaluate the expression and function of HIF-1α in human leukemia bone marrow stromal cells (BMSCs and to identify the downstream targets of HIF-1α. HIF-1α expression was detected at both the RNA and protein levels using real-time PCR and immunohistochemistry, respectively. Vascular endothelial growth factor (VEGF and stromal cell-derived factor-1α (SDF-1α were detected in stromal cells by enzyme-linked immunosorbent assay. HIF-1α was blocked by constructing the lentiviral RNAi vector system and infecting the BMSCs. The Jurkat cell/BMSC co-cultured system was constructed by putting the two cells into the same suitable cultured media and conditions. Cell adhesion and secretion functions of stromal cells were evaluated after transfection with the lentiviral RNAi vector of HIF-1α. Increased HIF-1α mRNA and protein was detected in the nucleus of the acute myeloblastic and acute lymphoblastic leukemia compared with normal BMSCs. The lentiviral RANi vector for HIF-1α was successfully constructed and was applied to block the expression of HIF-1α. When HIF-1α of BMSCs was blocked, the expression of VEGF and SDF-1 secreted by stromal cells were decreased. When HIF-1α was blocked, the co-cultured Jurkat cell’s adhesion and migration functions were also decreased. Taken together, these results suggest that HIF-1α acts as an important transcription factor and can significantly affect the secretion and adhesion functions of leukemia BMSCs.

  4. Silencing HIF-1α reduces the adhesion and secretion functions of acute leukemia hBMSCs

    International Nuclear Information System (INIS)

    Zeng, Dong-Feng; Liu, Ting; Chang, Cheng; Zhang, Xi; Liang, Xue; Chen, Xing-Hua; Kong, Pei-Yan

    2012-01-01

    Hypoxia inducible factor-1α (HIF-1α) is an important transcription factor, which plays a critical role in the formation of solid tumor and its microenvironment. The objective of the present study was to evaluate the expression and function of HIF-1α in human leukemia bone marrow stromal cells (BMSCs) and to identify the downstream targets of HIF-1α. HIF-1α expression was detected at both the RNA and protein levels using real-time PCR and immunohistochemistry, respectively. Vascular endothelial growth factor (VEGF) and stromal cell-derived factor-1α (SDF-1α) were detected in stromal cells by enzyme-linked immunosorbent assay. HIF-1α was blocked by constructing the lentiviral RNAi vector system and infecting the BMSCs. The Jurkat cell/BMSC co-cultured system was constructed by putting the two cells into the same suitable cultured media and conditions. Cell adhesion and secretion functions of stromal cells were evaluated after transfection with the lentiviral RNAi vector of HIF-1α. Increased HIF-1α mRNA and protein was detected in the nucleus of the acute myeloblastic and acute lymphoblastic leukemia compared with normal BMSCs. The lentiviral RANi vector for HIF-1α was successfully constructed and was applied to block the expression of HIF-1α. When HIF-1α of BMSCs was blocked, the expression of VEGF and SDF-1α secreted by stromal cells was decreased. When HIF-1α was blocked, the co-cultured Jurkat cell's adhesion and migration functions were also decreased. Taken together, these results suggest that HIF-1α acts as an important transcription factor and can significantly affect the secretion and adhesion functions of leukemia BMSCs

  5. Function of adhesion molecules lymphocyte function-associated antigen-3 and intercellular adhesion molecule-1 on human epidermal Langerhans cells in antigen-specific T cell activation

    NARCIS (Netherlands)

    Teunissen, M. B.; Rongen, H. A.; Bos, J. D.

    1994-01-01

    In addition to the interaction between the TCR and the MHC/Ag complex on the APC, optimal T cell activation also requires interaction between adhesion molecules on the APC and their ligands on T cells. We determined the presence of adhesion molecules on human epidermal Langerhans cells (LC) and

  6. Conformational transitions and interactions underlying the function of membrane embedded receptor protein kinases.

    Science.gov (United States)

    Bocharov, Eduard V; Sharonov, Georgy V; Bocharova, Olga V; Pavlov, Konstantin V

    2017-09-01

    Among membrane receptors, the single-span receptor protein kinases occupy a broad but specific functional niche determined by distinctive features of the underlying transmembrane signaling mechanisms that are briefly overviewed on the basis of some of the most representative examples, followed by a more detailed discussion of several hierarchical levels of organization and interactions involved. All these levels, including single-molecule interactions (e.g., dimerization, liganding, chemical modifications), local processes (e.g. lipid membrane perturbations, cytoskeletal interactions), and larger scale phenomena (e.g., effects of membrane surface shape or electrochemical potential gradients) appear to be closely integrated to achieve the observed diversity of the receptor functioning. Different species of receptor protein kinases meet their specific functional demands through different structural features defining their responses to stimulation, but certain common patterns exist. Signaling by receptor protein kinases is typically associated with the receptor dimerization and clustering, ligand-induced rearrangements of receptor domains through allosteric conformational transitions with involvement of lipids, release of the sequestered lipids, restriction of receptor diffusion, cytoskeleton and membrane shape remodeling. Understanding of complexity and continuity of the signaling processes can help identifying currently neglected opportunities for influencing the receptor signaling with potential therapeutic implications. This article is part of a Special Issue entitled: Interactions between membrane receptors in cellular membranes edited by Kalina Hristova. Copyright © 2017 Elsevier B.V. All rights reserved.

  7. HAM-5 Functions As a MAP Kinase Scaffold during Cell Fusion in Neurospora crassa

    Science.gov (United States)

    Jonkers, Wilfried; Leeder, Abigail C.; Ansong, Charles; Wang, Yuexi; Yang, Feng; Starr, Trevor L.; Camp, David G.; Smith, Richard D.; Glass, N. Louise

    2014-01-01

    Cell fusion in genetically identical Neurospora crassa germlings and in hyphae is a highly regulated process involving the activation of a conserved MAP kinase cascade that includes NRC-1, MEK-2 and MAK-2. During chemotrophic growth in germlings, the MAP kinase cascade members localize to conidial anastomosis tube (CAT) tips every ∼8 minutes, perfectly out of phase with another protein that is recruited to the tip: SOFT, a recently identified scaffold for the MAK-1 MAP kinase pathway in Sordaria macrospora. How the MAK-2 oscillation process is initiated, maintained and what proteins regulate the MAP kinase cascade is currently unclear. A global phosphoproteomics approach using an allele of mak-2 (mak-2Q100G) that can be specifically inhibited by the ATP analog 1NM-PP1 was utilized to identify MAK-2 kinase targets in germlings that were potentially involved in this process. One such putative target was HAM-5, a protein of unknown biochemical function. Previously, Δham-5 mutants were shown to be deficient for hyphal fusion. Here we show that HAM-5-GFP co-localized with NRC-1, MEK-2 and MAK-2 and oscillated with identical dynamics from the cytoplasm to CAT tips during chemotropic interactions. In the Δmak-2 strain, HAM-5-GFP localized to punctate complexes that did not oscillate, but still localized to the germling tip, suggesting that MAK-2 activity influences HAM-5 function/localization. However, MAK-2-GFP showed cytoplasmic and nuclear localization in a Δham-5 strain and did not localize to puncta. Via co-immunoprecipitation experiments, HAM-5 was shown to physically interact with NRC-1, MEK-2 and MAK-2, suggesting that it functions as a scaffold/transport hub for the MAP kinase cascade members for oscillation and chemotropic interactions during germling and hyphal fusion in N. crassa. The identification of HAM-5 as a scaffold-like protein will help to link the activation of MAK-2 cascade to upstream factors and proteins involved in this intriguing process of

  8. Adhesion of alkane as a functional group on muscovite and quartz

    DEFF Research Database (Denmark)

    Juhl, Klaus; Pedersen, Christian Schack; Bovet, Nicolas Emile

    2014-01-01

    The interactions between mineral surfaces and organic molecules in water control many processes in nature and in the production of modern materials. To improve the understanding of fluid-surface interactions, we investigated the interface behavior of quartz and muscovite, a model for clay minerals...... experiments and DLVO calculations, we were able to determine the surface charge density. We observed increased adhesion between the mineral surface and the hydrophobic tips as the contact time increased from 7 ms to ?2 s. The di ffusion of dissolved ions takes time, and density functional theory (DFT...

  9. A density functional theory-based investigation of adhesion of poly(butylene terephthalate) on aluminum

    International Nuclear Information System (INIS)

    David, Melanie; Roman, Tanglaw; Nakanishi, Hiroshi; Kasai, Hideaki; Ando, Naoki; Naritomi, Masanori

    2006-01-01

    We investigate the adhesion of PBT on aluminum using density functional theory-based calculations. The geometric structure of the PBT monomer is first relaxed then an aluminum atom is connected to the monomer in different orientations. We calculate their total energies and determine the orientation that gives the strongest binding between the monomer and the aluminum atom. Binding is strongest when the Al connects linearly with the carbonyl oxygen in the ester group. We present binding mechanisms and total energy relationships for the different orientations

  10. Surfactant functionalization induces robust, differential adhesion of tumor cells and blood cells to charged nanotube-coated biomaterials under flow.

    Science.gov (United States)

    Mitchell, Michael J; Castellanos, Carlos A; King, Michael R

    2015-07-01

    The metastatic spread of cancer cells from the primary tumor to distant sites leads to a poor prognosis in cancers originating from multiple organs. Increasing evidence has linked selectin-based adhesion between circulating tumor cells (CTCs) and endothelial cells of the microvasculature to metastatic dissemination, in a manner similar to leukocyte adhesion during inflammation. Functionalized biomaterial surfaces hold promise as a diagnostic tool to separate CTCs and potentially treat metastasis, utilizing antibody and selectin-mediated interactions for cell capture under flow. However, capture at high purity levels is challenged by the fact that CTCs and leukocytes both possess selectin ligands. Here, a straightforward technique to functionalize and alter the charge of naturally occurring halloysite nanotubes using surfactants is reported to induce robust, differential adhesion of tumor cells and blood cells to nanotube-coated surfaces under flow. Negatively charged sodium dodecanoate-functionalized nanotubes simultaneously enhanced tumor cell capture while negating leukocyte adhesion, both in the presence and absence of adhesion proteins, and can be utilized to isolate circulating tumor cells regardless of biomarker expression. Conversely, diminishing nanotube charge via functionalization with decyltrimethylammonium bromide both abolished tumor cell capture while promoting leukocyte adhesion. Copyright © 2015 Elsevier Ltd. All rights reserved.

  11. Enteric glia promote intestinal mucosal healing via activation of focal adhesion kinase and release of proEGF

    OpenAIRE

    Van Landeghem, Laurianne; Chevalier, Julien; Mahé, Maxime M.; Wedel, Thilo; Urvil, Petri; Derkinderen, Pascal; Savidge, Tor; Neunlist, Michel

    2011-01-01

    Wound healing of the gastrointestinal mucosa is essential for the maintenance of gut homeostasis and integrity. Enteric glial cells play a major role in regulating intestinal barrier function, but their role in mucosal barrier repair remains unknown. The impact of conditional ablation of enteric glia on dextran sodium sulfate (DSS)-induced mucosal damage and on healing of diclofenac-induced mucosal ulcerations was evaluated in vivo in GFAP-HSVtk transgenic mice. A mechanically induced model o...

  12. Grafting of phosphorylcholine functional groups on polycarbonate urethane surface for resisting platelet adhesion

    Energy Technology Data Exchange (ETDEWEB)

    Gao, Bin [School of Chemical Engineering and Technology, Tianjin University, Weijin Road 92, Tianjin 300072 (China); Feng, Yakai, E-mail: yakaifeng@hotmail.com [School of Chemical Engineering and Technology, Tianjin University, Weijin Road 92, Tianjin 300072 (China); Tianjin University-Helmholtz-Zentrum Geesthacht, Joint Laboratory for Biomaterials and Regenerative Medicine, Weijin Road 92, 300072 Tianjin (China); Key Laboratory of Systems Bioengineering, Ministry of Education, Tianjin University, Weijin Road 92, Tianjin 300072 (China); Lu, Jian; Zhang, Li; Zhao, Miao; Shi, Changcan; Khan, Musammir [School of Chemical Engineering and Technology, Tianjin University, Weijin Road 92, Tianjin 300072 (China); Guo, Jintang [School of Chemical Engineering and Technology, Tianjin University, Weijin Road 92, Tianjin 300072 (China); Tianjin University-Helmholtz-Zentrum Geesthacht, Joint Laboratory for Biomaterials and Regenerative Medicine, Weijin Road 92, 300072 Tianjin (China)

    2013-07-01

    In order to improve the resistance of platelet adhesion on material surface, 2-methacryloyloxyethyl phosphorylcholine (MPC) was grafted onto polycarbonate urethane (PCU) surface via Michael reaction to create biomimetic structure. After introducing primary amine groups via coupling tris(2-aminoethyl)amine (TAEA) onto the polymer surface, the double bond of MPC reacted with the amino group to obtain MPC modified PCU. The modified surface was characterized by Fourier transform infrared (FTIR) spectroscopy and X-ray photoelectron spectroscopy (XPS). The results verified that MPC was grafted onto PCU surface by Michael reaction method. The MPC grafted PCU surface had a low water contact angle and a high water uptake. This means that the hydrophilic PC functional groups improved the surface hydrophilicity significantly. In addition, surface morphology of MPC grafted PCU film was imaged by atomic force microscope (AFM). The results showed that the grafted surface was rougher than the blank PCU surface. In addition, platelet adhesion study was evaluated by scanning electron microscopy (SEM) observation. The PCU films after treated with platelet-rich plasma demonstrated that much fewer platelets adhered to the MPC-grafted PCU surface than to the blank PCU surface. The antithrombogenicity of the MPC-grafted PCU surface was determined by the activated partial thromboplastin time (APTT). The result suggested that the MPC modified PCU may have potential application as biomaterials in blood-contacting and some subcutaneously implanted devices. - Highlights: • MPC was successfully grafted onto polycarbonate urethane surface via Michael reaction. • High concentration of PC functional groups on the surface via TAEA molecule • Biomimetic surface modification • The modified surface showed high hydrophilicity and anti-platelet adhesion.

  13. Characterization and functional analyses of the human G protein-coupled receptor kinase 4 gene promoter.

    Science.gov (United States)

    Hasenkamp, Sandra; Telgmann, Ralph; Staessen, Jan A; Hagedorn, Claudia; Dördelmann, Corinna; Bek, Martin; Brand-Herrmann, Stefan-Martin; Brand, Eva

    2008-10-01

    The G protein-coupled receptor kinase 4 is involved in renal sodium handling and blood pressure regulation. Missense variants have already been tested functionally and are associated with hypertension, but no data on promoter analyses are yet available. We scanned 94 hypertensive white subjects for genetic variation and performed promoter reporter gene analyses in HEK293T, COS7, and SaOs-2 cells. Transient transfections with various full lengths and wild-type deletion constructs revealed that 1851 bp of the flanking region and 275 bp of the 5'-untranslated region were sufficient for transcriptional activities and composed a powerful cis-active element in the distal 293 bp. The -1702T and +2T alleles resulted in drastic general reductions of promoter function, whereas an activity increasing effect of +268C was cell type specific. Electrophoretic mobility-shift assay, supershift, and cotransfection analyses of transcription factor binding sites predicted in silico (Alibaba2.1/Transfac7) resulted in allele-specific binding patterns of nuclear proteins and identified the participation of CCAAT/enhancer-binding protein transcription factor family members. The G protein-coupled receptor kinase 4 core promoter resides in the first 1851 bp upstream of its transcription start site. The 4 identified genetic variants within this region exert allele-specific impact on both cell type- and stimulation-dependent transcription and may affect the expression balance of renal G protein-coupled receptor kinase 4.

  14. Conformational dynamics of human protein kinase CK2α and its effect on function and inhibition.

    Science.gov (United States)

    Srivastava, Ashutosh; Hirota, Tsuyoshi; Irle, Stephan; Tama, Florence

    2018-03-01

    Protein kinase, casein kinase II (CK2), is ubiquitously expressed and highly conserved protein kinase that shows constitutive activity. It phosphorylates a diverse set of proteins and plays crucial role in several cellular processes. The catalytic subunit of this enzyme (CK2α) shows remarkable flexibility as evidenced in numerous crystal structures determined till now. Here, using analysis of multiple crystal structures and long timescale molecular dynamics simulations, we explore the conformational flexibility of CK2α. The enzyme shows considerably higher flexibility in the solution as compared to that observed in crystal structure ensemble. Multiple conformations of hinge region, located near the active site, were observed during the dynamics. We further observed that among these multiple conformations, the most populated conformational state was inadequately represented in the crystal structure ensemble. The catalytic spine, was found to be less dismantled in this state as compared to the "open" hinge/αD state crystal structures. The comparison of dynamics in unbound (Apo) state and inhibitor (CX4945) bound state exhibits inhibitor induced suppression in the overall dynamics of the enzyme. This is especially true for functionally important glycine-rich loop above the active site. Together, this work gives novel insights into the dynamics of CK2α in solution and relates it to the function. This work also explains the effect of inhibitor on the dynamics of CK2α and paves way for development of better inhibitors. © 2017 Wiley Periodicals, Inc.

  15. The systematic functional analysis of plasmodium protein kinases identifies essential regulators of mosquito transmission

    KAUST Repository

    Tewari, Rita

    2010-10-21

    Although eukaryotic protein kinases (ePKs) contribute to many cellular processes, only three Plasmodium falciparum ePKs have thus far been identified as essential for parasite asexual blood stage development. To identify pathways essential for parasite transmission between their mammalian host and mosquito vector, we undertook a systematic functional analysis of ePKs in the genetically tractable rodent parasite Plasmodium berghei. Modeling domain signatures of conventional ePKs identified 66 putative Plasmodium ePKs. Kinomes are highly conserved between Plasmodium species. Using reverse genetics, we show that 23 ePKs are redundant for asexual erythrocytic parasite development in mice. Phenotyping mutants at four life cycle stages in Anopheles stephensi mosquitoes revealed functional clusters of kinases required for sexual development and sporogony. Roles for a putative SR protein kinase (SRPK) in microgamete formation, a conserved regulator of clathrin uncoating (GAK) in ookinete formation, and a likely regulator of energy metabolism (SNF1/KIN) in sporozoite development were identified. 2010 Elsevier Inc.

  16. GTP binding to the ROC domain of DAP-kinase regulates its function through intramolecular signalling

    Science.gov (United States)

    Carlessi, Rodrigo; Levin-Salomon, Vered; Ciprut, Sara; Bialik, Shani; Berissi, Hanna; Albeck, Shira; Peleg, Yoav; Kimchi, Adi

    2011-01-01

    Death-associated protein kinase (DAPk) was recently suggested by sequence homology to be a member of the ROCO family of proteins. Here, we show that DAPk has a functional ROC (Ras of complex proteins) domain that mediates homo-oligomerization and GTP binding through a defined P-loop motif. Upon binding to GTP, the ROC domain negatively regulates the catalytic activity of DAPk and its cellular effects. Mechanistically, GTP binding enhances an inhibitory autophosphorylation at a distal site that suppresses kinase activity. This study presents a new mechanism of intramolecular signal transduction, by which GTP binding operates in cis to affect the catalytic activity of a distal domain in the protein. PMID:21738225

  17. Calcium is the switch in the moonlighting dual function of the ligand-activated receptor kinase phytosulfokine receptor 1

    KAUST Repository

    Muleya, Victor

    2014-09-23

    Background: A number of receptor kinases contain guanylate cyclase (GC) catalytic centres encapsulated in the cytosolic kinase domain. A prototypical example is the phytosulfokine receptor 1 (PSKR1) that is involved in regulating growth responses in plants. PSKR1 contains both kinase and GC activities however the underlying mechanisms regulating the dual functions have remained elusive. Findings: Here, we confirm the dual activity of the cytoplasmic domain of the PSKR1 receptor. We show that mutations within the guanylate cyclase centre modulate the GC activity while not affecting the kinase catalytic activity. Using physiologically relevant Ca2+ levels, we demonstrate that its GC activity is enhanced over two-fold by Ca2+ in a concentration-dependent manner. Conversely, increasing Ca2+ levels inhibits kinase activity up to 500-fold at 100 nM Ca2+. Conclusions: Changes in calcium at physiological levels can regulate the kinase and GC activities of PSKR1. We therefore propose a functional model of how calcium acts as a bimodal switch between kinase and GC activity in PSKR1 that could be relevant to other members of this novel class of ligand-activated receptor kinases.

  18. Use of integrin-linked kinase to extend function of encapsulated pancreatic tissue

    Energy Technology Data Exchange (ETDEWEB)

    Blanchette, James O [Department of Chemical Engineering, University of South Carolina, Columbia, SC (United States); Langer, Steven J; Leinwand, Leslie L [Department of Molecular, Cellular and Developmental Biology, University of Colorado, Boulder, CO (United States); Sahai, Suchit; Topiwala, Pritesh S [Biomedical Engineering Program, University of South Carolina, Columbia, SC (United States); Anseth, Kristi S, E-mail: blanchej@cec.sc.ed [Howard Hughes Medical Institute, Boulder, CO (United States)

    2010-12-15

    We have studied the impact of overexpression of an intracellular signaling protein, integrin-linked kinase (ILK), on the survival and function of encapsulated islet tissue used for the treatment of type 1 diabetes. The dimensions of the encapsulated tissue can impact the stresses placed on the tissue and ILK overexpression shows the ability to extend function of dissociated cells as well as intact islets. These results suggest that lost cell-extracellular matrix interactions in cell encapsulation systems can lead to decreased insulin secretion and ILK signaling is a target to overcome this phenomenon. (communication)

  19. Selective adhesion of intestinal epithelial cells on patterned films with amine functionalities formed by plasma enhanced chemical vapor deposition

    Energy Technology Data Exchange (ETDEWEB)

    Kim, Kyung Seop; Choi, Changrok; Kim, Soo Heon; Choi, Kun oh [Department of Physics, Brain Korea 21 Physics Research Division and Institute of Basic Science, Sungkyunkwan University, Suwon 440-746 (Korea, Republic of); Kim, Jeong Min [Department of Molecular Biology and Institute of Nanosensor and Biotechnology, BK21 Graduate Program for RNA Biology, Dankook University, Yongin 448-701 (Korea, Republic of); Kim, Hong Ja [Department of Internal Medicine, Dankook University College of Medicine, Cheonan 330-715 (Korea, Republic of); Yeo, Sanghak [R and D Center, ELBIO Incorporation, 426-5 Gasan-dong Geumchun-gu, Seoul (Korea, Republic of); Park, Heonyong [Department of Molecular Biology and Institute of Nanosensor and Biotechnology, BK21 Graduate Program for RNA Biology, Dankook University, Yongin 448-701 (Korea, Republic of); Jung, Donggeun, E-mail: djung@skku.ac.kr [Department of Physics, Brain Korea 21 Physics Research Division and Institute of Basic Science, Sungkyunkwan University, Suwon 440-746 (Korea, Republic of)

    2010-11-01

    Control of cell adhesion to surfaces is important to develop analytical tools in the areas of biomedical engineering. To control cell adhesiveness of the surface, we constructed a variety of plasma polymerized hexamethyldisiloxane (PPHMDSO) thin films deposited at the plasma power range of 10-100 W by plasma enhanced chemical vapor deposition (PECVD). The PPHMDSO film that was formed at 10 W was revealed to be resistant to cell adhesion. The resistance to cell adhesion is closely related to physicochemical properties of the film. Atomic force microscopic data show an increase in surface roughness from 0.52 nm to 0.74 nm with increasing plasma power. From Fourier transform infrared (FT-IR) absorption spectroscopy data, it was also determined that the methyl (-CH{sub 3}) peak intensity increases with increasing plasma power, whereas the hydroxyl (-OH) peak decreases. X-ray photoelectron spectroscopy data reveal an increase in C-O bonding with increasing plasma power. These results suggest that C-O bonding and hydroxyl (-OH) and methyl (-CH{sub 3}) functional groups play a critical part in cell adhesion. Furthermore, to enhance a diversity of film surface, we accumulated the patterned plasma polymerized ethylenediamine (PPEDA) thin film on the top of the PPHMDSO thin film. The PPEDA film is established to be strongly cell-adherent. This patterned two-layer film stacking method can be used to form the selectively limited cell-adhesive PPEDA spots over the adhesion-resistant surface.

  20. Venus Kinase Receptors: prospects in signalling and biological functions of these invertebrate receptors

    Directory of Open Access Journals (Sweden)

    Colette eDissous

    2014-05-01

    Full Text Available Venus Kinase Receptors (VKRs form a family of invertebrate receptor tyrosine kinases (RTKs initially discovered in the parasitic platyhelminth Schistosoma mansoni. VKRs are single transmembrane receptors which contain an extracellular Venus Flytrap (VFT structure similar to the ligand binding domain of G Protein Coupled Receptors of class C, and an intracellular Tyrosine Kinase domain close to that of Insulin Receptors. VKRs are found in a large variety of invertebrates from cnidarians to echinoderms, and are highly expressed in larval stages and in gonads, suggesting a role of these proteins in embryonic and larval development as well as in reproduction. Vkr gene silencing could demonstrate the function of these receptors in oogenesis as well as in spermatogenesis in Schistosoma .mansoni. VKRs are activated by amino-acids, and highly responsive to arginine. As many other RTKs, they form dimers when activated by ligands and induce intracellular pathways involved in protein synthesis and cellular growth, such as MAPK and PI3K/Akt/S6K pathways. VKRs are not present in vertebrates, nor in some invertebrate species. Questions remain open about the origin of this little-known RTK family in evolution and its role in emergence and specialization of Metazoa. What is the meaning of maintenance or loss of VKR in some phyla or species in terms of development and physiological functions? The presence of VKRs in invertebrates of economical and medical importance, such as pests, vectors of pathogens and platyhelminth parasites, and the implication of these RTKs in gametogenesis and reproduction processes are valuable reasons to consider VKRs as interesting targets in new programs for eradication/ control of pests and infectious diseases, with the main advantage in the case of parasite targeting that VKR counterparts are absent from the vertebrate host kinase panel.

  1. Mechanical stimuli on C2C12 myoblasts affect myoblast differentiation, focal adhesion kinase phosphorylation and galectin-1 expression

    DEFF Research Database (Denmark)

    Grossi, Alberto Blak; Lametsch, Rene; Karlsson, Anders H

    2011-01-01

    Mechanical forces are crucial in the regulation of cell morphology and function. At the cellular level, these forces influence myoblast differentiation and fusion. In this study we applied mechanical stimuli to embryonic muscle cells using magnetic microbeads, a method shown to apply stress...... by mechanical stimulation including Galectin-1, Annexin III, and RhoGDI. In this study we demonstrate how the combination of this method of mechanical stimuli and proteomic analysis can be a powerful tool to detect proteins that are potentially interacting in biochemical pathways or complex cellular mechanisms...... during the process of myoblast differentiation. We determined an increase in expression and changes in cellular localization of Galectin-1, in mechanically stimulated myoblasts. A potential involvement of Galectin-1 in myoblast differentiation is presented....

  2. Advanced adhesives in electronics

    CERN Document Server

    Bailey, C

    2011-01-01

    Adhesives are widely used in the manufacture of electronic devices to act as passive and active components. Recently there has been considerable interest in the use of conductive adhesives. This book reviews key types of conductive adhesives, processing methods, properties and the way they can be modelled as well as potential applications.$bAdhesives for electronic applications serve important functional and structural purposes in electronic components and packaging, and have developed significantly over the last few decades. Advanced adhesives in electronics reviews recent developments in adhesive joining technology, processing and properties. The book opens with an introduction to adhesive joining technology for electronics. Part one goes on to cover different types of adhesive used in electronic systems, including thermally conductive adhesives, isotropic and anisotropic conductive adhesives and underfill adhesives for flip-chip applications. Part two focuses on the properties and processing of electronic ...

  3. Peptide-functionalized poly[oligo(ethylene glycol) methacrylate] brushes on dopamine-coated stainless steel for controlled cell adhesion.

    Science.gov (United States)

    Alas, Guillermo R; Agarwal, Rachit; Collard, David M; García, Andrés J

    2017-09-01

    The modification of the surface of surgical implants with cell adhesion ligands has emerged as a promising approach to improve biomaterial-host interactions. However, these approaches are limited by the non-specific adsorption of biomolecules and uncontrolled presentation of desired bioactive ligands on implant surfaces. This leads to sub-optimal integration with host tissue and delayed healing. Here we present a strategy to grow non-fouling polymer brushes of oligo(ethylene glycol) methacrylate by atom transfer radical polymerization from dopamine-functionalized clinical grade 316 stainless steel. These brushes prevent non-specific adsorption of proteins and attachment of cells. Subsequently, the brushes can be modified with covalently tethered adhesive peptides that provide controlled cell adhesion. This approach may therefore have broad application to promote bone growth and improvements in osseointegration. Stainless steel (SS) implants are widely used clinically for orthopaedic, spinal, dental and cardiovascular applications. However, non-specific adsorption of biomolecules onto implant surfaces results in sub-optimal integration with host tissue. To allow controlled cell-SS interactions, we have developed a strategy to grow non-fouling polymer brushes that prevent protein adsorption and cell adhesion and can be subsequently functionalized with adhesive peptides to direct cell adhesion and signaling. This approach has broad application to improve osseointegration onto stainless steel implants in bone repair. Copyright © 2017 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

  4. Molecular and Functional Characterization of a Trypanosoma cruzi Nuclear Adenylate Kinase Isoform

    Science.gov (United States)

    Cámara, María de los Milagros; Bouvier, León A.; Canepa, Gaspar E.; Miranda, Mariana R.; Pereira, Claudio A.

    2013-01-01

    Trypanosoma cruzi, the etiological agent of Chagas' disease, is an early divergent eukaryote in which control of gene expression relies mainly in post-transcriptional mechanisms. Transcription levels are globally up and down regulated during the transition between proliferating and non-proliferating life-cycle stages. In this work we characterized a nuclear adenylate kinase isoform (TcADKn) that is involved in ribosome biogenesis. Nuclear adenylate kinases have been recently described in a few organisms, being all related to RNA metabolism. Depending on active transcription and translation, TcADKn localizes in the nucleolus or the cytoplasm. A non-canonical nuclear localization signal was mapped towards the N-terminal of the protein, being the phosphate-binding loop essential for its localization. In addition, TcADKn nuclear exportation depends on the nuclear exportation adapter CRM1. TcADKn nuclear shuttling is governed by nutrient availability, oxidative stress and by the equivalent in T. cruzi of the mammalian TOR (Target of Rapamycin) pathway. One of the biological functions of TcADKn is ribosomal 18S RNA processing by direct interaction with ribosomal protein TcRps14. Finally, TcADKn expression is regulated by its 3′ UTR mRNA. Depending on extracellular conditions, cells modulate protein translation rates regulating ribosome biogenesis and nuclear adenylate kinases are probably key components in these processes. PMID:23409202

  5. RET Functions as a Dual-Specificity Kinase that Requires Allosteric Inputs from Juxtamembrane Elements

    Directory of Open Access Journals (Sweden)

    Iván Plaza-Menacho

    2016-12-01

    Full Text Available Receptor tyrosine kinases exhibit a variety of activation mechanisms despite highly homologous catalytic domains. Such diversity arises through coupling of extracellular ligand-binding portions with highly variable intracellular sequences flanking the tyrosine kinase domain and specific patterns of autophosphorylation sites. Here, we show that the juxtamembrane (JM segment enhances RET catalytic domain activity through Y687. This phospho-site is also required by the JM region to rescue an otherwise catalytically deficient RET activation-loop mutant lacking tyrosines. Structure-function analyses identified interactions between the JM hinge, αC helix, and an unconventional activation-loop serine phosphorylation site that engages the HRD motif and promotes phospho-tyrosine conformational accessibility and regulatory spine assembly. We demonstrate that this phospho-S909 arises from an intrinsic RET dual-specificity kinase activity and show that an equivalent serine is required for RET signaling in Drosophila. Our findings reveal dual-specificity and allosteric components for the mechanism of RET activation and signaling with direct implications for drug discovery.

  6. Circulating Cellular Adhesion Molecules and Cognitive Function: The Coronary Artery Risk Development in Young Adults Study.

    Science.gov (United States)

    Yoon, Cynthia Yursun; Steffen, Lyn M; Gross, Myron D; Launer, Lenore J; Odegaard, Andrew; Reiner, Alexander; Sanchez, Otto; Yaffe, Kristine; Sidney, Stephen; Jacobs, David R

    2017-01-01

    Higher circulating concentrations of cellular adhesion molecules (CAMs) can be used as markers of endothelial dysfunction. Given that the brain is highly vascularized, we assessed whether endothelial function is associated with cognitive performance. Within the Coronary Artery Risk Development in Young Adults (CARDIA) Study, excluding N  = 54 with stroke before year 25, we studied CAMs among N  = 2,690 black and white men and women in CARDIA year 7 (1992-1993, ages 25-37) and N  = 2,848 in CARDIA year 15 (2000-2001, ages 33-45). We included subjects with levels of circulating soluble CAMs measured in year 7 or 15 and cognitive function testing in year 25 (2010-2011, ages 43-55). Using multiple regression analysis, we evaluated the association between CAMs and year 25 cognitive test scores: Rey Auditory Verbal Learning Test (RAVLT, memory), Digit Symbol Substitution Test (DSST, speed of processing), and the Stroop Test (executive function). All CAM concentrations were greater in year 15 vs. year 7. Adjusting for age, race, sex, education, smoking, alcohol, diet, physical activity, participants in the fourth vs. the first quartile of CARDIA year 7 of circulating intercellular adhesion molecule-1 (ICAM-1) scored worse on RAVLT, DSST, and Stroop Test ( p  ≤ 0.05) in CARDIA year 25. Other CAMs showed little association with cognitive test scores. Findings were similar for ICAM-1 assessed at year 15. Adjustment for possibly mediating physical factors attenuated the findings. Higher circulating ICAM-1 at average ages 32 and 40 was associated with lower cognitive skills at average age 50. The study is consistent with the hypothesis that endothelial dysfunction is associated with worse short-term memory, speed of processing, and executive function.

  7. Circulating Cellular Adhesion Molecules and Cognitive Function: The Coronary Artery Risk Development in Young Adults Study

    Directory of Open Access Journals (Sweden)

    Cynthia Yursun Yoon

    2017-05-01

    Full Text Available ObjectiveHigher circulating concentrations of cellular adhesion molecules (CAMs can be used as markers of endothelial dysfunction. Given that the brain is highly vascularized, we assessed whether endothelial function is associated with cognitive performance.MethodWithin the Coronary Artery Risk Development in Young Adults (CARDIA Study, excluding N = 54 with stroke before year 25, we studied CAMs among N = 2,690 black and white men and women in CARDIA year 7 (1992–1993, ages 25–37 and N = 2,848 in CARDIA year 15 (2000–2001, ages 33–45. We included subjects with levels of circulating soluble CAMs measured in year 7 or 15 and cognitive function testing in year 25 (2010–2011, ages 43–55. Using multiple regression analysis, we evaluated the association between CAMs and year 25 cognitive test scores: Rey Auditory Verbal Learning Test (RAVLT, memory, Digit Symbol Substitution Test (DSST, speed of processing, and the Stroop Test (executive function.ResultAll CAM concentrations were greater in year 15 vs. year 7. Adjusting for age, race, sex, education, smoking, alcohol, diet, physical activity, participants in the fourth vs. the first quartile of CARDIA year 7 of circulating intercellular adhesion molecule-1 (ICAM-1 scored worse on RAVLT, DSST, and Stroop Test (p ≤ 0.05 in CARDIA year 25. Other CAMs showed little association with cognitive test scores. Findings were similar for ICAM-1 assessed at year 15. Adjustment for possibly mediating physical factors attenuated the findings.ConclusionHigher circulating ICAM-1 at average ages 32 and 40 was associated with lower cognitive skills at average age 50. The study is consistent with the hypothesis that endothelial dysfunction is associated with worse short-term memory, speed of processing, and executive function.

  8. Structural and Functional Characterization of the Protein Kinase Mps1 in Arabidopsis thaliana

    Science.gov (United States)

    de Oliveira, Eduardo Alves Gamosa; Romeiro, Nelilma Correia; Ribeiro, Elane da Silva; Santa-Catarina, Claudete; Oliveira, Antônia Elenir Amâncio; Silveira, Vanildo; de Souza Filho, Gonçalo Apolinário; Venancio, Thiago Motta; Cruz, Marco Antônio Lopes

    2012-01-01

    In eukaryotes, protein kinases catalyze the transfer of a gamma-phosphate from ATP (or GTP) to specific amino acids in protein targets. In plants, protein kinases have been shown to participate in signaling cascades driving responses to environmental stimuli and developmental processes. Plant meristems are undifferentiated tissues that provide the major source of cells that will form organs throughout development. However, non-dividing specialized cells can also dedifferentiate and re-initiate cell division if exposed to appropriate conditions. Mps1 (Monopolar spindle) is a dual-specificity protein kinase that plays a critical role in monitoring the accuracy of chromosome segregation in the mitotic checkpoint mechanism. Although Mps1 functions have been clearly demonstrated in animals and fungi, its role in plants is so far unclear. Here, using structural and biochemical analyses here we show that Mps1 has highly similar homologs in many plant genomes across distinct lineages (e.g. AtMps1 in Arabidopsis thaliana). Several structural features (i.e. catalytic site, DFG motif and threonine triad) are clearly conserved in plant Mps1 kinases. Structural and sequence analysis also suggest that AtMps1 interact with other cell cycle proteins, such as Mad2 and MAPK1. By using a very specific Mps1 inhibitor (SP600125) we show that compromised AtMps1 activity hampers the development of A. thaliana seedlings in a dose-dependent manner, especially in secondary roots. Moreover, concomitant administration of the auxin IAA neutralizes the AtMps1 inhibition phenotype, allowing secondary root development. These observations let us to hypothesize that AtMps1 might be a downstream regulator of IAA signaling in the formation of secondary roots. Our results indicate that Mps1 might be a universal component of the Spindle Assembly Checkpoint machinery across very distant lineages of eukaryotes. PMID:23049844

  9. Protein Kinase C Inhibitors as Modulators of Vascular Function and Their Application in Vascular Disease

    Directory of Open Access Journals (Sweden)

    Raouf A. Khalil

    2013-03-01

    Full Text Available Blood pressure (BP is regulated by multiple neuronal, hormonal, renal and vascular control mechanisms. Changes in signaling mechanisms in the endothelium, vascular smooth muscle (VSM and extracellular matrix cause alterations in vascular tone and blood vessel remodeling and may lead to persistent increases in vascular resistance and hypertension (HTN. In VSM, activation of surface receptors by vasoconstrictor stimuli causes an increase in intracellular free Ca2+ concentration ([Ca2+]i, which forms a complex with calmodulin, activates myosin light chain (MLC kinase and leads to MLC phosphorylation, actin-myosin interaction and VSM contraction. Vasoconstrictor agonists could also increase the production of diacylglycerol which activates protein kinase C (PKC. PKC is a family of Ca2+-dependent and Ca2+-independent isozymes that have different distributions in various blood vessels, and undergo translocation from the cytosol to the plasma membrane, cytoskeleton or the nucleus during cell activation. In VSM, PKC translocation to the cell surface may trigger a cascade of biochemical events leading to activation of mitogen-activated protein kinase (MAPK and MAPK kinase (MEK, a pathway that ultimately increases the myofilament force sensitivity to [Ca2+]i, and enhances actin-myosin interaction and VSM contraction. PKC translocation to the nucleus may induce transactivation of various genes and promote VSM growth and proliferation. PKC could also affect endothelium-derived relaxing and contracting factors as well as matrix metalloproteinases (MMPs in the extracellular matrix further affecting vascular reactivity and remodeling. In addition to vasoactive factors, reactive oxygen species, inflammatory cytokines and other metabolic factors could affect PKC activity. Increased PKC expression and activity have been observed in vascular disease and in certain forms of experimental and human HTN. Targeting of vascular PKC using PKC inhibitors may function in

  10. The oncogenic tyrosine kinase Lyn impairs the pro-apoptotic function of Bim.

    Science.gov (United States)

    Aira, Lazaro E; Villa, Elodie; Colosetti, Pascal; Gamas, Parvati; Signetti, Laurie; Obba, Sandrine; Proics, Emma; Gautier, Fabien; Bailly-Maitre, Béatrice; Jacquel, Arnaud; Robert, Guillaume; Luciano, Frédéric; Juin, Philippe P; Ricci, Jean-Ehrland; Auberger, Patrick; Marchetti, Sandrine

    2018-02-02

    Phosphorylation of Ser/Thr residues is a well-established modulating mechanism of the pro-apoptotic function of the BH3-only protein Bim. However, nothing is known about the putative tyrosine phosphorylation of this Bcl-2 family member and its potential impact on Bim function and subsequent Bax/Bak-mediated cytochrome c release and apoptosis. As we have previously shown that the tyrosine kinase Lyn could behave as an anti-apoptotic molecule, we investigated whether this Src family member could directly regulate the pro-apoptotic function of Bim. In the present study, we show that Bim is phosphorylated onto tyrosine residues 92 and 161 by Lyn, which results in an inhibition of its pro-apoptotic function. Mechanistically, we show that Lyn-dependent tyrosine phosphorylation of Bim increases its interaction with anti-apoptotic members such as Bcl-xL, therefore limiting mitochondrial outer membrane permeabilization and subsequent apoptosis. Collectively, our data uncover one molecular mechanism through which the oncogenic tyrosine kinase Lyn negatively regulates the mitochondrial apoptotic pathway, which may contribute to the transformation and/or the chemotherapeutic resistance of cancer cells.

  11. Protein kinase Gin4 negatively regulates flippase function and controls plasma membrane asymmetry

    Science.gov (United States)

    Roelants, Françoise M.; Su, Brooke M.; von Wulffen, Joachim; Ramachandran, Subramaniam; Sartorel, Elodie; Trott, Amy E.

    2015-01-01

    Plasma membrane function requires distinct leaflet lipid compositions. Two of the P-type ATPases (flippases) in yeast, Dnf1 and Dnf2, translocate aminoglycerophospholipids from the outer to the inner leaflet, stimulated via phosphorylation by cortically localized protein kinase Fpk1. By monitoring Fpk1 activity in vivo, we found that Fpk1 was hyperactive in cells lacking Gin4, a protein kinase previously implicated in septin collar assembly. Gin4 colocalized with Fpk1 at the cortical site of future bud emergence and phosphorylated Fpk1 at multiple sites, which we mapped. As judged by biochemical and phenotypic criteria, a mutant (Fpk111A), in which 11 sites were mutated to Ala, was hyperactive, causing increased inward transport of phosphatidylethanolamine. Thus, Gin4 is a negative regulator of Fpk1 and therefore an indirect negative regulator of flippase function. Moreover, we found that decreasing flippase function rescued the growth deficiency of four different cytokinesis mutants, which suggests that the primary function of Gin4 is highly localized control of membrane lipid asymmetry and is necessary for optimal cytokinesis. PMID:25646086

  12. Investigating the effects of membrane deformability on artificial capsule adhesion to the functionalized surface.

    Science.gov (United States)

    Balsara, Hiren D; Banton, Rohan J; Eggleton, Charles D

    2016-10-01

    Understanding, manipulating and controlling cellular adhesion processes can be critical in developing biomedical technologies. Adhesive mechanisms can be used to the target, pattern and separate cells such as leukocytes from whole blood for biomedical applications. The deformability response of the cell directly affects the rolling and adhesion behavior under viscous linear shear flow conditions. To that end, the primary objective of the present study was to investigate numerically the influence of capsule membrane's nonlinear material behavior (i.e. elastic-plastic to strain hardening) on the rolling and adhesion behavior of representative artificial capsules. Specifically, spherical capsules with radius of [Formula: see text] were represented using an elastic membrane governed by a Mooney-Rivlin strain energy functions. The surfaces of the capsules were coated with P-selectin glycoprotein-ligand-1 to initiate binding interaction with P-selectin-coated planar surface with density of [Formula: see text] under linear shear flow varying from 100 to [Formula: see text]. The numerical model is based on the Immersed Boundary Method for rolling of deformable capsule in shear flow coupled with Monte Carlo simulation for receptor/ligand interaction modeled using Bell model. The results reveal that the mechanical properties of the capsule play an important role in the rolling behavior and the binding kinetics between the capsule contact surface and the substrate. The rolling behavior of the strain hardening capsules is relatively smoother and slower compared to the elastic-plastic capsules. The strain hardening capsules exhibits higher contact area at any given shear rate compared to elastic-plastic capsules. The increase in contact area leads to decrease in rolling velocity. The capsule contact surface is not in complete contact with the substrate because of thin lubrication film that is trapped between the capsule and substrate. This creates a concave shape on the bottom

  13. Structural and functional insights into the interaction between the Cas family scaffolding protein p130Cas and the focal adhesion-associated protein paxillin

    Energy Technology Data Exchange (ETDEWEB)

    Zhang, Chi; Miller, Darcie J.; Guibao, Cristina D.; Donato, Dominique M.; Hanks, Steven K.; Zheng, Jie J.

    2017-08-31

    The Cas family scaffolding protein p130Cas is a Src substrate localized in focal adhesions (FAs) and functions in integrin signaling to promote cell motility, invasion, proliferation, and survival. p130Cas targeting to FAs is essential for its tyrosine phosphorylation and downstream signaling. Although the N-terminal SH3 domain is important for p130Cas localization, it has also been reported that the C-terminal region is involved in p130Cas FA targeting. The C-terminal region of p130Cas or Cas family homology domain (CCHD) has been reported to adopt a structure similar to that of the focal adhesion kinase C-terminal focal adhesion-targeting domain. The mechanism by which the CCHD promotes FA targeting of p130Cas, however, remains unclear. In this study, using a calorimetry approach, we identified the first LD motif (LD1) of the FA-associated protein paxillin as the binding partner of the p130Cas CCHD (in a 1:1 stoichiometry with a Kd ~4.2 μM) and elucidated the structure of the p130Cas CCHD in complex with the paxillin LD1 motif by X-ray crystallography. Of note, a comparison of the CCHD/LD1 complex with a previously solved structure of CCHD in complex with the SH2-containing protein NSP3 revealed that LD1 had almost identical positioning of key hydrophobic and acidic residues relative to NSP3. Because paxillin is one of the key scaffold molecules in FAs, we propose that the interaction between the p130Cas CCHD and the LD1 motif of paxillin plays an important role in p130Cas FA targeting.

  14. Dark chocolate consumption improves leukocyte adhesion factors and vascular function in overweight men.

    Science.gov (United States)

    Esser, Diederik; Mars, Monica; Oosterink, Els; Stalmach, Angelique; Müller, Michael; Afman, Lydia A

    2014-03-01

    Flavanol-enriched chocolate consumption increases endothelium-dependent vasodilation. Most research so far has focused on flow-mediated dilation (FMD) only; the effects on other factors relevant to endothelial health, such as inflammation and leukocyte adhesion, have hardly been addressed. We investigated whether consumption of regular dark chocolate also affects other markers of endothelial health, and whether chocolate enrichment with flavanols has additional benefits. In a randomized double-blind crossover study, the effects of acute and of 4 wk daily consumption of high flavanol chocolate (HFC) and normal flavanol chocolate (NFC) on FMD, augmentation index (AIX), leukocyte count, plasma cytokines, and leukocyte cell surface molecules in overweight men (age 45-70 yr) were investigated. Sensory profiles and motivation scores to eat chocolate were also collected. Findings showed that a 4 wk chocolate intake increased FMD by 1%, which was paralleled by a decreased AIX of 1%, decreased leukocyte cell count, decreased plasma sICAM1 and sICAM3, and decreased leukocyte adhesion marker expression (Pchocolate. This study provides new insights on how chocolate affects endothelial health by demonstrating that chocolate consumption, besides improving vascular function, also lowers the adherence capacity of leukocytes in the circulation.

  15. Small Molecule Agonists of Cell Adhesion Molecule L1 Mimic L1 Functions In Vivo.

    Science.gov (United States)

    Kataria, Hardeep; Lutz, David; Chaudhary, Harshita; Schachner, Melitta; Loers, Gabriele

    2016-09-01

    Lack of permissive mechanisms and abundance of inhibitory molecules in the lesioned central nervous system of adult mammals contribute to the failure of functional recovery after injury, leading to severe disabilities in motor functions and pain. Peripheral nerve injury impairs motor, sensory, and autonomic functions, particularly in cases where nerve gaps are large and chronic nerve injury ensues. Previous studies have indicated that the neural cell adhesion molecule L1 constitutes a viable target to promote regeneration after acute injury. We screened libraries of known drugs for small molecule agonists of L1 and evaluated the effect of hit compounds in cell-based assays in vitro and in mice after femoral nerve and spinal cord injuries in vivo. We identified eight small molecule L1 agonists and showed in cell-based assays that they stimulate neuronal survival, neuronal migration, and neurite outgrowth and enhance Schwann cell proliferation and migration and myelination of neurons in an L1-dependent manner. In a femoral nerve injury mouse model, enhanced functional regeneration and remyelination after application of the L1 agonists were observed. In a spinal cord injury mouse model, L1 agonists improved recovery of motor functions, being paralleled by enhanced remyelination, neuronal survival, and monoaminergic innervation, reduced astrogliosis, and activation of microglia. Together, these findings suggest that application of small organic compounds that bind to L1 and stimulate the beneficial homophilic L1 functions may prove to be a valuable addition to treatments of nervous system injuries.

  16. The Rho kinases I and II regulate different aspects of myosin II activity

    DEFF Research Database (Denmark)

    Yoneda, Atsuko; Multhaupt, Hinke A B; Couchman, John R

    2005-01-01

    The homologous mammalian rho kinases (ROCK I and II) are assumed to be functionally redundant, based largely on kinase construct overexpression. As downstream effectors of Rho GTPases, their major substrates are myosin light chain and myosin phosphatase. Both kinases are implicated in microfilament...... bundle assembly and smooth muscle contractility. Here, analysis of fibroblast adhesion to fibronectin revealed that although ROCK II was more abundant, its activity was always lower than ROCK I. Specific reduction of ROCK I by siRNA resulted in loss of stress fibers and focal adhesions, despite...

  17. Separation of the gluconeogenic and mitochondrial functions of pgc-1α through s6 kinase

    DEFF Research Database (Denmark)

    Lustig, Y.; Ruas, J.L.; Estall, J.L.

    2011-01-01

    and gluconeogenesis and fatty acid oxidation in the liver. We show here that S6 kinase, activated in the liver upon feeding, can phosphorylate PGC-1α directly on two sites within its arginine/serine-rich (RS) domain. This phosphorylation significantly attenuates the ability of PGC-1α to turn on genes...... of gluconeogenesis in cultured hepatocytes and in vivo, while leaving the functions of PGC-1α as an activator of mitochondrial and fatty acid oxidation genes completely intact. These phosphorylations interfere with the ability of PGC-1α to bind to HNF4α, a transcription factor required for gluconeogenesis, while...

  18. Diacylglycerol Kinases: Regulated Controllers of T Cell Activation, Function, and Development

    Directory of Open Access Journals (Sweden)

    Gary A. Koretzky

    2013-03-01

    Full Text Available Diacylglycerol kinases (DGKs are a diverse family of enzymes that catalyze the conversion of diacylglycerol (DAG, a crucial second messenger of receptor-mediated signaling, to phosphatidic acid (PA. Both DAG and PA are bioactive molecules that regulate a wide set of intracellular signaling proteins involved in innate and adaptive immunity. Clear evidence points to a critical role for DGKs in modulating T cell activation, function, and development. More recently, studies have elucidated factors that control DGK function, suggesting an added complexity to how DGKs act during signaling. This review summarizes the available knowledge of the function and regulation of DGK isoforms in signal transduction with a particular focus on T lymphocytes.

  19. Functional roles of mannose-binding protein in the adhesion, cytotoxicity and phagocytosis of Acanthamoeba castellanii.

    Science.gov (United States)

    Kim, Jong-Hyun; Matin, Abdul; Shin, Ho-Joon; Park, Hyun; Yoo, Kyung-Tae; Yuan, Xi-Zhe; Kim, Kwang Sik; Jung, Suk-Yul

    2012-10-01

    Acanthamoeba castellanii is a single-celled protozoan that is widely distributed in the environment and is a well-known of causing human keratitis, a vision-threatening infection. In this study, an ethyl methane sulfonate (EMS) and a selection of saccharide were applied to A. castellanii by chemical mutagenesis. To understand the functional roles of a mannose-binding protein (MBP). A. castellanii were treated with methyl-alpha-D-mannopyranoside abbreviated Man, with and without the EMS pre-treatment, and their adhesion and cytotoxicity were analyzed, using a human brain microvascular endothelial cell (HBMEC) as the target cell. Both EMS and Man mutants exhibited significantly decreased levels of MBP expression and cytotoxicity to HBMEC, but showed similar levels of binding to HBMEC, as compared with the wild type. Of interest was that the exogenous mannose inhibited amoebae (i.e., Man mutant) binding to the HBMEC by castellanii. Copyright © 2012 Elsevier Inc. All rights reserved.

  20. Src Family Kinases Modulate the Loss of Endothelial Barrier Function in Response to TNF-α: Crosstalk with p38 Signaling.

    Directory of Open Access Journals (Sweden)

    Alejandro P Adam

    Full Text Available Activation of Src Family Kinase (SFK signaling is required for the increase in endothelial permeability induced by a variety of cytokines and growth factors. However, we previously demonstrated that activation of endogenous SFKs by expression of dominant negative C-terminal Src Kinase (DN-Csk is not sufficient to decrease endothelial adherens junction integrity. Basal SFK activity has been observed in normal venular endothelia and was not associated with increased basal permeability. The basal SFK activity however was found to contribute to increased sensitivity of the venular endothelium to inflammatory mediator-induced leakage. How SFK activation achieves this is still not well understood. Here, we show that SFK activation renders human dermal microvascular endothelial cells susceptible to low doses of TNF-α. Treatment of DN-Csk-expressing cells with 50 pg/ml TNF-α induced a loss of TEER as well as drastic changes in the actin cytoskeleton and focal adhesion proteins. This synergistic effect was independent of ROCK or NF-κB activity. TNF-α-induced p38 signaling was required for the synergistic effect on barrier function, and activation of the p38 MAPK alone was also able to induce changes in permeability only in monolayers with active SFKs. These results suggest that the activation of endogenous levels of SFK renders the endothelial barrier more susceptible to low, physiologic doses of TNF-α through activation of p38 which leads to a loss of endothelial tight junctions.

  1. Functional characterization of a constitutively active kinase variant of Arabidopsis phototropin 1.

    Science.gov (United States)

    Petersen, Jan; Inoue, Shin-Ichiro; Kelly, Sharon M; Sullivan, Stuart; Kinoshita, Toshinori; Christie, John M

    2017-08-18

    Phototropins (phots) are plasma membrane-associated serine/threonine kinases that coordinate a range of processes linked to optimizing photosynthetic efficiency in plants. These photoreceptors contain two light-, oxygen-, or voltage-sensing (LOV) domains within their N terminus, with each binding one molecule of flavin mononucleotide as a UV/blue light-absorbing chromophore. Although phots contain two LOV domains, light-induced activation of the C-terminal kinase domain and subsequent receptor autophosphorylation is controlled primarily by the A'α-LOV2-Jα photosensory module. Mutations that disrupt interactions between the LOV2 core and its flanking helical segments can uncouple this mode of light regulation. However, the impact of these mutations on phot function in Arabidopsis has not been explored. Here we report that histidine substitution of Arg-472 located within the A'α-helix of Arabidopsis phot1 constitutively activates phot1 kinase activity in vitro without affecting LOV2 photochemistry. Expression analysis of phot1 R472H in the phot-deficient mutant confirmed that it is autophosphorylated in darkness in vivo but unable to initiate phot1 signaling in the absence of light. Instead, we found that phot1 R472H is poorly functional under low-light conditions but can restore phototropism, chloroplast accumulation, stomatal opening, and leaf positioning and expansion at higher light intensities. Our findings suggest that Arabidopsis can adapt to the elevated phosphorylation status of the phot1 R472H mutant in part by reducing its stability, whereas the activity of the mutant under high-light conditions can be attributed to additional increases in LOV2-mediated photoreceptor autophosphorylation. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

  2. Phosphorylation of Staphylococcus aureus Protein-Tyrosine Kinase Affects the Function of Glucokinase and Biofilm Formation.

    Science.gov (United States)

    Vasu, Dudipeta; Kumar, Pasupuleti Santhosh; Prasad, Uppu Venkateswara; Swarupa, Vimjam; Yeswanth, Sthanikam; Srikanth, Lokanathan; Sunitha, Manne Mudhu; Choudhary, Abhijith; Sarma, Potukuchi Venkata Gurunadha Krishna

    2017-03-01

    When Staphylococcus aureus is grown in the presence of high concentration of external glucose, this sugar is phosphorylated by glucokinase (glkA) to form glucose-6-phosphate. This product subsequently enters into anabolic phase, which favors biofilm formation. The presence of ROK (repressor protein, open reading frame, sugar kinase) motif, phosphate-1 and -2 sites, and tyrosine kinase sites in glkA of S. aureus indicates that phosphorylation must regulate the glkA activity. The aim of the present study was to identify the effect of phosphorylation on the function of S. aureus glkA and biofilm formation. Pure glkA and protein-tyrosine kinase (BYK) of S. aureus ATCC 12600 were obtained by fractionating the cytosolic fractions of glkA1 and BYK-1 expressing recombinant clones through nickel metal chelate column. The pure glkA was used as a substrate for BYK and the phosphorylation of glkA was confirmed by treating with reagent A and resolving in SDS-PAGE, as well as staining with reagent A. The kinetic parameters of glkA and phosphorylated glkA were determined spectrophotometrically, and in silico tools were used for validation. S. aureus was grown in brain heart infusion broth, which was supplemented with glucose, and then biofilm units were calculated. Fourfold elevated glkA activity was observed upon the phosphorylation by BYK. Protein-protein docking analysis revealed that glkA structure docked close to the adenosine triphosphate-binding site of BYK structure corroborating the kinetic results. Further, S. aureus grown in the presence of elevated glucose concentration exhibited an increase in the rate of biofilm formation. The elevated function of glkA is an essential requirement for increased biofilm units in S. aureus, a key pathogenic factor that helps its survival and spread the infection.

  3. The NIMA Kinase Is Required To Execute Stage-Specific Mitotic Functions after Initiation of Mitosis

    Science.gov (United States)

    Govindaraghavan, Meera; Lad, Alisha A.

    2014-01-01

    The G2-M transition in Aspergillus nidulans requires the NIMA kinase, the founding member of the Nek kinase family. Inactivation of NIMA results in a late G2 arrest, while overexpression of NIMA is sufficient to promote mitotic events independently of cell cycle phase. Endogenously tagged NIMA-GFP has dynamic mitotic localizations appearing first at the spindle pole body and then at nuclear pore complexes before transitioning to within nuclei and the mitotic spindle and back at the spindle pole bodies at mitotic exit, suggesting that it functions sequentially at these locations. Since NIMA is indispensable for mitotic entry, it has been difficult to determine the requirement of NIMA for subaspects of mitosis. We show here that when NIMA is partially inactivated, although mitosis can be initiated, a proportion of cells fail to successfully generate two daughter nuclei. We further define the mitotic defects to show that normal NIMA function is required for the formation of a bipolar spindle, nuclear pore complex disassembly, completion of chromatin segregation, and the normal structural rearrangements of the nuclear envelope required to generate two nuclei from one. In the remaining population of cells that enter mitosis with inadequate NIMA, two daughter nuclei are generated in a manner dependent on the spindle assembly checkpoint, indicating highly penetrant defects in mitotic progression without sufficient NIMA activity. This study shows that NIMA is required not only for mitotic entry but also sequentially for successful completion of stage-specific mitotic events. PMID:24186954

  4. Distinctive expression and functional regulation of the maize (Zea mays L.) TOR kinase ortholog.

    Science.gov (United States)

    Agredano-Moreno, Lourdes Teresa; Reyes de la Cruz, Homero; Martínez-Castilla, León Patricio; Sánchez de Jiménez, Estela

    2007-11-01

    TOR (Target of rapamycin) kinase is a central component of a signal transduction pathway that regulates cellular growth in response to nutrients, mitogens and growth factors in eukaryotes. Knowledge of the TOR pathway in plants is scarce, and reports in agronomical relevant plants are lacking. Previous studies indicate that Arabidopsis thaliana TOR (AtTOR) activity is resistant to rapamycin whereas maize TOR (ZmTOR) is not, suggesting that plants might have different regulation mechanisms for this signal transduction pathway. In the present work maize ZmTOR cDNA was identified and its expression regulation was analyzed during germination on different tissues at various stages of differentiation and by the main ZmTOR regulators. Our results show that ZmTOR contains all functional domains characteristic of metazoan TOR kinase. ZmTOR expression is highly regulated during germination, a critical plant development period, but not on other tissues of contrasting physiological characteristics. Bioinformatic analyses indicated that maize FKBP12 and rapamycin form a functional structure capable of targeting the ZmTOR protein, similar to other non-plant eukaryotes, further supporting its regulation by rapamycin (in contrast with the rapamycin insensitivity of Arabidopsis thaliana) and the conservation of rapamycin regulation through plant evolution.

  5. Selective engagement of G protein coupled receptor kinases (GRKs) encodes distinct functions of biased ligands

    Science.gov (United States)

    Zidar, David A.; Violin, Jonathan D.; Whalen, Erin J.; Lefkowitz, Robert J.

    2009-01-01

    CCL19 and CCL21 are endogenous agonists for the seven-transmembrane receptor CCR7. They are equally active in promoting G protein stimulation and chemotaxis. Yet, we find that they result in striking differences in activation of the G protein-coupled receptor kinase (GRK)/ß-arrestin system. CCL19 leads to robust CCR7 phosphorylation and β-arrestin2 recruitment catalyzed by both GRK3 and GRK6 whereas CCL21 activates GRK6 alone. This differential GRK activation leads to distinct functional consequences. Although each ligand leads to β-arrestin2 recruitment, only CCL19 leads to redistribution of β-arrestin2-GFP into endocytic vesicles and classical receptor desensitization. In contrast, these agonists are both capable of signaling through GRK6 and β-arrestin2 to ERK kinases. Thus, this mechanism for “ligand bias” whereby endogenous agonists activate different GRK isoforms leads to functionally distinct pools of β-arrestin. PMID:19497875

  6. Adjustment of surface chemical and physical properties with functionalized polymers to control cell adhesion

    Science.gov (United States)

    Zhou, Zhaoli

    Cell-surface interaction is crucial in many cellular functions such as movement, growth, differentiation, proliferation and survival. In the present work, we have developed several strategies to design and prepare synthetic polymeric materials with selected cues to control cell attachment. To promote neuronal cell adhesion on the surfaces, biocompatible, non-adhesive PEG-based materials were modified with neurotransmitter acetylcholine functionalities to produce hydrogels with a range of porous structures, swollen states, and mechanical strengths. Mice hippocampal cells cultured on the hydrogels showed differences in number, length of processes and exhibited different survival rates, thereby highlighting the importance of chemical composition and structure in biomaterials. Similar strategies were used to prepare polymer brushes to assess how topographical cues influence neuronal cell behaviors. The brushes were prepared using the "grown from" method through surface-initiated atom transfer radical polymerization (SI-ATRP) reactions and further patterned via UV photolithography. Protein absorption tests and hippocampal neuronal cell culture of the brush patterns showed that both protein and neuronal cells can adhere to the patterns and therefore can be guided by the patterns at certain length scales. We also prepared functional polymers to discourage attachment of undesirable cells on the surfaces. For example, we synthesized PEG-perfluorinated alkyl amphiphilic surfactants to modify polystyrene-block-poly(ethylene-ran-butylene)- block-polyisoprene (SEBI or K3) triblock copolymers for marine antifouling/fouling release surface coatings. Initial results showed that the polymer coated surfaces can facilitate removal of Ulva sporelings on the surfaces. In addition, we prepared both bioactive and dual functional biopassive/bioactive antimicrobial coatings based on SEBI polymers. Incubating the polymer coated surfaces with gram-positive bacteria (S. aureus), gram

  7. TLR signalling affects sperm mitochondrial function and motility via phosphatidylinositol 3-kinase and glycogen synthase kinase-3α.

    Science.gov (United States)

    Zhu, Xingxing; Shi, Dongyan; Li, Xiaoqian; Gong, Weijuan; Wu, Fengjiao; Guo, Xuejiang; Xiao, Hui; Liu, Lixin; Zhou, Hong

    2016-03-01

    Infection in male and female genital tracts can lead to infertility. The underlying mechanisms of this process remain unclear. Toll-like receptors (TLRs) recognize conserved structures and respond to pathogens by initiating signals that activate inflammatory gene transcription. Here, we demonstrate that TLR activation in sperm reduces sperm motility via signalling through myeloid differentiation factor 88 (MyD88), phosphatidylinositol 3-kinase (PI3K), and glycogen synthase kinase (GSK)-3α. Upon TLR activation, phosphorylated forms of PI3K and GSK3α were detected in the mitochondria, and the mitochondrial membrane potential was impaired in sperm. In addition, mitochondrial ATP levels were decreased after TLR agonist stimulation. Furthermore, blocking PI3K or GSK3α activation abrogated these effects and reversed the TLR-induced reduction in sperm motility. These results identify a previously unrecognized TLR signalling pathway that leads to dysfunctional sperm mitochondria, which reduce sperm motility. Our study reveals a novel mechanism by which pathogenic infection affects sperm motility and possibly leads to infertility. Copyright © 2015. Published by Elsevier Inc.

  8. Proteome-wide dataset supporting functional study of tyrosine kinases in breast cancer

    Directory of Open Access Journals (Sweden)

    Nicos Angelopoulos

    2016-06-01

    Full Text Available Tyrosine kinases (TKs play an essential role in regulating various cellular activities and dysregulation of TK signaling contributes to oncogenesis. However, less than half of the TKs have been thoroughly studied. Through a combined use of RNAi and stable isotope labeling with amino acids in cell culture (SILAC-based quantitative proteomics, a global functional proteomic landscape of TKs in breast cancer was recently revealed highlighting a comprehensive and highly integrated signaling network regulated by TKs (Stebbing et al., 2015 [1]. We collate the enormous amount of the proteomic data in an open access platform, providing a valuable resource for studying the function of TKs in cancer and benefiting the science community. Here we present a detailed description related to this study (Stebbing et al., 2015 [1] and the raw data have been deposited to the ProteomeXchange Consortium via the PRIDE partner repository with the identifier http://www.ebi.ac.uk/pride/archive/projects/PXD002065.

  9. Modulation of Tight Junction Structure and Function by Kinases and Phosphatases Targeting Occludin

    Directory of Open Access Journals (Sweden)

    Max Johannes Dörfel

    2012-01-01

    Full Text Available Tight junctions (TJs typically represent the most apical contacts in epithelial and endothelial cell layers where they play an essential role in the separation of extracellular or luminal spaces from underlying tissues in the body. Depending on the protein composition, TJs define the barrier characteristics and in addition maintain cell polarity. Two major families of integral membrane proteins form the typical TJ strand network, the tight junction-associated MARVEL protein (TAMP family members occludin, tricellulin, and MarvelD3 as well as a specific set of claudins. Occludin was the first identified member of these tetraspanins and is now widely accepted as a regulator of TJ assembly and function. Therefore, occludin itself has to be tightly regulated. Phosphorylation of occludin appears to be of central importance in this context. Here we want to summarize current knowledge on the kinases and phosphatases directly modifying occludin, and their role in the regulation of TJ structure, function, and dynamics.

  10. Energetics and Structural Characterization of the large-scale Functional Motion of Adenylate Kinase

    Science.gov (United States)

    Formoso, Elena; Limongelli, Vittorio; Parrinello, Michele

    2015-02-01

    Adenylate Kinase (AK) is a signal transducing protein that regulates cellular energy homeostasis balancing between different conformations. An alteration of its activity can lead to severe pathologies such as heart failure, cancer and neurodegenerative diseases. A comprehensive elucidation of the large-scale conformational motions that rule the functional mechanism of this enzyme is of great value to guide rationally the development of new medications. Here using a metadynamics-based computational protocol we elucidate the thermodynamics and structural properties underlying the AK functional transitions. The free energy estimation of the conformational motions of the enzyme allows characterizing the sequence of events that regulate its action. We reveal the atomistic details of the most relevant enzyme states, identifying residues such as Arg119 and Lys13, which play a key role during the conformational transitions and represent druggable spots to design enzyme inhibitors. Our study offers tools that open new areas of investigation on large-scale motion in proteins.

  11. Roles of acidic functional groups of carbon fiber surfaces in enhancing interfacial adhesion behavior

    International Nuclear Information System (INIS)

    Park, Soo-Jin; Kim, Byung-Joo

    2005-01-01

    The gas phase ozone treatment was used as a method to bind acidic oxygen functional groups on carbon fiber surfaces. The ozone treatment on carbon fibers was varied with the ozone concentration and treatment time. Surface analyses of the carbon fibers before and after treatments were performed by FT-IR, X-ray photoelectron spectrometer (XPS), and dynamic contact angle measurements. Mechanical interfacial properties of the fibers/polymer composites were investigated by using critical stress intensity factor (K IC ) and critical energy release rate (G IC ) measurements. From the results of FT-IR and XPS, it was observed that the oxygen functional groups, such as -OH, O-C=O, C=O, and C-O, were attached on the carbon fiber surfaces after the ozone treatment. The mechanical interfacial properties of the composites also showed higher values than those of untreated composites. Ozone treatment is attributed to the increase of both the acidic functional groups and the degree of adhesion at interfaces between the fibers and polymeric resin in composites

  12. Functionally distinct PI 3-kinase pathways regulate myelination in the peripheral nervous system

    Science.gov (United States)

    Heller, Bradley A.; Ghidinelli, Monica; Voelkl, Jakob; Einheber, Steven; Smith, Ryan; Grund, Ethan; Morahan, Grant; Chandler, David; Kalaydjieva, Luba; Giancotti, Filippo; King, Rosalind H.; Fejes-Toth, Aniko Naray; Fejes-Toth, Gerard; Feltri, Maria Laura; Lang, Florian

    2014-01-01

    The PI 3-kinase (PI 3-K) signaling pathway is essential for Schwann cell myelination. Here we have characterized PI 3-K effectors activated during myelination by probing myelinating cultures and developing nerves with an antibody that recognizes phosphorylated substrates for this pathway. We identified a discrete number of phospho-proteins including the S6 ribosomal protein (S6rp), which is down-regulated at the onset of myelination, and N-myc downstream-regulated gene-1 (NDRG1), which is up-regulated strikingly with myelination. We show that type III Neuregulin1 on the axon is the primary activator of S6rp, an effector of mTORC1. In contrast, laminin-2 in the extracellular matrix (ECM), signaling through the α6β4 integrin and Sgk1 (serum and glucocorticoid-induced kinase 1), drives phosphorylation of NDRG1 in the Cajal bands of the abaxonal compartment. Unexpectedly, mice deficient in α6β4 integrin signaling or Sgk1 exhibit hypermyelination during development. These results identify functionally and spatially distinct PI 3-K pathways: an early, pro-myelinating pathway driven by axonal Neuregulin1 and a later-acting, laminin–integrin-dependent pathway that negatively regulates myelination. PMID:24687281

  13. LIM domains regulate protein kinase C activity: a novel molecular function.

    Science.gov (United States)

    Maturana, Andrés D; Nakagawa, Noritaka; Yoshimoto, Nobuo; Tatematsu, Kenji; Hoshijima, Masahiko; Tanizawa, Katsuyuki; Kuroda, Shun'ichi

    2011-05-01

    Enigma homolog protein 1 (ENH1) acts as a scaffold that selectively associates protein kinases and transcription factors with cytoskeletal elements. ENH1 comprises an N-terminal PDZ domain and three C-terminal LIM domains. Through the LIM domains ENH1 interacts with the N-terminal region of protein kinase C βI (PKCβI). Here, we show that when ENH1 is co-expressed, PKCβI is translocated from the cytoplasm to the plasma membrane in the absence of any other stimulation. Moreover expression of ENH1 markedly increases PKCβI activity in the absence of PKC activators. A similar activation of PKCβI was observed with co-expression of Cypher1 or Enigma, but not other LIM proteins. The region including the three LIM domains of ENH1 (residues 415-591) appears to be sufficient for this PKCβI activation. Finally, interaction with ENH1 also increases the activity of PKCα and PKCγ, whereas it reduces PKCζ activity. These findings provide strong evidence that ENH1 activates conventional PKCs by directly binding through its LIM domains. Thus, LIM domains have a novel molecular function: the regulation of PKC activities in a PKC isoform-specific manner. Copyright © 2011 Elsevier Inc. All rights reserved.

  14. Functional Divergence of Poplar Histidine-Aspartate Kinase HK1 Paralogs in Response to Osmotic Stress

    Directory of Open Access Journals (Sweden)

    François Héricourt

    2016-12-01

    Full Text Available Previous works have shown the existence of protein partnerships belonging to a MultiStep Phosphorelay (MSP in Populus putatively involved in osmosensing. This study is focused on the identification of a histidine-aspartate kinase, HK1b, paralog of HK1a. The characterization of HK1b showed its ability to homo- and hetero-dimerize and to interact with a few Histidine-containing Phosphotransfer (HPt proteins, suggesting a preferential partnership in poplar MSP linked to drought perception. Furthermore, determinants for interaction specificity between HK1a/1b and HPts were studied by mutagenesis analysis, identifying amino acids involved in this specificity. The HK1b expression analysis in different poplar organs revealed its co-expression with three HPts, reinforcing the hypothesis of partnership participation in the MSP in planta. Moreover, HK1b was shown to act as an osmosensor with kinase activity in a functional complementation assay of an osmosensor deficient yeast strain. These results revealed that HK1b showed a different behaviour for canonical phosphorylation of histidine and aspartate residues. These phosphorylation modularities of canonical amino acids could explain the improved osmosensor performances observed in yeast. As conserved duplicates reflect the selective pressures imposed by the environmental requirements on the species, our results emphasize the importance of HK1 gene duplication in poplar adaptation to drought stress.

  15. Functional and Structural Mimicry of Cellular Protein Kinase A Anchoring Proteins by a Viral Oncoprotein.

    Directory of Open Access Journals (Sweden)

    Cason R King

    2016-05-01

    Full Text Available The oncoproteins of the small DNA tumor viruses interact with a plethora of cellular regulators to commandeer control of the infected cell. During infection, adenovirus E1A deregulates cAMP signalling and repurposes it for activation of viral gene expression. We show that E1A structurally and functionally mimics a cellular A-kinase anchoring protein (AKAP. E1A interacts with and relocalizes protein kinase A (PKA to the nucleus, likely to virus replication centres, via an interaction with the regulatory subunits of PKA. Binding to PKA requires the N-terminus of E1A, which bears striking similarity to the amphipathic α-helical domain present in cellular AKAPs. E1A also targets the same docking-dimerization domain of PKA normally bound by cellular AKAPs. In addition, the AKAP like motif within E1A could restore PKA interaction to a cellular AKAP in which its normal interaction motif was deleted. During infection, E1A successfully competes with endogenous cellular AKAPs for PKA interaction. E1A's role as a viral AKAP contributes to viral transcription, protein expression and progeny production. These data establish HAdV E1A as the first known viral AKAP. This represents a unique example of viral subversion of a crucial cellular regulatory pathway via structural mimicry of the PKA interaction domain of cellular AKAPs.

  16. Pathogenesis of RON receptor tyrosine kinase in cancer cells: activation mechanism, functional crosstalk, and signaling addiction.

    Science.gov (United States)

    Wang, Ming-Hai; Zhang, Ruiwen; Zhou, Yong-Qing; Yao, Hang-Ping

    2013-09-01

    The RON receptor tyrosine kinase, a member of the MET proto-oncogene family, is a pathogenic factor implicated in tumor malignancy. Specifically, aberrations in RON signaling result in increased cancer cell growth, survival, invasion, angiogenesis, and drug resistance. Biochemical events such as ligand binding, receptor overexpression, generation of structure-defected variants, and point mutations in the kinase domain contribute to RON signaling activation. Recently, functional crosstalk between RON and signaling proteins such as MET and EFGR has emerged as an additional mechanism for RON activation, which is critical for tumorigenic development. The RON signaling crosstalk acts either as a regulatory feedback loop that strengthens or enhances tumorigenic phenotype of cancer cells or serves as a signaling compensatory pathway providing a growth/survival advantage for cancer cells to escape targeted therapy. Moreover, viral oncoproteins derived from Friend leukemia or Epstein-Barr viruses interact with RON to drive viral oncogenesis. In cancer cells, RON signaling is integrated into cellular signaling network essential for cancer cell growth and survival. These activities provide the molecular basis of targeting RON for cancer treatment. In this review, we will discuss recent data that uncover the mechanisms of RON activation in cancer cells, review evidence of RON signaling crosstalk relevant to cancer malignancy, and emphasize the significance of the RON signaling addiction by cancer cells for tumor therapy. Understanding aberrant RON signaling will not only provide insight into the mechanisms of tumor pathogenesis, but also lead to the development of novel strategies for molecularly targeted cancer treatment.

  17. Functional adhesive surfaces with “gecko” effect: the concept of contact splitting

    NARCIS (Netherlands)

    Kamperman, M.M.G.; Kroner, E.; Campo, del A.; McMeeking, R.M.; Arzt, E.

    2010-01-01

    Nature has developed reversibly adhesive surfaces whose stickiness has attracted much research attention over the last decade. The central lesson from nature is that “patterned” or “fibrillar” surfaces can produce higher adhesion forces to flat and rough substrates than smooth surfaces. This paper

  18. Improved adhesion of Ag NPs to the polyethylene terephthalate surface via atmospheric plasma treatment and surface functionalization

    Energy Technology Data Exchange (ETDEWEB)

    Shen, Tao [Faculty of Materials Science and Engineering, Kunming University of Science and Technology, 253 Xuefu Rd, Kunming, Yunnan, 650093 (China); Liu, Yong [Faculty of Materials Science and Engineering, Kunming University of Science and Technology, 253 Xuefu Rd, Kunming, Yunnan, 650093 (China); Solmont Technology Wuxi Co., Ltd. 228 Linghu Blvd. Tianan Tech Park, A1-602, Xinwu District, Wuxi, Jiangsu 214135 (China); Zhu, Yan, E-mail: zhuyan@kmust.edu.cn [Faculty of Materials Science and Engineering, Kunming University of Science and Technology, 253 Xuefu Rd, Kunming, Yunnan, 650093 (China); Yang, De-Quan, E-mail: dequan.yang@gmail.com [Faculty of Materials Science and Engineering, Kunming University of Science and Technology, 253 Xuefu Rd, Kunming, Yunnan, 650093 (China); Solmont Technology Wuxi Co., Ltd. 228 Linghu Blvd. Tianan Tech Park, A1-602, Xinwu District, Wuxi, Jiangsu 214135 (China); Sacher, Edward [Regroupement Québécois de Matériaux de Pointe, Department of Engineering Physics, École Polytechnique de Montréal, Case Postale 6079, succursale Centre-Ville, Montréal, Québec H3C 3A7 (Canada)

    2017-07-31

    Highlights: • A two-step process has been developed to enhance the adhesion of immobilized Ag NPs to the PET surface. • The method is simple, easy to use and low-cost for mass production. • The increased density of active sites (−OH, −CH=O and COOH) at the PET surface, after plasma treatment, permits increased reaction with 3-aminopropyltriethoxysilane (APTES). • The presence of APTES with high surface density permits −NH{sub 2}-Ag complex formation, increasing the adhesion of the Ag NPs. - Abstract: Ag nanoparticles (NPs) have been widely applied, as important antibacterial materials, on textile and polymer surfaces. However, their adhesion to nonreactive polymer surfaces is generally too weak for many applications. Here, we propose a two-step process, atmospheric plasma treatment followed by a surface chemical modification process, which enhances their adhesion to polyethylene terephthalate (PET) surfaces. We found that, compared to either plasma treatments or surface chemical functionalizations, alone, this combination greatly enhanced their adhesion. The plasma treatment resulted in an increase of active sites (−OH, −CH=O and COOH) at the PET surface, permitting increased bonding to 3-aminopropyltriethoxysilane (APTES), whose −NH{sub 2} groups were then able to form a bonding complex with the Ag NPs.

  19. Improved adhesion of Ag NPs to the polyethylene terephthalate surface via atmospheric plasma treatment and surface functionalization

    Science.gov (United States)

    Shen, Tao; Liu, Yong; Zhu, Yan; Yang, De-Quan; Sacher, Edward

    2017-07-01

    Ag nanoparticles (NPs) have been widely applied, as important antibacterial materials, on textile and polymer surfaces. However, their adhesion to nonreactive polymer surfaces is generally too weak for many applications. Here, we propose a two-step process, atmospheric plasma treatment followed by a surface chemical modification process, which enhances their adhesion to polyethylene terephthalate (PET) surfaces. We found that, compared to either plasma treatments or surface chemical functionalizations, alone, this combination greatly enhanced their adhesion. The plasma treatment resulted in an increase of active sites (sbnd OH, sbnd CHdbnd O and COOH) at the PET surface, permitting increased bonding to 3-aminopropyltriethoxysilane (APTES), whose sbnd NH2 groups were then able to form a bonding complex with the Ag NPs.

  20. Functional Role and Therapeutic Potential of the Pim-1 Kinase in Colon Carcinoma

    Directory of Open Access Journals (Sweden)

    Ulrike Weirauch

    2013-07-01

    Full Text Available PURPOSE: The provirus integration site for Moloney murine leukemia virus 1 (Pim-1 kinase is overexpressed in various tumors and has been linked to poor prognosis. Its role as proto-oncogene is based on several Pim-1 target proteins involved in pivotal cellular processes. Here, we explore the functional relevance of Pim-1 in colon carcinoma. EXPERIMENTAL DESIGN: RNAi-based knockdown approaches, as well as a specific small molecule inhibitor, were used to inhibit Pim-1 in colon carcinoma cells. The effects were analyzed regarding proliferation, apoptosis, sensitization toward cytostatic treatment, and overall antitumor effect in vitro and in mouse tumor models in vivo. RESULTS: We demonstrate antiproliferative, proapoptotic, and overall antitumor effects of Pim-1 inhibition. The sensitization to 5-fluorouracil (5-FU treatment upon Pim-1 knockdown offers new possibilities for combinatorial treatment approaches. Importantly, this also antagonizes a 5-FU-triggered Pim-1 up-regulation, which is mediated by decreased levels of miR-15b, a microRNA we newly identify to regulate Pim-1. The analysis of the molecular effects of Pim-1 inhibition reveals a complex regulatory network, with therapeutic Pim-1 repression leading to major changes in oncogenic signal transduction with regard to p21Cip1/WAF1, STAT3, c-jun-N-terminal kinase (JNK, c-Myc, and survivin and in the levels of apoptosis-related proteins Puma, Bax, and Bcl-xL. CONCLUSIONS: We demonstrate that Pim-1 plays a pivotal role in several tumor-relevant signaling pathways and establish the functional relevance of Pim-1 in colon carcinoma. Our results also substantiate the RNAi-mediated Pim-1 knockdown based on polymeric polyethylenimine/ small interfering RNA nanoparticles as a promising therapeutic approach.

  1. Calcium-dependent protein kinases in plants: evolution, expression and function.

    Science.gov (United States)

    Valmonte, Gardette R; Arthur, Kieren; Higgins, Colleen M; MacDiarmid, Robin M

    2014-03-01

    Calcium-dependent protein kinases (CPKs) are plant proteins that directly bind calcium ions before phosphorylating substrates involved in metabolism, osmosis, hormone response and stress signaling pathways. CPKs are a large multigene family of proteins that are present in all plants studied to date, as well as in protists, oomycetes and green algae, but are not found in animals and fungi. Despite the increasing evidence of the importance of CPKs in developmental and stress responses from various plants, a comprehensive genome-wide analysis of CPKs from algae to higher plants has not been undertaken. This paper describes the evolution of CPKs from green algae to plants using a broadly sampled phylogenetic analysis and demonstrates the functional diversification of CPKs based on expression and functional studies in different plant species. Our findings reveal that CPK sequence diversification into four major groups occurred in parallel with the terrestrial transition of plants. Despite significant expansion of the CPK gene family during evolution from green algae to higher plants, there is a high level of sequence conservation among CPKs in all plant species. This sequence conservation results in very little correlation between CPK evolutionary groupings and functional diversity, making the search for CPK functional orthologs a challenge.

  2. Adhesion molecules

    CERN Document Server

    Preedy, Victor R

    2016-01-01

    This book covers the structure and classification of adhesion molecules in relation to signaling pathways and gene expression. It discusses immunohistochemical localization, neutrophil migration, and junctional, functional, and inflammatory adhesion molecules in pathologies such as leukocyte decompression sickness and ischemia reperfusion injury. Highlighting the medical applications of current research, chapters cover diabetes, obesity, and metabolic syndrome; hypoxia; kidney disease; smoking, atrial fibrillation, and heart disease, the brain and dementia; and tumor proliferation. Finally, it looks at molecular imaging and bioinformatics, high-throughput technologies, and chemotherapy.

  3. HGF induces FAK activation and integrin-mediated adhesion in MTLn3 breast carcinoma cells.

    Science.gov (United States)

    Beviglia, L; Kramer, R H

    1999-11-26

    Expression of hepatocyte growth factor (HGF) and its tyrosine kinase receptor, c-Met, is positively correlated with breast carcinoma progression. We found that in invasive and metastatic MTLn3 breast carcinoma cells, HGF stimulated both initial adhesion to and motility on the extracellular matrix (ECM) ligands laminin 1, type I collagen, and fibronectin. Next, analysis with function-perturbing antibodies showed that adhesion to the different ECM proteins was mediated through specific beta1 integrins. In MTLn3 cells, HGF induced rapid tyrosine phosphorylation and activation of both c-Met and focal adhesion kinase (FAK). Cell anchorage and adhesion to the ECM substrates was required for HGF-induced FAK activation, since HGF failed to trigger tyrosine phosphorylation of FAK in suspended cells. Our results provide evidence that the 2 signaling pathways, integrin/ECM and c-Met/HGF, cooperate synergistically to induce FAK activation in an adhesion-dependent manner, leading to enhanced cell adhesion and motility. Moreover, we found that a FRNK (the FAK-related non-kinase)-like molecule is expressed in MTLn3 cells. Since FRNK acts as a competitive inhibitor of FAK function, our results suggest that a FRNK-like protein could facilitate disassembly of focal adhesions and likely be responsible for the HGF-induced scattering and motility of MTLn3 cells. Copyright 1999 Wiley-Liss, Inc.

  4. Nano Enabled Thermo-Mechanical Materials in Adhesive Joints: A New Paradigm to Materials Functionality (Preprint)

    National Research Council Canada - National Science Library

    Roy, Ajit K; Ganguli, Sabyasachi; Sihn, Sangwook; Qu, Liangti; Dai, Liming

    2006-01-01

    One of the barriers in achieving adequate through-thickness thermal conductivity in composite materials and also in composite joints is the extremely low thermal conductivity of resins (polymer) or adhesives (typically 0.3 W/mK...

  5. Characterization of Spindle Checkpoint Kinase Mps1 Reveals Domain with Functional and Structural Similarities to Tetratricopeptide Repeat Motifs of Bub1 and BubR1 Checkpoint Kinases*

    Science.gov (United States)

    Lee, Semin; Thebault, Philippe; Freschi, Luca; Beaufils, Sylvie; Blundell, Tom L.; Landry, Christian R.; Bolanos-Garcia, Victor M.; Elowe, Sabine

    2012-01-01

    Kinetochore targeting of the mitotic kinases Bub1, BubR1, and Mps1 has been implicated in efficient execution of their functions in the spindle checkpoint, the self-monitoring system of the eukaryotic cell cycle that ensures chromosome segregation occurs with high fidelity. In all three kinases, kinetochore docking is mediated by the N-terminal region of the protein. Deletions within this region result in checkpoint failure and chromosome segregation defects. Here, we use an interdisciplinary approach that includes biophysical, biochemical, cell biological, and bioinformatics methods to study the N-terminal region of human Mps1. We report the identification of a tandem repeat of the tetratricopeptide repeat (TPR) motif in the N-terminal kinetochore binding region of Mps1, with close homology to the tandem TPR motif of Bub1 and BubR1. Phylogenetic analysis indicates that TPR Mps1 was acquired after the split between deutorostomes and protostomes, as it is distinguishable in chordates and echinoderms. Overexpression of TPR Mps1 resulted in decreased efficiency of both chromosome alignment and mitotic arrest, likely through displacement of endogenous Mps1 from the kinetochore and decreased Mps1 catalytic activity. Taken together, our multidisciplinary strategy provides new insights into the evolution, structural organization, and function of Mps1 N-terminal region. PMID:22187426

  6. Functional groups grafted nonwoven fabrics for blood filtration-The effects of functional groups and wettability on the adhesion of leukocyte and platelet

    Energy Technology Data Exchange (ETDEWEB)

    Yang Chao [State Key Lab of Metal Matrix Composites, Shanghai Jiao Tong University, 800 Dongchuan Road, Shanghai 200240 (China); Cao Ye [Institute of Blood Transfusion, Chinese Academy of Medical Sciences and Peking Union Medical College, Chengdu 610081 (China); Sun Kang, E-mail: ksun@sjtu.edu.cn [State Key Lab of Metal Matrix Composites, Shanghai Jiao Tong University, 800 Dongchuan Road, Shanghai 200240 (China); Liu Jiaxin; Wang Hong [Institute of Blood Transfusion, Chinese Academy of Medical Sciences and Peking Union Medical College, Chengdu 610081 (China)

    2011-01-15

    In this work, the effects of grafted functional groups and surface wettability on the adhesion of leukocyte and platelet were investigated by the method of blood filtration. The filter materials, poly(butylene terephthalate) nonwoven fabrics bearing different functional groups including hydroxyl (OH), carboxyl (COOH), sulfonic acid group (SO{sub 3}H) and zwitterionic sulfobetaine group ({sup +}N((CH{sub 3}){sub 2})(CH{sub 2}){sub 3}SO{sub 3}{sup Circled-Minus }) with controllable wettability were prepared by UV radiation grafting vinyl monomers with these functional groups. Our results emphasized that both surface functional groups and surface wettability had significant effects on the adhesion of leukocyte and platelet. In the case of filter materials with the same wettability, leukocytes adhering to filter materials decreased in the order: the surface bearing OH only > the surface bearing both OH and COOH > the surface bearing sulfobetaine group > the surface bearing SO{sub 3}H, while platelets adhering to filter materials decreased as the following order: the surface bearing SO{sub 3}H > the surface bearing both OH and COOH > the surface bearing OH only > the surface bearing sulfobetaine group. As the wettability of filter materials increased, both leukocyte and platelet adhesion to filter materials declined, except that leukocyte adhesion to the surface bearing OH only remained unchanged.

  7. Functional groups grafted nonwoven fabrics for blood filtration-The effects of functional groups and wettability on the adhesion of leukocyte and platelet

    Science.gov (United States)

    Yang, Chao; Cao, Ye; Sun, Kang; Liu, Jiaxin; Wang, Hong

    2011-01-01

    In this work, the effects of grafted functional groups and surface wettability on the adhesion of leukocyte and platelet were investigated by the method of blood filtration. The filter materials, poly(butylene terephthalate) nonwoven fabrics bearing different functional groups including hydroxyl (OH), carboxyl (COOH), sulfonic acid group (SO3H) and zwitterionic sulfobetaine group (⊕N((CH3)2)(CH2)3SO3⊖) with controllable wettability were prepared by UV radiation grafting vinyl monomers with these functional groups. Our results emphasized that both surface functional groups and surface wettability had significant effects on the adhesion of leukocyte and platelet. In the case of filter materials with the same wettability, leukocytes adhering to filter materials decreased in the order: the surface bearing OH only > the surface bearing both OH and COOH > the surface bearing sulfobetaine group > the surface bearing SO3H, while platelets adhering to filter materials decreased as the following order: the surface bearing SO3H > the surface bearing both OH and COOH > the surface bearing OH only > the surface bearing sulfobetaine group. As the wettability of filter materials increased, both leukocyte and platelet adhesion to filter materials declined, except that leukocyte adhesion to the surface bearing OH only remained unchanged.

  8. Functional groups grafted nonwoven fabrics for blood filtration-The effects of functional groups and wettability on the adhesion of leukocyte and platelet

    International Nuclear Information System (INIS)

    Yang Chao; Cao Ye; Sun Kang; Liu Jiaxin; Wang Hong

    2011-01-01

    In this work, the effects of grafted functional groups and surface wettability on the adhesion of leukocyte and platelet were investigated by the method of blood filtration. The filter materials, poly(butylene terephthalate) nonwoven fabrics bearing different functional groups including hydroxyl (OH), carboxyl (COOH), sulfonic acid group (SO 3 H) and zwitterionic sulfobetaine group ( + N((CH 3 ) 2 )(CH 2 ) 3 SO 3 ⊖ ) with controllable wettability were prepared by UV radiation grafting vinyl monomers with these functional groups. Our results emphasized that both surface functional groups and surface wettability had significant effects on the adhesion of leukocyte and platelet. In the case of filter materials with the same wettability, leukocytes adhering to filter materials decreased in the order: the surface bearing OH only > the surface bearing both OH and COOH > the surface bearing sulfobetaine group > the surface bearing SO 3 H, while platelets adhering to filter materials decreased as the following order: the surface bearing SO 3 H > the surface bearing both OH and COOH > the surface bearing OH only > the surface bearing sulfobetaine group. As the wettability of filter materials increased, both leukocyte and platelet adhesion to filter materials declined, except that leukocyte adhesion to the surface bearing OH only remained unchanged.

  9. Leucine-rich repeat kinase-1 regulates osteoclast function by modulating RAC1/Cdc42 Small GTPase phosphorylation and activation.

    Science.gov (United States)

    Zeng, Canjun; Goodluck, Helen; Qin, Xuezhong; Liu, Bo; Mohan, Subburaman; Xing, Weirong

    2016-10-01

    Leucine-rich repeat kinase-1 (Lrrk1) consists of ankyrin repeats (ANK), leucine-rich repeats (LRR), a GTPase-like domain of Roc (ROC), a COR domain, a serine/threonine kinase domain (KD), and WD40 repeats (WD40). Previous studies have revealed that knockout (KO) of Lrrk1 in mice causes severe osteopetrosis, and a human mutation of Lrrk1 leads to osteosclerotic metaphysial dysplasia. The molecular mechanism by which Lrrk1 regulates osteoclast function is unknown. In this study, we generated a series of Lrrk1 mutants and evaluated their ability to rescue defective bone resorption in Lrrk1-deficient osteoclasts by use of pit formation assays. Overexpression of Lrrk1 or LRR-truncated Lrrk1, but not ANK-truncated Lrrk1, WD40-truncated Lrrk1, Lrrk1-KD, or K651A mutant Lrrk1, rescued bone resorption function of Lrrk1 KO osteoclasts. We next examined whether RAC1/Cdc42 small GTPases are direct substrates of Lrrk1 in osteoclasts. Western blot and pull-down assays revealed that Lrrk1 deficiency in osteoclasts resulted in reduced phosphorylation and activation of RAC1/Cdc42. In vitro kinase assays confirmed that recombinant Lrrk1 phosphorylated RAC1-GST protein, and immunoprecipitation showed that the interaction of Lrrk1 with RAC1 occurred within 10 min after RANKL treatment. Overexpression of constitutively active Q61L RAC1 partially rescued the resorptive function of Lrrk1-deficient osteoclasts. Furthermore, lack of Lrrk1 in osteoclasts led to reduced autophosphorylation of p21 protein-activated kinase-1 at Ser 144 , catalyzed by RAC1/Cdc42 binding and activation. Our data indicate that Lrrk1 regulates osteoclast function by directly modulating phosphorylation and activation of small GTPase RAC1/Cdc42 and that its function depends on ANK, ROC, WD40, and kinase domains. Copyright © 2016 the American Physiological Society.

  10. Distinct and Overlapping Functions of TEC Kinase and BTK in B Cell Receptor Signaling.

    Science.gov (United States)

    de Bruijn, Marjolein J W; Rip, Jasper; van der Ploeg, Esmee K; van Greuningen, Lars W; Ta, Van T B; Kil, Laurens P; Langerak, Anton W; Rimmelzwaan, Guus F; Ellmeier, Wilfried; Hendriks, Rudi W; Corneth, Odilia B J

    2017-04-15

    The Tec tyrosine kinase is expressed in many cell types, including hematopoietic cells, and is a member of the Tec kinase family that also includes Btk. Although the role of Btk in B cells has been extensively studied, the role of Tec kinase in B cells remains largely unclear. It was previously shown that Tec kinase has the ability to partly compensate for loss of Btk activity in B cell differentiation, although the underlying mechanism is unknown. In this study, we confirm that Tec kinase is not essential for normal B cell development when Btk is present, but we also found that Tec-deficient mature B cells showed increased activation, proliferation, and survival upon BCR stimulation, even in the presence of Btk. Whereas Tec deficiency did not affect phosphorylation of phospholipase Cγ or Ca 2+ influx, it was associated with significantly increased activation of the intracellular Akt/S6 kinase signaling pathway upon BCR and CD40 stimulation. The increased S6 kinase phosphorylation in Tec-deficient B cells was dependent on Btk kinase activity, as ibrutinib treatment restored pS6 to wild-type levels, although Btk protein and phosphorylation levels were comparable to controls. In Tec-deficient mice in vivo, B cell responses to model Ags and humoral immunity upon influenza infection were enhanced. Moreover, aged mice lacking Tec kinase developed a mild autoimmune phenotype. Taken together, these data indicate that in mature B cells, Tec and Btk may compete for activation of the Akt signaling pathway, whereby the activating capacity of Btk is limited by the presence of Tec kinase. Copyright © 2017 by The American Association of Immunologists, Inc.

  11. Signaling through intercellular adhesion molecule 1 (ICAM-1) in a B cell lymphoma line

    DEFF Research Database (Denmark)

    Holland, J; Owens, T

    1997-01-01

    Intercellular adhesion molecule 1 (ICAM-1) (CD54) is an adhesion molecule of the immunoglobulin superfamily. The interaction between ICAM-1 on B lymphocytes and leukocyte function-associated antigen 1 on T cells plays a major role in several aspects of the immune response, including T-dependent B...... cell activation. While it was originally believed that ICAM-1 played a purely adhesive role, recent evidence suggests that it can itself transduce biochemical signals. We demonstrate that cross-linking of ICAM-1 results in the up-regulation of class II major histocompatibility complex, and we...... investigate the biochemical mechanism for the signaling role of ICAM-1. We show that cross-linking of ICAM-1 on the B lymphoma line A20 induces an increase in tyrosine phosphorylation of several cellular proteins, including the Src family kinase p53/p56(lyn). In vitro kinase assays showed that Lyn kinase...

  12. Functional analysis of Casein Kinase 1 in a minimal circadian system.

    Directory of Open Access Journals (Sweden)

    Gerben van Ooijen

    Full Text Available The Earth's rotation has driven the evolution of cellular circadian clocks to facilitate anticipation of the solar cycle. Some evidence for timekeeping mechanism conserved from early unicellular life through to modern organisms was recently identified, but the components of this oscillator are currently unknown. Although very few clock components appear to be shared across higher species, Casein Kinase 1 (CK1 is known to affect timekeeping across metazoans and fungi, but has not previously been implicated in the circadian clock in the plant kingdom. We now show that modulation of CK1 function lengthens circadian rhythms in Ostreococcustauri, a unicellular marine algal species at the base of the green lineage, separated from humans by ~1.5 billion years of evolution. CK1 contributes to timekeeping in a phase-dependent manner, indicating clock-mediated gating of CK1 activity. Label-free proteomic analyses upon overexpression as well as inhibition revealed CK1-responsive phosphorylation events on a set of target proteins, including highly conserved potentially clock-relevant cellular regulator proteins. These results have major implications for our understanding of cellular timekeeping and can inform future studies in any circadian organism.

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

    Directory of Open Access Journals (Sweden)

    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.

  14. A GFP trap study uncovers the functions of Gilgamesh protein kinase in Drosophila melanogaster spermatogenesis.

    Science.gov (United States)

    Nerusheva, O O; Dorogova, N V; Gubanova, N V; Yudina, O S; Omelyanchuk, L V

    2009-05-01

    The function of the gene gilgamesh (89B9-12) encoding a casein kinase in Drosophila spermatogenesis was studied. The chimeric Gilgamesh-GFP protein in spermatocytes is cortically located. In the polar and apolar spermatocytes, it concentrates at the terminal ends of the fusome, the organelle that passes through the system of ring canals of the spermatocyte cyst. At the stage of spermatid elongation, the protein associates with the nucleus. A spot of the highest Gilgamesh-GFP concentration in the nucleus co-localizes with gamma-tubulin in the basal body. At later stages, Gilgamesh is localized to the individualization complex (IC), leaving the nuclei somewhat before the IC investment cones, as detected by actin binding. The sterile mutation due to the gilgamesh gene leads to the phenotype of scattered nuclei and altered structure of actin cones in the individualizing spermatid cyst. Ultrastructural evidence confirmed defective spermatid individualization due to the mutation. The phylogenetic origin of the protein, and the connection between vesicular trafficking and spermatid individualization, are discussed.

  15. A-RAF kinase functions in ARF6 regulated endocytic membrane traffic.

    Directory of Open Access Journals (Sweden)

    Elena Nekhoroshkova

    Full Text Available BACKGROUND: RAF kinases direct ERK MAPK signaling to distinct subcellular compartments in response to growth factor stimulation. METHODOLOGY/PRINCIPAL FINDINGS: Of the three mammalian isoforms A-RAF is special in that one of its two lipid binding domains mediates a unique pattern of membrane localization. Specific membrane binding is retained by an N-terminal fragment (AR149 that corresponds to a naturally occurring splice variant termed DA-RAF2. AR149 colocalizes with ARF6 on tubular endosomes and has a dominant negative effect on endocytic trafficking. Moreover actin polymerization of yeast and mammalian cells is abolished. AR149/DA-RAF2 does not affect the internalization step of endocytosis, but trafficking to the recycling compartment. CONCLUSIONS/SIGNIFICANCE: A-RAF induced ERK activation is required for this step by activating ARF6, as A-RAF depletion or inhibition of the A-RAF controlled MEK-ERK cascade blocks recycling. These data led to a new model for A-RAF function in endocytic trafficking.

  16. Proteomic analysis reveals a novel function of the kinase Sat4p in Saccharomyces cerevisiae mitochondria.

    Directory of Open Access Journals (Sweden)

    Uta Gey

    Full Text Available The Saccharomyces cerevisiae kinase Sat4p has been originally identified as a protein involved in salt tolerance and stabilization of plasma membrane transporters, implicating a cytoplasmic localization. Our study revealed an additional mitochondrial (mt localization, suggesting a dual function for Sat4p. While no mt related phenotype was observed in the absence of Sat4p, its overexpression resulted in significant changes of a specific mitochondrial subproteome. As shown by a comparative two dimensional difference gel electrophoresis (2D-DIGE approach combined with mass spectrometry, particularly two groups of proteins were affected: the iron-sulfur containing aconitase-type proteins (Aco1p, Lys4p and the lipoamide-containing subproteome (Lat1p, Kgd2p and Gcv3p. The lipoylation sites of all three proteins could be assigned by nanoLC-MS/MS to Lys75 (Lat1p, Lys114 (Kgd2p and Lys102 (Gcv3p, respectively. Sat4p overexpression resulted in accumulation of the delipoylated protein variants and in reduced levels of aconitase-type proteins, accompanied by a decrease in the activities of the respective enzyme complexes. We propose a regulatory role of Sat4p in the late steps of the maturation of a specific subset of mitochondrial iron-sulfur cluster proteins, including Aco1p and lipoate synthase Lip5p. Impairment of the latter enzyme may account for the observed lipoylation defects.

  17. Curcumin inhibits development and cell adhesion in Dictyostelium discoideum: Implications for YakA signaling and GST enzyme function

    International Nuclear Information System (INIS)

    Garige, Mamatha; Walters, Eric

    2015-01-01

    The molecular basis for nutraceutical properties of the polyphenol curcumin (Curcuma longa, Turmeric) is complex, affecting multiple factors that regulate cell signaling and homeostasis. Here, we report the effect of curcumin on cellular and developmental mechanisms in the eukaryotic model, Dictyostelium discoideum. Dictyostelium proliferation was inhibited in the presence of curcumin, which also suppressed the prestarvation marker, discoidin I, members of the yakA-mediated developmental signaling pathway, and expression of the extracellular matrix/cell adhesion proteins (DdCAD and csA). This resulted in delayed chemotaxis, adhesion, and development of the organism. In contrast to the inhibitory effects on developmental genes, curcumin induced gstA gene expression, overall GST activity, and generated production of reactive oxygen species. These studies expand our knowledge of developmental and biochemical signaling influenced by curcumin, and lends greater consideration of GST enzyme function in eukaryotic cell signaling, development, and differentiation.

  18. Curcumin inhibits development and cell adhesion in Dictyostelium discoideum: Implications for YakA signaling and GST enzyme function

    Energy Technology Data Exchange (ETDEWEB)

    Garige, Mamatha; Walters, Eric, E-mail: ewalters@howard.edu

    2015-11-13

    The molecular basis for nutraceutical properties of the polyphenol curcumin (Curcuma longa, Turmeric) is complex, affecting multiple factors that regulate cell signaling and homeostasis. Here, we report the effect of curcumin on cellular and developmental mechanisms in the eukaryotic model, Dictyostelium discoideum. Dictyostelium proliferation was inhibited in the presence of curcumin, which also suppressed the prestarvation marker, discoidin I, members of the yakA-mediated developmental signaling pathway, and expression of the extracellular matrix/cell adhesion proteins (DdCAD and csA). This resulted in delayed chemotaxis, adhesion, and development of the organism. In contrast to the inhibitory effects on developmental genes, curcumin induced gstA gene expression, overall GST activity, and generated production of reactive oxygen species. These studies expand our knowledge of developmental and biochemical signaling influenced by curcumin, and lends greater consideration of GST enzyme function in eukaryotic cell signaling, development, and differentiation.

  19. Dark chocolate consumption improves leukocyte adhesion factors and vascular function in overweight men

    NARCIS (Netherlands)

    Esser, D.; Mars, M.; Oosterink, E.; Stalmach, A.; Müller, M.R.; Afman, L.A.

    2014-01-01

    Flavanol-enriched chocolate consumption increases endothelium-dependent vasodilation. Most research so far has focused on flow-mediated dilation (FMD) only; the effects on other factors relevant to endothelial health, such as inflammation and leukocyte adhesion, have hardly been addressed. We

  20. Analysis of p21-Activated Kinase Function in Neurofibromatosis Type 2

    Science.gov (United States)

    2010-01-01

    mediated activation ( Abo et al., 1998; Cau et al., 2001; Lee et al., 2002), the basal kinase activity of the group II Paks was not stimulated by Cdc42...Received: November 15, 2007 Revised: February 25, 2008 Accepted: March 3, 2008 Published: April 18, 2008 REFERENCES Abo , A., Qu, J., Cammarano, M.S...et al. Role of p21-activated kinase pathway defects in the cognitive deficits of Alzheimer disease. Nat Neurosci 2006;9(2):234-42. 53. Reutershan J

  1. Inhibition of protein kinase Cbeta does not improve endothelial function in type 2 diabetes.

    Science.gov (United States)

    Beckman, Joshua A; Goldfine, Allison B; Goldin, Alison; Prsic, Adnan; Kim, Sora; Creager, Mark A

    2010-08-01

    Antagonism of protein kinase Cbeta (PKCbeta) restores endothelial function in experimental models of diabetes and prevents vascular dysfunction in response to hyperglycemia in healthy humans. We tested the hypothesis that PKCbeta antagonism would improve vascular function in subjects with type 2 diabetes compared with healthy control subjects. The effect of PKCbeta was evaluated in a randomized, placebo-controlled, double-blinded crossover trial. The study was performed in the outpatient setting of a university medical center. Thirteen subjects with type 2 diabetes without evidence of cardiovascular disease and 15 healthy control subjects were recruited via newspaper advertisement. Subjects underwent a randomized, double-blind, crossover, placebo-controlled trial of the selective PKCbeta antagonist ruboxistaurin mesylate. Subjects received each treatment for 14 d. Endothelium-dependent and endothelium-independent vasodilation of forearm resistance vessels was measured with mercury-in-silastic, strain-gauge plethysmography during intraarterial administration of methacholine chloride and verapamil, respectively. Markers of inflammation, fibrinolysis, endothelial damage, and oxidative stress were measured after each treatment. Endothelium-dependent vasodilation of forearm resistance vessels was attenuated in diabetic subjects when compared with healthy subjects (P=0.001). Endothelium-independent vasodilation did not differ between groups (P value not significant). Ruboxistaurin did not significantly change endothelium-dependent or endothelium-independent vasodilation or blood-based markers of inflammation, fibrinolysis, endothelial damage, and oxidative stress in either diabetic or healthy subjects. Endothelial dysfunction of forearm resistance vessels was not improved by 2 wk of selective PKCbeta inhibition in patients with diabetes. These results suggest that PKCbeta does not contribute significantly to vascular dysfunction in otherwise healthy patients with type 2

  2. Independence of protein kinase C-delta activity from activation loop phosphorylation: structural basis and altered functions in cells.

    Science.gov (United States)

    Liu, Yin; Belkina, Natalya V; Graham, Caroline; Shaw, Stephen

    2006-04-28

    Activation loop phosphorylation plays critical regulatory roles for many kinases. Unlike other protein kinase Cs (PKC), PKC-delta does not require phosphorylation of its activation loop (Thr-507) for in vitro activity. We investigated the structural basis for this unusual capacity and its relevance to PKC-delta function in intact cells. Mutational analysis demonstrated that activity without Thr-507 phosphorylation depends on 20 residues N-terminal to the kinase domain and a pair of phenylalanines (Phe-500/Phe-527) unique to PKC-delta in/near the activation loop. Molecular modeling demonstrated that these elements stabilize the activation loop by forming a hydrophobic chain of interactions from the C-lobe to activation loop to N-terminal (helical) extension. In cells PKC-delta mediates both apoptosis and transcription regulation. We found that the T507A mutant of the PKC-delta kinase domain resembled the corresponding wild type in mediating apoptosis in transfected HEK293T cells. But the T507A mutant was completely defective in AP-1 and NF-kappaB reporter assays. A novel assay in which the kinase domain of PKC-delta and its substrate (a fusion protein of PKC substrate peptide with green fluorescent protein) were co-targeted to lipid rafts revealed a major substrate-selective defect of the T507A mutant in phosphorylating the substrate in cells. In vitro analysis showed strong product inhibition on the T507A mutant with particular substrates whose characteristics suggest it contributes to the substrate selective defect of the PKC-delta T507A mutant in cells. Thus, activation loop phosphorylation of PKC-delta may regulate its function in cells in a novel way.

  3. A Global Analysis of Kinase Function in Candida albicans Hyphal Morphogenesis Reveals a Role for the Endocytosis Regulator Akl1

    Directory of Open Access Journals (Sweden)

    Hagit Bar-Yosef

    2018-02-01

    Full Text Available The human pathogenic fungus Candida albicans can switch between yeast and hyphal morphologies as a function of environmental conditions and cellular physiology. The yeast-to-hyphae morphogenetic switch is activated by well-established, kinase-based signal transduction pathways that are induced by extracellular stimuli. In order to identify possible inhibitory pathways of the yeast-to-hyphae transition, we interrogated a collection of C. albicans protein kinases and phosphatases ectopically expressed under the regulation of the TETon promoter. Proportionately more phosphatases than kinases were identified that inhibited hyphal morphogenesis, consistent with the known role of protein phosphorylation in hyphal induction. Among the kinases, we identified AKL1 as a gene that significantly suppressed hyphal morphogenesis in serum. Akl1 specifically affected hyphal elongation rather than initiation: overexpression of AKL1 repressed hyphal growth, and deletion of AKL1 resulted in acceleration of the rate of hyphal elongation. Akl1 suppressed fluid-phase endocytosis, probably via Pan1, a putative clathrin-mediated endocytosis scaffolding protein. In the absence of Akl1, the Pan1 patches were delocalized from the sub-apical region, and fluid-phase endocytosis was intensified. These results underscore the requirement of an active endocytic pathway for hyphal morphogenesis. Furthermore, these results suggest that under standard conditions, endocytosis is rate-limiting for hyphal elongation.

  4. FERMT2 links cortical actin structures, plasma membrane tension and focal adhesion function to stabilize podocyte morphology.

    Science.gov (United States)

    Yasuda-Yamahara, M; Rogg, M; Frimmel, J; Trachte, P; Helmstaedter, M; Schroder, P; Schiffer, M; Schell, C; Huber, T B

    2018-01-11

    Simplification and retraction of podocyte protrusions, generally termed as foot process effacement, is a uniform pathological pattern observed in the majority of glomerular disease, including focal segmental glomerulosclerosis. However, it is still incompletely understood how the interaction of cortical actin structures, actomyosin contractility and focal adhesions, is being orchestrated to control foot process morphology in health and disease. By uncovering the functional role of fermitin family member 2 (FERMT2 or kindlin-2) in podocytes, we provide now evidence, how cell-extracellular matrix (ECM) interactions modulate membrane tension and actomyosin contractility. A genetic modeling approach was applied by deleting FERMT2 in a set of in vivo systems as well as in CRISPR/Cas9 modified human podocytes. Loss of FERMT2 results in altered cortical actin composition, cell cortex destabilization associated with plasma membrane blebbing and a remodeling of focal adhesions. We further show that FERMT2 knockout podocytes have high levels of RhoA activation and concomitantly increased actomyosin contractility. Inhibition of actomyosin tension reverses the membrane blebbing phenotype. Thus, our findings establish a direct link between cell-matrix adhesions, cortical actin structures and plasma membrane tension allowing to better explain cell morphological changes in foot process effacement. Copyright © 2018 The Authors. Published by Elsevier B.V. All rights reserved.

  5. Effect of a functional monomer (MDP) on the enamel bond durability of single-step self-etch adhesives.

    Science.gov (United States)

    Tsuchiya, Kenji; Takamizawa, Toshiki; Barkmeier, Wayne W; Tsubota, Keishi; Tsujimoto, Akimasa; Berry, Thomas P; Erickson, Robert L; Latta, Mark A; Miyazaki, Masashi

    2016-02-01

    The present study aimed to determine the effect of the functional monomer, 10-methacryloxydecyl dihydrogen phosphate (MDP), on the enamel bond durability of single-step self-etch adhesives through integrating fatigue testing and long-term water storage. An MDP-containing self-etch adhesive, Clearfil Bond SE ONE (SE), and an experimental adhesive, MDP-free (MF), which comprised the same ingredients as SE apart from MDP, were used. Shear bond strength (SBS) and shear fatigue strength (SFS) were measured with or without phosphoric acid pre-etching. The specimens were stored in distilled water for 24 h, 6 months, or 1 yr. Although similar SBS and SFS values were obtained for SE with pre-etching and for MF after 24 h of storage in distilled water, SE with pre-etching showed higher SBS and SFS values than MF after storage in water for 6 months or 1 yr. Regardless of the pre-etching procedure, SE showed higher SBS and SFS values after 6 months of storage in distilled water than after 24 h or 1 yr. To conclude, MDP might play an important role in enhancing not only bond strength but also bond durability with respect to repeated subcritical loading after long-term water storage. © 2015 Eur J Oral Sci.

  6. Role of Interaction and Nucleoside Diphosphate Kinase B in Regulation of the Cystic Fibrosis Transmembrane Conductance Regulator Function by cAMP-Dependent Protein Kinase A.

    Directory of Open Access Journals (Sweden)

    Lee A Borthwick

    Full Text Available Cystic fibrosis results from mutations in the cystic fibrosis transmembrane conductance regulator (CFTR, a cAMP-dependent protein kinase A (PKA and ATP-regulated chloride channel. Here, we demonstrate that nucleoside diphosphate kinase B (NDPK-B, NM23-H2 forms a functional complex with CFTR. In airway epithelia forskolin/IBMX significantly increases NDPK-B co-localisation with CFTR whereas PKA inhibitors attenuate complex formation. Furthermore, an NDPK-B derived peptide (but not its NDPK-A equivalent disrupts the NDPK-B/CFTR complex in vitro (19-mers comprising amino acids 36-54 from NDPK-B or NDPK-A. Overlay (Far-Western and Surface Plasmon Resonance (SPR analysis both demonstrate that NDPK-B binds CFTR within its first nucleotide binding domain (NBD1, CFTR amino acids 351-727. Analysis of chloride currents reflective of CFTR or outwardly rectifying chloride channels (ORCC, DIDS-sensitive showed that the 19-mer NDPK-B peptide (but not its NDPK-A equivalent reduced both chloride conductances. Additionally, the NDPK-B (but not NDPK-A peptide also attenuated acetylcholine-induced intestinal short circuit currents. In silico analysis of the NBD1/NDPK-B complex reveals an extended interaction surface between the two proteins. This binding zone is also target of the 19-mer NDPK-B peptide, thus confirming its capability to disrupt NDPK-B/CFTR complex. We propose that NDPK-B forms part of the complex that controls chloride currents in epithelia.

  7. Functional, genetic and bioinformatic characterization of a calcium/calmodulin kinase gene in Sporothrix schenckii

    Directory of Open Access Journals (Sweden)

    Rodriguez-del Valle Nuri

    2007-11-01

    Full Text Available Abstract Background Sporothrix schenckii is a pathogenic, dimorphic fungus, the etiological agent of sporotrichosis, a subcutaneous lymphatic mycosis. Dimorphism in S. schenckii responds to second messengers such as cAMP and calcium, suggesting the possible involvement of a calcium/calmodulin kinase in its regulation. In this study we describe a novel calcium/calmodulin-dependent protein kinase gene in S. schenckii, sscmk1, and the effects of inhibitors of calmodulin and calcium/calmodulin kinases on the yeast to mycelium transition and the yeast cell cycle. Results Using the PCR homology approach a new member of the calcium/calmodulin kinase family, SSCMK1, was identified in this fungus. The cDNA sequence of sscmk1 revealed an open reading frame of 1,221 nucleotides encoding a 407 amino acid protein with a predicted molecular weight of 45.6 kDa. The genomic sequence of sscmk1 revealed the same ORF interrupted by five introns. Bioinformatic analyses of SSCMK1 showed that this protein had the distinctive features that characterize a calcium/calmodulin protein kinase: a serine/threonine protein kinase domain and a calmodulin-binding domain. When compared to homologues from seven species of filamentous fungi, SSCMK1 showed substantial similarities, except for a large and highly variable region that encompasses positions 330 – 380 of the multiple sequence alignment. Inhibition studies using calmodulin inhibitor W-7, and calcium/calmodulin kinase inhibitors, KN-62 and lavendustin C, were found to inhibit budding by cells induced to re-enter the yeast cell cycle and to favor the yeast to mycelium transition. Conclusion This study constitutes the first evidence of the presence of a calcium/calmodulin kinase-encoding gene in S. schenckii and its possible involvement as an effector of dimorphism in this fungus. These results suggest that a calcium/calmodulin dependent signaling pathway could be involved in the regulation of dimorphism in this fungus

  8. Lactobacillus reuteri glyceraldehyde-3-phosphate dehydrogenase functions in adhesion to intestinal epithelial cells.

    Science.gov (United States)

    Zhang, Wen-Ming; Wang, Hai-Feng; Gao, Kan; Wang, Cong; Liu, Li; Liu, Jian-Xin

    2015-05-01

    This study was aimed to identify key surface proteins mediating the adhesion of lactobacilli to intestinal epithelial cells. By using Caco-2 and IPEC-J2 cells labeled with sulfo-NHS-biotin in the western blotting, a protein band of an approximately 37 kDa was detected on the surface layer of Lactobacillus reuteri strains ZJ616, ZJ617, ZJ621, and ZJ623 and Lactobacillus rhamnosus GG. Mass spectrometry analysis using the adhesion-related protein from L. reuteri ZJ617 showed that it was 100% homologous to the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) of L. reuteri JCM 1112 (GenBank: YP_001841377). The ability of L. reuteri ZJ617 to adhere to epithelial cells decreased significantly by treatment with LiCl or by blocking with an anti-GAPDH antibody, in comparison with the untreated strain (p reuteri ZJ617. The results indicated that the GAPDH protein of L. reuteri ZJ617 acts as an adhesion component that plays an important role in binding to the intestinal epithelial cells.

  9. Structure-function analysis of STRUBBELIG, an Arabidopsis atypical receptor-like kinase involved in tissue morphogenesis.

    Directory of Open Access Journals (Sweden)

    Prasad Vaddepalli

    Full Text Available Tissue morphogenesis in plants requires the coordination of cellular behavior across clonally distinct histogenic layers. The underlying signaling mechanisms are presently being unraveled and are known to include the cell surface leucine-rich repeat receptor-like kinase STRUBBELIG in Arabidopsis. To understand better its mode of action an extensive structure-function analysis of STRUBBELIG was performed. The phenotypes of 20 EMS and T-DNA-induced strubbelig alleles were assessed and homology modeling was applied to rationalize their possible effects on STRUBBELIG protein structure. The analysis was complemented by phenotypic, cell biological, and pharmacological investigations of a strubbelig null allele carrying genomic rescue constructs encoding fusions between various mutated STRUBBELIG proteins and GFP. The results indicate that STRUBBELIG accepts quite some sequence variation, reveal the biological importance for the STRUBBELIG N-capping domain, and reinforce the notion that kinase activity is not essential for its function in vivo. Furthermore, individual protein domains of STRUBBELIG cannot be related to specific STRUBBELIG-dependent biological processes suggesting that process specificity is mediated by factors acting together with or downstream of STRUBBELIG. In addition, the evidence indicates that biogenesis of a functional STRUBBELIG receptor is subject to endoplasmic reticulum-mediated quality control, and that an MG132-sensitive process regulates its stability. Finally, STRUBBELIG and the receptor-like kinase gene ERECTA interact synergistically in the control of internode length. The data provide genetic and molecular insight into how STRUBBELIG regulates intercellular communication in tissue morphogenesis.

  10. Dry adhesives with sensing features

    International Nuclear Information System (INIS)

    Krahn, J; Menon, C

    2013-01-01

    Geckos are capable of detecting detachment of their feet. Inspired by this basic observation, a novel functional dry adhesive is proposed, which can be used to measure the instantaneous forces and torques acting on an adhesive pad. Such a novel sensing dry adhesive could potentially be used by climbing robots to quickly realize and respond appropriately to catastrophic detachment conditions. The proposed torque and force sensing dry adhesive was fabricated by mixing Carbon Black (CB) and Polydimethylsiloxane (PDMS) to form a functionalized adhesive with mushroom caps. The addition of CB to PDMS resulted in conductive PDMS which, when under compression, tension or torque, resulted in a change in the resistance across the adhesive patch terminals. The proposed design of the functionalized dry adhesive enables distinguishing an applied torque from a compressive force in a single adhesive pad. A model based on beam theory was used to predict the change in resistance across the terminals as either a torque or compressive force was applied to the adhesive patch. Under a compressive force, the sensing dry adhesive was capable of measuring compression stresses from 0.11 Pa to 20.9 kPa. The torque measured by the adhesive patch ranged from 2.6 to 10 mN m, at which point the dry adhesives became detached. The adhesive strength was 1.75 kPa under an applied preload of 1.65 kPa for an adhesive patch with an adhesive contact area of 7.07 cm 2 . (paper)

  11. Dry adhesives with sensing features

    Science.gov (United States)

    Krahn, J.; Menon, C.

    2013-08-01

    Geckos are capable of detecting detachment of their feet. Inspired by this basic observation, a novel functional dry adhesive is proposed, which can be used to measure the instantaneous forces and torques acting on an adhesive pad. Such a novel sensing dry adhesive could potentially be used by climbing robots to quickly realize and respond appropriately to catastrophic detachment conditions. The proposed torque and force sensing dry adhesive was fabricated by mixing Carbon Black (CB) and Polydimethylsiloxane (PDMS) to form a functionalized adhesive with mushroom caps. The addition of CB to PDMS resulted in conductive PDMS which, when under compression, tension or torque, resulted in a change in the resistance across the adhesive patch terminals. The proposed design of the functionalized dry adhesive enables distinguishing an applied torque from a compressive force in a single adhesive pad. A model based on beam theory was used to predict the change in resistance across the terminals as either a torque or compressive force was applied to the adhesive patch. Under a compressive force, the sensing dry adhesive was capable of measuring compression stresses from 0.11 Pa to 20.9 kPa. The torque measured by the adhesive patch ranged from 2.6 to 10 mN m, at which point the dry adhesives became detached. The adhesive strength was 1.75 kPa under an applied preload of 1.65 kPa for an adhesive patch with an adhesive contact area of 7.07 cm2.

  12. Arabidopsis mitogen-activated protein kinase kinases MKK1 and MKK2 have overlapping functions in defense signaling mediated by MEKK1, MPK4, and MKS1

    DEFF Research Database (Denmark)

    Qiu, Jin-Long; Zhou, Lu; Yun, Byung-Wook

    2008-01-01

    by biochemical and molecular analyses implicating the kinases in jasmonate- and salicylate-dependent defense responses, mediated in part via the MPK4 substrate MKS1. In addition, transcriptome analyses delineate overlapping and specific effects of the kinases on global gene expression patterns demonstrating both...

  13. Effects of cryopreservation on excretory function, cellular adhesion molecules and vessel lumen formation in human umbilical vein endothelial cells.

    Science.gov (United States)

    Cai, Guoping; Lai, Binbin; Hong, Huaxing; Lin, Peng; Chen, Weifu; Zhu, Zhong; Chen, Haixiao

    2017-07-01

    Cryopreservation is widely used in regenerative medicine for tissue preservation. In the present study, the effects of cryopreservation on excretory function, cellular adhesion molecules and vessel lumen formation in human umbilical vein endothelial cells (HUVECs) were investigated. After 0, 4, 8, 12 or 24 weeks of cryopreservation in liquid nitrogen, the HUVECs were thawed. The excretory functions markers (endothelin‑1, prostaglandin E1, von Willebrand factor and nitric oxide) of HUVECs were measured by ELISA assay. The expression of intercellular adhesion molecule‑1 (ICAM‑1) in HUVECs was analyzed using flow cytometry. An angiogenesis assay was used to determine the angiogeneic capabilities of the thawed HUVECs. The results demonstrated that cryopreserved/thawed and recultivated HUVECs were unsuitable for tissue‑engineered microvascular construction. Specifically, the excretory function of the cells was significantly decreased in the post‑cryopreserved HUVECs at 24 weeks. In addition, the level of ICAM‑1 in HUVECs was significantly upregulated from the fourth week of cryopreservation. Furthermore, the tube‑like structure‑forming potential was weakened with increasing cryopreservation duration, and the numbers of lumen and the length of the pipeline were decreased in the thawed HUVECs, in a time‑dependent manner. In conclusion, the results of the present study revealed that prolonged cryopreservation may lead to HUVEC dysfunction and did not create stable cell lines for tissue‑engineered microvascular construction.

  14. Coordinating structural and functional synapse development: postsynaptic p21-activated kinase independently specifies glutamate receptor abundance and postsynaptic morphology.

    Science.gov (United States)

    Albin, Stephanie D; Davis, Graeme W

    2004-08-04

    Here, we show that postsynaptic p21-activated kinase (Pak) signaling diverges into two genetically separable pathways at the Drosophila neuromuscular junction. One pathway controls glutamate receptor abundance. Pak signaling within this pathway is specified by a required interaction with the adaptor protein Dreadlocks (Dock). We demonstrate that Dock is localized to the synapse via an Src homology 2-mediated protein interaction. Dock is not necessary for Pak localization but is necessary to restrict Pak signaling to control glutamate receptor abundance. A second genetically separable function of Pak kinase signaling controls muscle membrane specialization through the regulation of synaptic Discs-large. In this pathway, Dock is dispensable. We present a model in which divergent Pak signaling is able to coordinate two different features of postsynaptic maturation, receptor abundance, and muscle membrane specialization.

  15. Functional polyaniline nanofibre mats for human adipose-derived stem cell proliferation and adhesion

    Energy Technology Data Exchange (ETDEWEB)

    Abdul Rahman, Norizah, E-mail: norizah@science.putra.edu.my [Polymer Electronics Research Centre, School of Chemical Sciences, The University of Auckland, Private Bag 92019, Auckland (New Zealand); Department of Chemistry, University of Putra Malaysia, 43400 UPM Serdang, Selangor Darul Ehsan (Malaysia); Feisst, Vaughan [School of Biological Sciences, The University of Auckland, Private Bag 92019, Auckland (New Zealand); Dickinson, Michelle E. [Department of Chemical and Materials Engineering, The University of Auckland, Private Bag 92019, Auckland (New Zealand); Malmström, Jenny [Polymer Electronics Research Centre, School of Chemical Sciences, The University of Auckland, Private Bag 92019, Auckland (New Zealand); Dunbar, P. Rod [School of Biological Sciences, The University of Auckland, Private Bag 92019, Auckland (New Zealand); Maurice Wilkins Centre, Private Bag 92019, Auckland (New Zealand); Travas-Sejdic, Jadranka, E-mail: j.travas-sejdic@auckland.ac.nz [Polymer Electronics Research Centre, School of Chemical Sciences, The University of Auckland, Private Bag 92019, Auckland (New Zealand); MacDiarmid Institute for Advanced Materials and Nanotechnology, P.O. Box 600, Wellington 6140 (New Zealand)

    2013-02-15

    Conductive polymer poly(aniline-co-m-aminobenzoic acid) (P(ANI-co-m-ABA)) and polyaniline (PANI) were blended with a biodegradable, biocompatible polymer, poly(L-lactic acid) and were electrospun into nanofibres to investigate their potential application as a scaffold for human adipose-derived stem cells (hASCs). These polymers, in both conductive and non-conductive form, were electrospun with average fibre diameters of less than 400 nm. Novel nanoindentation results obtained on the individual nanofibres revealed that the elastic moduli of the nanofibres are much higher at the surface (4–10 GPa, h{sub max} <75 nm) than in the inner fibre core (2–4 GPa, h{sub max} >75 nm). The composite nanofibres showed great promise as a scaffold for hASCs as they supported the cell adhesion and proliferation. After 1 week of cell culture hASCs were well spread on the substrates with abundant focal adhesions. The electrospun mats provide the cells with comparably stiff, sub-micron sized fibres as anchoring points on a substrate of high porosity. The conductive nature of these composite nanofibres offers exciting opportunities for electrical stimulation of the cells. - Highlights: ► Polyaniline and its copolymer's nanofibres were prepared by electrospinning. ► The elastic modulus of a single polyaniline composite nanofibres were determined. ► Elastic moduli of the nanofibres are much higher at the surface than the inner core. ► The electrospun mats supported the cell adhesion and proliferation. ► The nanofibres show great promise as a scaffold for adipose derived stem cells.

  16. New functions and signaling mechanisms for the class of adhesion G protein-coupled receptors

    DEFF Research Database (Denmark)

    Liebscher, Ines; Ackley, Brian; Araç, Demet

    2014-01-01

    The class of adhesion G protein-coupled receptors (aGPCRs), with 33 human homologs, is the second largest family of GPCRs. In addition to a seven-transmembrane α-helix-a structural feature of all GPCRs-the class of aGPCRs is characterized by the presence of a large N-terminal extracellular region....... In addition, all aGPCRs but one (GPR123) contain a GPCR autoproteolysis-inducing (GAIN) domain that mediates autoproteolytic cleavage at the GPCR autoproteolysis site motif to generate N- and a C-terminal fragments (NTF and CTF, respectively) during protein maturation. Subsequently, the NTF and CTF...

  17. Leucine-rich repeat kinase 2 (LRRK2) cellular biology: a review of recent advances in identifying physiological substrates and cellular functions.

    Science.gov (United States)

    Drolet, Robert E; Sanders, John M; Kern, Jonathan T

    2011-12-01

    Mutations in the leucine-rich repeat kinase 2 (LRRK2) gene are the most common forms of inheritable Parkinson's disease and likely play a role in sporadic disease as well. LRRK2 is a large multidomain protein containing two key groups, a Ras-like GTP binding domain and a serine, threonine kinase domain. Mutations in the LRRK2 gene that associate with Parkinson's disease reside primarily within the two functional domains of the protein, suggesting that LRRK2 function is critical to the pathogenesis of the disease. The most common LRRK2 mutation increases kinase activity, making LRRK2 kinase inhibition an attractive target for small molecule drug development. However, the physiological function of LRRK2 kinase as well as its endogenous protein substrates remains poorly understood and has hindered drug development efforts. Recent advances in LRRK2 biology have revealed several potential cellular roles, interacting proteins, and putative physiological substrates. Together, a picture emerges of a complex multifunctional protein that exists in multiple cellular compartments. Through unclear mechanisms, LRRK2 kinase regulates cytoskeleton architecture through control of protein translation, phosphorylation of cytoskeletal proteins, and response to cellular stressors. This article will briefly cover some interesting recent studies in LRRK2 cellular biology and highlight emerging cellular models of LRRK2 kinase function.

  18. Protein kinase G1 α overexpression increases stem cell survival and cardiac function after myocardial infarction.

    Directory of Open Access Journals (Sweden)

    Linlin Wang

    Full Text Available We hypothesized that overexpression of cGMP-dependent protein kinase type 1α (PKG1α could mimic the effect of tadalafil on the survival of bone marrow derived mesenchymal stem cells (MSCs contributing to regeneration of the ischemic heart.MSCs from male rats were transduced with adenoviral vector encoding for PKG1α ((PKG1αMSCs.Controls included native MSCs ((NatMSCs and MSCs transduced with an empty vector ((NullMSCs. PKG1α activity was increased approximately 20, 5 and 16 fold respectively in (PKG1αMSCs. (PKG1αMSCs showed improved survival under oxygen and glucose deprivation (OGD which was evidenced by lower LDH release, caspase-3/7 activity and number of positive TUNEL cells. Anti-apoptotic proteins pAkt, pGSK3β, and Bcl-2 were significantly increased in (PKG1αMSCs compared to (NatMSCs and (NullMSCs. Higher release of multiple prosurvival and angiogenic factors such as HGF, bFGF, SDF-1 and Ang-1 was observed in (PKG1αMSCs before and after OGD. In a female rat model of acute myocardial infarction, (PKG1αMSCs group showed higher survival compared with (NullMSCs group at 3 and 7 days after transplantation as determined by TUNEL staining and sry-gene quantitation by real-time PCR. Increased anti-apoptotic proteins and paracrine factors in vitro were also identified. Immunostaining for cardiac troponin I combined with GFP showed increased myogenic differentiation of (PKG1αMSCs. At 4 weeks after transplantation, compared to DMEM group and (NullMSCs group, (PKG1αMSCs group showed increased blood vessel density in infarct and peri-infarct areas (62.5±7.7; 68.8±7.3 per microscopic view, p<0.05 and attenuated infarct size (27.2±2.5%, p<0.01. Heart function indices including ejection fraction (52.1±2.2%, p<0.01 and fractional shortening (24.8%±1.3%, p<0.01 were improved significantly in (PKG1αMSCs group.Overexpression of PKG1α transgene could be a powerful approach to improve MSCs survival and their angiomyogenic potential in the

  19. Streptococcus pyogenes CAMP factor promotes bacterial adhesion and invasion in pharyngeal epithelial cells without serum via PI3K/Akt signaling pathway.

    Science.gov (United States)

    Kurosawa, Mie; Oda, Masataka; Domon, Hisanori; Isono, Toshihito; Nakamura, Yuki; Saitoh, Issei; Hayasaki, Haruaki; Yamaguchi, Masaya; Kawabata, Shigetada; Terao, Yutaka

    2018-01-01

    Streptococcus pyogenes is a bacterium that causes systemic diseases, such as pharyngitis and toxic shock syndrome, via oral- or nasal-cavity infection. S. pyogenes produces various molecules known to function with serum components that lead to bacterial adhesion and invasion in human tissues. In this study, we identified a novel S. pyogenes adhesin/invasin. Our results revealed that CAMP factor promoted streptococcal adhesion and invasion in pharyngeal epithelial Detroit562 cells without serum. Recombinant CAMP factor initially localized on the membranes of cells and then became internalized in the cytosol following S. pyogenes infection. Additionally, CAMP factor phosphorylated phosphoinositide 3-kinase and serine-threonine kinase in the cells. ELISA results demonstrate that CAMP factor affected the amount of phosphorylated phosphoinositide 3-kinase and serine-threonine kinase in Detroit562 cells. Furthermore, CAMP factor did not reverse the effect of phosphoinositide 3-kinase knockdown by small interfering RNA in reducing the level of adhesion and invasion of S. pyogenes isogenic cfa-deficient mutant. These results suggested that S. pyogenes CAMP factor activated the phosphoinositide 3-kinase/serine-threonine kinase signaling pathway, promoting S. pyogenes invasion of Detroit562 cells without serum. Our findings suggested that CAMP factor played an important role on adhesion and invasion in pharyngeal epithelial cells. Copyright © 2017 Institut Pasteur. Published by Elsevier Masson SAS. All rights reserved.

  20. Prevention of bacterial adhesion

    DEFF Research Database (Denmark)

    Klemm, Per; Vejborg, Rebecca Munk; Hancock, Viktoria

    2010-01-01

    Management of bacterial infections is becoming increasingly difficult due to the emergence and increasing prevalence of bacterial pathogens that are resistant to available antibiotics. Conventional antibiotics generally kill bacteria by interfering with vital cellular functions, an approach...... that imposes selection pressure for resistant bacteria. New approaches are urgently needed. Targeting bacterial virulence functions directly is an attractive alternative. An obvious target is bacterial adhesion. Bacterial adhesion to surfaces is the first step in colonization, invasion, and biofilm formation....... As such, adhesion represents the Achilles heel of crucial pathogenic functions. It follows that interference with adhesion can reduce bacterial virulence. Here, we illustrate this important topic with examples of techniques being developed that can inhibit bacterial adhesion. Some of these will become...

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

    Directory of Open Access Journals (Sweden)

    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.

  2. How tyrosine kinase inhibitors impair metabolism and endocrine system function: a systematic updated review.

    Science.gov (United States)

    Breccia, Massimo; Molica, Matteo; Alimena, Giuliana

    2014-12-01

    Tyrosine kinase inhibitors (TKIs) advent has deeply changed the outcome of chronic myeloid leukemia (CML) patients, with improved rates of response and overall survival. However, for this success some patients paid the price of a number of peculiar side effects, the so-called off-target side effects, specific for each one TKI. These effects are due to non-selective inhibition of other tyrosine kinase receptors, such as PDGFR, c-KIT, Src, VEGF. Consequences of this inhibition, some metabolic changes during the treatment with TKIs are reported. Aim of present review is to report metabolic changes and potential mechanisms involved in the pathogenesis related to imatinib, second (nilotinib and dasatinib) and third generation (bosutinib and ponatinib) TKIs. Copyright © 2014 Elsevier Ltd. All rights reserved.

  3. G-Protein Gα13Functions with Abl Kinase to Regulate Actin Cytoskeletal Reorganization.

    Science.gov (United States)

    Wang, Limin; Wang, Dawei; Xing, Bowen; Tan, Ying-Cai; Huang, Jianyun; Liu, Bingqian; Syrovatkina, Viktoriya; Espenel, Cedric; Kreitzer, Geri; Guo, Lin; Zhang, J Jillian; Huang, Xin-Yun

    2017-12-08

    Heterotrimeric G-proteins are essential cellular signal transducers. One of the G-proteins, Gα 13 , is critical for actin cytoskeletal reorganization, cell migration, cell proliferation, and apoptosis. Previously, we have shown that Gα 13 is essential for both G-protein-coupled receptor and receptor tyrosine kinase-induced actin cytoskeletal reorganization such as dynamic dorsal ruffle turnover and cell migration. However, the mechanism by which Gα 13 signals to actin cytoskeletal reorganization is not completely understood. Here we show that Gα 13 directly interacts with Abl tyrosine kinase, which is a critical regulator of actin cytoskeleton. This interaction is critical for Gα 13 -induced dorsal ruffle turnover, endothelial cell remodeling, and cell migration. Our data uncover a new molecular signaling pathway by which Gα 13 controls actin cytoskeletal reorganization. Copyright © 2017 Elsevier Ltd. All rights reserved.

  4. Physical and functional interactions between ZIP kinase and UbcH5

    International Nuclear Information System (INIS)

    Ohbayashi, Norihiko; Okada, Katsuya; Kawakami, Shiho; Togi, Sumihito; Sato, Noriko; Ikeda, Osamu; Kamitani, Shinya; Muromoto, Ryuta; Sekine, Yuichi; Kawai, Taro; Akira, Shizuo; Matsuda, Tadashi

    2008-01-01

    Zipper-interacting protein kinase (ZIPK) is a widely expressed serine/threonine kinase that has been implicated in cell death and transcriptional regulation, but its mechanism of regulation remains unknown. In our previous study, we showed that leukemia inhibitory factor stimulated threonine-265 phosphorylation of ZIPK, thereby leading to phosphorylation and activation of signal transducer and activator of transcription 3. Here, we identified UbcH5c as a novel ZIPK-binding partner by yeast two-hybrid screening. Importantly, we found that UbcH5c induced ubiquitination of ZIPK. Small-interfering RNA-mediated reduction of endogenous UbcH5 expression decreased ZIPK ubiquitination. Furthermore, coexpression of UbcH5c with ZIPK influenced promyelocytic leukemia protein nuclear body (PML-NB) formation. These results suggest that UbcH5 regulates ZIPK accumulation in PML-NBs by interacting with ZIPK and stimulating its ubiquitination

  5. Protein-Tyrosine Kinase Signaling in the Biological Functions Associated with Sperm

    Directory of Open Access Journals (Sweden)

    Takashi W. Ijiri

    2012-01-01

    Full Text Available In sexual reproduction, two gamete cells (i.e., egg and sperm fuse (fertilization to create a newborn with a genetic identity distinct from those of the parents. In the course of these developmental processes, a variety of signal transduction events occur simultaneously in each of the two gametes, as well as in the fertilized egg/zygote/early embryo. In particular, a growing body of knowledge suggests that the tyrosine kinase Src and/or other protein-tyrosine kinases are important elements that facilitate successful implementation of the aforementioned processes in many animal species. In this paper, we summarize recent findings on the roles of protein-tyrosine phosphorylation in many sperm-related processes (from spermatogenesis to epididymal maturation, capacitation, acrosomal exocytosis, and fertilization.

  6. Improvement of Interfacial Adhesion by Bio-Inspired Catechol-Functionalized Soy Protein with Versatile Reactivity: Preparation of Fully Utilizable Soy-Based Film

    Directory of Open Access Journals (Sweden)

    Zhong Wang

    2017-03-01

    Full Text Available The development of materials based on renewable resources with enhanced mechanical and physicochemical properties is hampered by the abundance of hydrophilic groups because of their structural instability. Bio-inspired from the strong adhesion ability of mussel proteins, renewable and robust soy-based composite films were fabricated from two soybean-derived industrial materials: soluble soybean polysaccharide (SSPS and catechol-functionalized soy protein isolate (SPI-CH. The conjugation of SPI with multiple catechol moieties as a versatile adhesive component for SSPS matrix efficiently improved the interfacial adhesion between each segment of biopolymer. The biomimetic adherent catechol moieties were successfully bonded in the polymeric network based on catechol crosslinking chemistry through simple oxidative coupling and/or coordinative interaction. A combination of H-bonding, strong adhesion between the SPI-CH conjugation and SSPS matrix resulted in remarkable enhancements for mechanical properties. It was found that the tensile strength and Young’s modulus was improved from 2.80 and 17.24 MPa of unmodified SP film to 4.04 and 97.22 MPa of modified one, respectively. More importantly, the resultant films exhibited favorable water resistance and gas (water vapor barrier performances. The results suggested that the promising way improved the phase adhesion of graft copolymers using catechol-functionalized polymers as versatile adhesive components.

  7. Functional Analysis of Lactobacillus rhamnosus GG Pili in Relation to Adhesion and Immunomodulatory Interactions with Intestinal Epithelial Cells

    Science.gov (United States)

    Claes, Ingmar; Tytgat, Hanne L. P.; Verhoeven, Tine L. A.; Marien, Eyra; von Ossowski, Ingemar; Reunanen, Justus; Palva, Airi; de Vos, Willem M.; De Keersmaecker, Sigrid C. J.; Vanderleyden, Jos

    2012-01-01

    Lactobacillus rhamnosus GG, a probiotic with good survival capacity in the human gut, has well-documented adhesion properties and health effects. Recently, spaCBA-encoded pili that bind to human intestinal mucus were identified on its cell surface. Here, we report on the phenotypic analysis of a spaCBA pilus knockout mutant in comparison with the wild type and other adhesin mutants. The SpaCBA pilus of L. rhamnosus GG showed to be key for efficient adherence to the Caco-2 intestinal epithelial cell (IEC) line and biofilm formation. Moreover, the spaCBA mutant induces an elevated level of interleukin-8 (IL-8) mRNA in Caco-2 cells compared to the wild type, possibly involving an interaction of lipoteichoic acid with Toll-like receptor 2. In contrast, an L. rhamnosus GG mutant without exopolysaccharides but with an increased exposure of pili leads to the reduced expression of IL-8. Using Transwells to partition bacteria from Caco-2 cells, IL-8 induction is blocked completely regardless of whether wild-type or mutant L. rhamnosus GG cells are used. Taken together, our data suggest that L. rhamnosus GG SpaCBA pili, while promoting strong adhesive interactions with IECs, have a functional role in balancing IL-8 mRNA expression induced by surface molecules such as lipoteichoic acid. PMID:22020518

  8. Functional analysis of Lactobacillus rhamnosus GG pili in relation to adhesion and immunomodulatory interactions with intestinal epithelial cells.

    Science.gov (United States)

    Lebeer, Sarah; Claes, Ingmar; Tytgat, Hanne L P; Verhoeven, Tine L A; Marien, Eyra; von Ossowski, Ingemar; Reunanen, Justus; Palva, Airi; Vos, Willem M de; Keersmaecker, Sigrid C J De; Vanderleyden, Jos

    2012-01-01

    Lactobacillus rhamnosus GG, a probiotic with good survival capacity in the human gut, has well-documented adhesion properties and health effects. Recently, spaCBA-encoded pili that bind to human intestinal mucus were identified on its cell surface. Here, we report on the phenotypic analysis of a spaCBA pilus knockout mutant in comparison with the wild type and other adhesin mutants. The SpaCBA pilus of L. rhamnosus GG showed to be key for efficient adherence to the Caco-2 intestinal epithelial cell (IEC) line and biofilm formation. Moreover, the spaCBA mutant induces an elevated level of interleukin-8 (IL-8) mRNA in Caco-2 cells compared to the wild type, possibly involving an interaction of lipoteichoic acid with Toll-like receptor 2. In contrast, an L. rhamnosus GG mutant without exopolysaccharides but with an increased exposure of pili leads to the reduced expression of IL-8. Using Transwells to partition bacteria from Caco-2 cells, IL-8 induction is blocked completely regardless of whether wild-type or mutant L. rhamnosus GG cells are used. Taken together, our data suggest that L. rhamnosus GG SpaCBA pili, while promoting strong adhesive interactions with IECs, have a functional role in balancing IL-8 mRNA expression induced by surface molecules such as lipoteichoic acid.

  9. Structural and Functional Studies Indicate That the EPEC Effector, EspG, Directly Binds p21-Activated Kinase

    Energy Technology Data Exchange (ETDEWEB)

    Germane, Katherine L.; Spiller, Benjamin W. (Vanderbilt)

    2011-09-20

    Bacterial pathogens secrete effectors into their hosts that subvert host defenses and redirect host processes. EspG is a type three secretion effector with a disputed function that is found in enteropathogenic Escherichia coli. Here we show that EspG is structurally similar to VirA, a Shigella virulence factor; EspG has a large, conserved pocket on its surface; EspG binds directly to the amino-terminal inhibitory domain of human p21-activated kinase (PAK); and mutations to conserved residues in the surface pocket disrupt the interaction with PAK.

  10. The kinase TBK1 functions in dendritic cells to regulate T cell homeostasis, autoimmunity, and antitumor immunity.

    Science.gov (United States)

    Xiao, Yichuan; Zou, Qiang; Xie, Xiaoping; Liu, Ting; Li, Haiyan S; Jie, Zuliang; Jin, Jin; Hu, Hongbo; Manyam, Ganiraju; Zhang, Li; Cheng, Xuhong; Wang, Hui; Marie, Isabelle; Levy, David E; Watowich, Stephanie S; Sun, Shao-Cong

    2017-05-01

    Dendritic cells (DCs) are crucial for mediating immune responses but, when deregulated, also contribute to immunological disorders, such as autoimmunity. The molecular mechanism underlying the function of DCs is incompletely understood. In this study, we have identified TANK-binding kinase 1 (TBK1), a master innate immune kinase, as an important regulator of DC function. DC-specific deletion of Tbk1 causes T cell activation and autoimmune symptoms and also enhances antitumor immunity in animal models of cancer immunotherapy. The TBK1-deficient DCs have up-regulated expression of co-stimulatory molecules and increased T cell-priming activity. We further demonstrate that TBK1 negatively regulates the induction of a subset of genes by type I interferon receptor (IFNAR). Deletion of IFNAR1 could largely prevent aberrant T cell activation and autoimmunity in DC-conditional Tbk1 knockout mice. These findings identify a DC-specific function of TBK1 in the maintenance of immune homeostasis and tolerance. © 2017 Xiao et al.

  11. Inducible Activation of ERK5 MAP Kinase Enhances Adult Neurogenesis in the Olfactory Bulb and Improves Olfactory Function

    Science.gov (United States)

    Wang, Wenbin; Lu, Song; Li, Tan; Pan, Yung-Wei; Zou, Junhui; Abel, Glen M.; Xu, Lihong; Storm, Daniel R.

    2015-01-01

    Recent discoveries have suggested that adult neurogenesis in the subventricular zone (SVZ) and olfactory bulb (OB) may be required for at least some forms of olfactory behavior in mice. However, it is unclear whether conditional and selective enhancement of adult neurogenesis by genetic approaches is sufficient to improve olfactory function under physiological conditions or after injury. Furthermore, specific signaling mechanisms regulating adult neurogenesis in the SVZ/OB are not fully defined. We previously reported that ERK5, a MAP kinase selectively expressed in the neurogenic regions of the adult brain, plays a critical role in adult neurogenesis in the SVZ/OB. Using a site-specific knock-in mouse model, we report here that inducible and targeted activation of the endogenous ERK5 in adult neural stem/progenitor cells enhances adult neurogenesis in the OB by increasing cell survival and neuronal differentiation. This conditional ERK5 activation also improves short-term olfactory memory and odor-cued associative olfactory learning under normal physiological conditions. Furthermore, these mice show enhanced recovery of olfactory function and have more adult-born neurons after a zinc sulfate-induced lesion of the main olfactory epithelium. We conclude that ERK5 MAP kinase is an important endogenous signaling pathway regulating adult neurogenesis in the SVZ/OB, and that conditional activation of endogenous ERK5 is sufficient to enhance adult neurogenesis in the OB thereby improving olfactory function both under normal conditions and after injury. PMID:25995470

  12. The Cav1.2 N terminus contains a CaM kinase site that modulates channel trafficking and function.

    Science.gov (United States)

    Simms, Brett A; Souza, Ivana A; Rehak, Renata; Zamponi, Gerald W

    2015-04-01

    The L-type voltage-gated calcium channel Cav1.2 and the calcium-activated CaM kinase cascade both regulate excitation transcription coupling in the brain. CaM kinase is known to associate with the C terminus of Cav1.2 in a region called the PreIQ-IQ domain, which also binds multiple calmodulin molecules. Here we identify and characterize a second CaMKII binding site in the N terminus of Cav1.2 that is formed by a stretch of four amino residues (cysteine-isoleucine-serine-isoleucine) and which regulates channel expression and function. By using live cell imaging of tsA-201 cells we show that GFP fusion constructs of the CaMKII binding region, termed N2B-II co-localize with mCherry-CaMKII. Mutating CISI to AAAA ablates binding to and colocalization with CaMKII. Cav1.2-AAAA channels show reduced cell surface expression in tsA-201 cells, but interestingly, display an increase in channel function that offsets the trafficking deficit. Altogether our data reveal that the proximal N terminus of Cav1.2 contains a CaMKII binding region which contributes to channel surface expression and function.

  13. Identification and Structure-Function Analysis of Subfamily Selective G Protein-Coupled Receptor Kinase Inhibitors

    Energy Technology Data Exchange (ETDEWEB)

    Homan, Kristoff T.; Larimore, Kelly M.; Elkins, Jonathan M.; Szklarz, Marta; Knapp, Stefan; Tesmer, John J.G. [Michigan; (Oxford)

    2015-02-13

    Selective inhibitors of individual subfamilies of G protein-coupled receptor kinases (GRKs) would serve as useful chemical probes as well as leads for therapeutic applications ranging from heart failure to Parkinson’s disease. To identify such inhibitors, differential scanning fluorimetry was used to screen a collection of known protein kinase inhibitors that could increase the melting points of the two most ubiquitously expressed GRKs: GRK2 and GRK5. Enzymatic assays on 14 of the most stabilizing hits revealed that three exhibit nanomolar potency of inhibition for individual GRKs, some of which exhibiting orders of magnitude selectivity. Most of the identified compounds can be clustered into two chemical classes: indazole/dihydropyrimidine-containing compounds that are selective for GRK2 and pyrrolopyrimidine-containing compounds that potently inhibit GRK1 and GRK5 but with more modest selectivity. The two most potent inhibitors representing each class, GSK180736A and GSK2163632A, were cocrystallized with GRK2 and GRK1, and their atomic structures were determined to 2.6 and 1.85 Å spacings, respectively. GSK180736A, developed as a Rho-associated, coiled-coil-containing protein kinase inhibitor, binds to GRK2 in a manner analogous to that of paroxetine, whereas GSK2163632A, developed as an insulin-like growth factor 1 receptor inhibitor, occupies a novel region of the GRK active site cleft that could likely be exploited to achieve more selectivity. However, neither compound inhibits GRKs more potently than their initial targets. This data provides the foundation for future efforts to rationally design even more potent and selective GRK inhibitors.

  14. HER2 oncogenic function escapes EGFR tyrosine kinase inhibitors via activation of alternative HER receptors in breast cancer cells.

    Directory of Open Access Journals (Sweden)

    Anthony Kong

    2008-08-01

    Full Text Available The response rate to EGFR tyrosine kinase inhibitors (TKIs may be poor and unpredictable in cancer patients with EGFR expression itself being an inadequate response indicator. There is limited understanding of the mechanisms underlying this resistance. Furthermore, although TKIs suppress the growth of HER2-overexpressing breast tumor cells, they do not fully inhibit HER2 oncogenic function at physiological doses.Here we have provided a molecular mechanism of how HER2 oncogenic function escapes TKIs' inhibition via alternative HER receptor activation as a result of autocrine ligand release. Using both Förster Resonance Energy Transfer (FRET which monitors in situ HER receptor phosphorylation as well as classical biochemical analysis, we have shown that the specific tyrosine kinase inhibitors (TKIs of EGFR, AG1478 and Iressa (Gefitinib decreased EGFR and HER3 phosphorylation through the inhibition of EGFR/HER3 dimerization. Consequent to this, we demonstrate that cleavage of HER4 and dimerization of HER4/HER2 occur together with reactivation of HER3 via HER2/HER3, leading to persistent HER2 phosphorylation in the now resistant, surviving cells. These drug treatment-induced processes were found to be mediated by the release of ligands including heregulin and betacellulin that activate HER3 and HER4 via HER2. Whereas an anti-betacellulin antibody in combination with Iressa increased the anti-proliferative effect in resistant cells, ligands such as heregulin and betacellulin rendered sensitive SKBR3 cells resistant to Iressa.These results demonstrate the role of drug-induced autocrine events leading to the activation of alternative HER receptors in maintaining HER2 phosphorylation and in mediating resistance to EGFR tyrosine kinase inhibitors (TKIs in breast cancer cells, and hence specify treatment opportunities to overcome resistance in patients.

  15. Structural and Functional Aspects of the Sensor Histidine Kinase PrrB from Mycobacterium tuberculosis

    DEFF Research Database (Denmark)

    Nowak, E.; Panjikar, S.; Morth, J.P.

    2006-01-01

    We describe the solution structures of two- and three-domain constructs of the sensor histidine kinase PrrB from Mycobacterium tuberculosis, which allow us to locate the HAMP linker relative to the ATP binding and dimerization domains. We show that the three-domain construct is active both...... for autophosphorylation and for phosphotransfer to the cognate response regulator, PrrA. We also describe the high-resolution crystal structure of the catalytic domain alone, and we show that, in solution, it binds ATP. The conformational flexibility of this domain is discussed and related to other structural information....

  16. Adhesive Categories

    DEFF Research Database (Denmark)

    Lack, Stephen; Sobocinski, Pawel

    2003-01-01

    We introduce adhesive categories, which are categories with structure ensuring that pushouts along monomorphisms are well-behaved. Many types of graphical structures used in computer science are shown to be examples of adhesive categories. Double-pushout graph rewriting generalises well...... to rewriting on arbitrary adhesive categories....

  17. Protein adhesives

    Science.gov (United States)

    Charles R. Frihart; Linda F. Lorenz

    2018-01-01

    Nature uses a wide variety of chemicals for providing adhesion internally (e.g., cell to cell) and externally (e.g., mussels to ships and piers). This adhesive bonding is chemically and mechanically complex, involving a variety of proteins, carbohydrates, and other compounds.Consequently,the effect of protein structures on adhesive properties is only partially...

  18. RP1 Is a Phosphorylation Target of CK2 and Is Involved in Cell Adhesion

    Science.gov (United States)

    Göttig, Stephan; Henschler, Reinhard; Markuly, Norbert; Kleber, Sascha; Faust, Michael; Mischo, Axel; Bauer, Stefan; Zweifel, Martin; Knuth, Alexander; Renner, Christoph; Wadle, Andreas

    2013-01-01

    RP1 (synonym: MAPRE2, EB2) is a member of the microtubule binding EB1 protein family, which interacts with APC, a key regulatory molecule in the Wnt signalling pathway. While the other EB1 proteins are well characterized the cellular function and regulation of RP1 remain speculative to date. However, recently RP1 has been implicated in pancreatic cancerogenesis. CK2 is a pleiotropic kinase involved in adhesion, proliferation and anti-apoptosis. Overexpression of protein kinase CK2 is a hallmark of many cancers and supports the malignant phenotype of tumor cells. In this study we investigate the interaction of protein kinase CK2 with RP1 and demonstrate that CK2 phosphorylates RP1 at Ser236 in vitro. Stable RP1 expression in cell lines leads to a significant cleavage and down-regulation of N-cadherin and impaired adhesion. Cells expressing a Phospho-mimicking point mutant RP1-ASP236 show a marked decrease of adhesion to endothelial cells under shear stress. Inversely, we found that the cells under shear stress downregulate endogenous RP1, most likely to improve cellular adhesion. Accordingly, when RP1 expression is suppressed by shRNA, cells lacking RP1 display significantly increased cell adherence to surfaces. In summary, RP1 phosphorylation at Ser236 by CK2 seems to play a significant role in cell adhesion and might initiate new insights in the CK2 and EB1 family protein association. PMID:23844040

  19. Loss-of-Function Mutations in SERPINB8 Linked to Exfoliative Ichthyosis with Impaired Mechanical Stability of Intercellular Adhesions.

    Science.gov (United States)

    Pigors, Manuela; Sarig, Ofer; Heinz, Lisa; Plagnol, Vincent; Fischer, Judith; Mohamad, Janan; Malchin, Natalia; Rajpopat, Shefali; Kharfi, Monia; Lestringant, Giles G; Sprecher, Eli; Kelsell, David P; Blaydon, Diana C

    2016-08-04

    SERPINS comprise a large and functionally diverse family of serine protease inhibitors. Here, we report three unrelated families with loss-of-function mutations in SERPINB8 in association with an autosomal-recessive form of exfoliative ichthyosis. Whole-exome sequencing of affected individuals from a consanguineous Tunisian family and a large Israeli family revealed a homozygous frameshift mutation, c.947delA (p.Lys316Serfs(∗)90), and a nonsense mutation, c.850C>T (p.Arg284(∗)), respectively. These two mutations are located in the last exon of SERPINB8 and, hence, would not be expected to lead to nonsense-mediated decay of the mRNA; nonetheless, both mutations are predicted to lead to loss of the reactive site loop of SERPINB8, which is crucial for forming the SERPINB8-protease complex. Using Sanger sequencing, a homozygous missense mutation, c.2T>C (p.Met1?), predicted to result in an N-terminal truncated protein, was identified in an additional family from UAE. Histological analysis of a skin biopsy from an individual homozygous for the variant p.Arg284(∗) showed disadhesion of keratinocytes in the lower epidermal layers plus decreased SERPINB8 levels compared to control. In vitro studies utilizing siRNA-mediated knockdown of SERPINB8 in keratinocytes demonstrated that in the absence of the protein, there is a cell-cell adhesion defect, particularly when cells are subjected to mechanical stress. In addition, immunoblotting and immunostaining revealed an upregulation of desmosomal proteins. In conclusion, we report mutations in SERPINB8 that are associated with exfoliative ichthyosis and provide evidence that SERPINB8 contributes to the mechanical stability of intercellular adhesions in the epidermis. Copyright © 2016 American Society of Human Genetics. Published by Elsevier Inc. All rights reserved.

  20. Effect on push-out bond strength of glass-fiber posts functionalized with polydopamine using different adhesives.

    Science.gov (United States)

    Chen, Qian; Cai, Qing; Li, Yan; Wei, Xu-Yi; Huang, Zhi; Wang, Xin-Zhi

    2014-04-01

    To evaluate the push-out bond strengths of prefabricated glass-fiber posts (Beijing Oya Biomaterials) with polydopamine functionalized to root dentin using two different resin cements (Paracore and RelyX Unicem) in different root regions (cervical, middle, and apical). Forty extracted human, single-rooted teeth were endodontically treated and a 9-mm post space was prepared in each tooth with post drills provided by the manufacturer. Specimens were then randomly assigned into four groups (n = 10 per group), depending on the adhesive system and post surface treatment used: group IA (Paracore + polydopamine); group IB (Paracore + control); group IIA (RelyX Unicem + polydopamine); group IIB (RelyX Unicem + control). Following post cementation, the specimens were stored in distilled water at 37°C for 7 days. The push-out test was performed using a universal testing machine (0.5 mm/ min), and the failure modes were examined with a stereomicroscope. Data were statistically analyzed using twoway ANOVA (p = 0.05). Bond strengths (mean ± SD) were: 7.909 ± 3.166 MPa (group IA), 4.675 ± 2.170 MPa (group IB), 8.186 ± 2.766 MPa (group IIA), 4.723 ± 2.084 MPa (group IIB). The bond strength of polydopamine groups was significantly higher than one of the control groups (p 0.05). Stereomicroscopic analysis showed a higher percentage of adhesive than cohesive failures in all groups. Surface polydopamine functionalization was confirmed to be a reliable method for improving the bond strength of resin luting agents to fiber posts. The bond strength of Paracore to fiber posts was not significantly different from that of RelyX Unicem, and considering its convenient application, Paracore can be recommended.

  1. Functional interaction between nonreceptor tyrosine kinase c-Abl and SR-Rich protein RBM39

    Energy Technology Data Exchange (ETDEWEB)

    Mai, Sanyue [Beijing Institute of Biotechnology, 27 Taiping Rd, Haidian District, Beijing 100850 (China); Qu, Xiuhua [General Navy Hospital of PLA, 6 Fucheng Rd, Haidian District, Beijing 100037 (China); Li, Ping; Ma, Qingjun [Beijing Institute of Biotechnology, 27 Taiping Rd, Haidian District, Beijing 100850 (China); Liu, Xuan, E-mail: liux931932@163.com [Beijing Institute of Biotechnology, 27 Taiping Rd, Haidian District, Beijing 100850 (China); Cao, Cheng, E-mail: cao_c@sohu.com [Beijing Institute of Biotechnology, 27 Taiping Rd, Haidian District, Beijing 100850 (China)

    2016-04-22

    RBM39, also known as splicing factor HCC1.4, acts as a transcriptional coactivator for the steroid nuclear receptors JUN/AP-1, ESR1/ER-α and ESR2/ER-β. RBM39 is involved in the regulation of the transcriptional responses of these steroid nuclear receptors and promotes transcriptional initiation. In this paper, we report that RBM39 interacts with the nonreceptor tyrosine kinase c-Abl. Both the Src homology (SH) 2 and SH3 domains of c-Abl interact with RBM39. The major tyrosine phosphorylation sites on RBM39 that are phosphorylated by c-Abl are Y95 and Y99, as demonstrated by liquid chromatography coupled with tandem mass spectrometry (LC/MS/MS) and mutational analysis. c-Abl was shown boost the transcriptional coactivation activity of RBM39 for ERα and PRβ in a tyrosine kinase-dependent manner. The results suggest that mammalian c-Abl plays an important role in steroid hormone receptor-mediated transcription by regulating RBM39. - Highlights: • c-Abl interacts with RBM39. • RBM39 is phosphorylated by c-Abl. • c-Abl regulates transcriptional coactivation activity of RBM39 on the ERα and PRβ.

  2. Functions of Calcium-Dependent Protein Kinases in Plant Innate Immunity

    Directory of Open Access Journals (Sweden)

    Xiquan Gao

    2014-03-01

    Full Text Available An increase of cytosolic Ca2+ is generated by diverse physiological stimuli and stresses, including pathogen attack. Plants have evolved two branches of the immune system to defend against pathogen infections. The primary innate immune response is triggered by the detection of evolutionarily conserved pathogen-associated molecular pattern (PAMP, which is called PAMP-triggered immunity (PTI. The second branch of plant innate immunity is triggered by the recognition of specific pathogen effector proteins and known as effector-triggered immunity (ETI. Calcium (Ca2+ signaling is essential in both plant PTI and ETI responses. Calcium-dependent protein kinases (CDPKs have emerged as important Ca2+ sensor proteins in transducing differential Ca2+ signatures, triggered by PAMPs or effectors and activating complex downstream responses. CDPKs directly transmit calcium signals by calcium binding to the elongation factor (EF-hand domain at the C-terminus and substrate phosphorylation by the catalytic kinase domain at the N-terminus. Emerging evidence suggests that specific and overlapping CDPKs phosphorylate distinct substrates in PTI and ETI to regulate diverse plant immune responses, including production of reactive oxygen species, transcriptional reprogramming of immune genes, and the hypersensitive response.

  3. Functional interaction between nonreceptor tyrosine kinase c-Abl and SR-Rich protein RBM39

    International Nuclear Information System (INIS)

    Mai, Sanyue; Qu, Xiuhua; Li, Ping; Ma, Qingjun; Liu, Xuan; Cao, Cheng

    2016-01-01

    RBM39, also known as splicing factor HCC1.4, acts as a transcriptional coactivator for the steroid nuclear receptors JUN/AP-1, ESR1/ER-α and ESR2/ER-β. RBM39 is involved in the regulation of the transcriptional responses of these steroid nuclear receptors and promotes transcriptional initiation. In this paper, we report that RBM39 interacts with the nonreceptor tyrosine kinase c-Abl. Both the Src homology (SH) 2 and SH3 domains of c-Abl interact with RBM39. The major tyrosine phosphorylation sites on RBM39 that are phosphorylated by c-Abl are Y95 and Y99, as demonstrated by liquid chromatography coupled with tandem mass spectrometry (LC/MS/MS) and mutational analysis. c-Abl was shown boost the transcriptional coactivation activity of RBM39 for ERα and PRβ in a tyrosine kinase-dependent manner. The results suggest that mammalian c-Abl plays an important role in steroid hormone receptor-mediated transcription by regulating RBM39. - Highlights: • c-Abl interacts with RBM39. • RBM39 is phosphorylated by c-Abl. • c-Abl regulates transcriptional coactivation activity of RBM39 on the ERα and PRβ.

  4. Functional Analysis of the Aurora Kinase A Ile31 Allelic Variant in Human Prostate

    Directory of Open Access Journals (Sweden)

    Noa Matarasso

    2007-09-01

    Full Text Available Overexpression of the centrosome-associated serine/ threonine kinase Aurora Kinase A (AURKA has been demonstrated in both advanced prostate cancer and high-grade prostatic intraepithelial neoplasia lesions. The single-nucleotide polymorphism T91A (Phe3lile has been implicated in AURKA overexpression and has been suggested as a low-penetrance susceptibility allele in multiple human cancers, including prostate cancer. We studied the transcriptional consequences of the AURKA Ile31 allele in 28 commercial normal prostate tissue RNA samples (median age, 27 years. Significant overexpression of AURKA was demonstrated in homozygous and heterozygous AURKA Ile31 prostate RNA (2.07-fold and 1.93-fold, respectively; P < .05. Expression levels of 1509 genes differentiated between samples homozygous for Phe31 alleles and samples homozygous for Ile31 alleles (P = .05. Gene Ontology classification revealed overrepresentation of cell cycle arrest, ubiquitin cycle, antiapoptosis, angiogenesisrelated genes. When these hypothesis-generating results were subjected to more stringent statistical criteria, overexpression of a novel transcript of the natural killer tumor recognition sequence (NKTR gene was revealed and validated in homozygous Ile31 samples (2.6-fold; P < .05. In summary, our data suggest an association between the AURKA Ile31 allele and an altered transcriptome in normal non-neopasic prostates.

  5. Bruton's tyrosine kinase and phospholipase C gamma 2 mediate chemokine-controlled B cell migration and homing

    NARCIS (Netherlands)

    de Gorter, David J. J.; Beuling, Esther A.; Kersseboom, Rogier; Middendorp, Sabine; van Gils, Janine M.; Hendriks, Rudolf W.; Pals, Steven T.; Spaargaren, Marcel

    2007-01-01

    Control of integrin-mediated adhesion and migration by chemokines plays a critical role in B cell development, differentiation, and function; however, the underlying signaling mechanisms are poorly defined. Here we show that the chemokine SDF-1 induced activation of Bruton's tyrosine kinase (Btk)

  6. Are pelvic adhesions associated with pain, physical, emotional and functional characteristics of women presenting with chronic pelvic pain? A cluster analysis.

    Science.gov (United States)

    Cheong, Ying; Saran, Mili; Hounslow, James William; Reading, Isabel Claire

    2018-01-08

    Chronic pelvic pain is a debilitating condition. It is unknown if there is a clinical phenotype for adhesive disorders. This study aimed to determine if the presence or absence, nature, severity and extent of adhesions correlated with demographic and patient reported clinical characteristics of women presenting with CPP. Women undergoing a laparoscopy for the investigation of chronic pelvic pain were recruited prospectively; their pain and phenotypic characteristics were entered into a hierarchical cluster analysis. The groups with differing baseline clinical and operative characteristics in terms of adhesions involvement were analyzed. Sixty two women were recruited where 37 had adhesions. A low correlation was found between women's reported current pain scores and that of most severe (r = 0.34) or average pain experienced (r = 0.44) in the last 6 months. Three main groups of women with CPP were identified: Cluster 1 (n = 35) had moderate severity of pain, with poor average and present pain intensity; Cluster 2 (n = 14) had a long duration of symptoms/diagnosis, the worst current pain and worst physical, emotional and social functions; Cluster 3 (n = 11) had the shortest duration of pain and showed the best evidence of coping with low (good) physical, social and emotional scores. This cluster also had the highest proportion of women with adhesions (82%) compared to 51% in Cluster 1 and 71% in Cluster 2. In this study, we found that there is little or no correlation between patient-reported pain, physical, emotional and functional characteristics scores with the presence or absence of intra-abdominal/pelvic adhesions found during investigative laparoscopy. Most women who had adhesions had the lowest reported current pain scores.

  7. Functional aspects of the EGF-induced MAP kinase cascade: a complex self-organizing system approach.

    Directory of Open Access Journals (Sweden)

    Efstratios K Kosmidis

    Full Text Available The EGF-induced MAP kinase cascade is one of the most important and best characterized networks in intracellular signalling. It has a vital role in the development and maturation of living organisms. However, when deregulated, it is involved in the onset of a number of diseases. Based on a computational model describing a "surface" and an "internalized" parallel route, we use systems biology techniques to characterize aspects of the network's functional organization. We examine the re-organization of protein groups from low to high external stimulation, define functional groups of proteins within the network, determine the parameter best encoding for input intensity and predict the effect of protein removal to the system's output response. Extensive functional re-organization of proteins is observed in the lower end of stimulus concentrations. As we move to higher concentrations the variability is less pronounced. 6 functional groups have emerged from a consensus clustering approach, reflecting different dynamical aspects of the network. Mutual information investigation revealed that the maximum activation rate of the two output proteins best encodes for stimulus intensity. Removal of each protein of the network resulted in a range of graded effects, from complete silencing to intense activation. Our results provide a new "vista" of the EGF-induced MAP kinase cascade, from the perspective of complex self-organizing systems. Functional grouping of the proteins reveals an organizational scheme contrasting the current understanding of modular topology. The six identified groups may provide the means to experimentally follow the dynamics of this complex network. Also, the vulnerability analysis approach may be used for the development of novel therapeutic targets in the context of personalized medicine.

  8. Germline deletion of AMP-activated protein kinase β subunits reduces bone mass without altering osteoclast differentiation or function

    Science.gov (United States)

    Quinn, Julian M. W.; Tam, Shanna; Sims, Natalie A.; Saleh, Hasnawati; McGregor, Narelle E.; Poulton, Ingrid J.; Scott, John W.; Gillespie, Matthew T.; Kemp, Bruce E.; van Denderen, B. J. W.

    2010-01-01

    Since AMP-activated protein kinase (AMPK) plays important roles in modulating metabolism in response to diet and exercise, both of which influence bone mass, we examined the influence of AMPK on bone mass in mice. AMPK is an αβγ heterotrimer where the β subunit anchors the α catalytic and γ regulatory subunits. Germline deletion of either AMPK β1 or β2 subunit isoforms resulted in reduced trabecular bone density and mass, but without effects on osteoclast (OC) or osteoblast (OB) numbers, as compared to wild-type littermate controls. We tested whether activating AMPK in vivo would enhance bone density but found AICA-riboside treatment caused a profound loss of trabecular bone volume (49.5%) and density and associated increased OC numbers. Consistent with this, AICA-riboside strongly stimulated OC differentiation in vitro, in an adenosine kinase-dependent manner. OCs and macrophages (unlike OBs) lacked AMPK β2 subunit expression, and when generated from AMPK β1−/− mice displayed no detectable AMPK activity. Nevertheless, AICA-riboside was equally effective at stimulating OC differentiation from wild-type or β1−/− progenitors, indicating that AMPK is not essential for OC differentiation or the stimulatory action of AICA-riboside. These results show that AMPK is required to maintain normal bone density, but not through bone cell differentiation, and does not mediate powerful osteolytic effects of AICA-riboside.—Quinn, J. M. W., Tam, S., Sims, N. A., Saleh, H., McGregor, N. E., Poulton, I. J., Scott, J. W., Gillespie, M. T., Kemp, B. E., van Denderen, B. J. W. Germline deletion of AMP-activated protein kinase β subunits reduces bone mass without altering osteoclast differentiation or function. PMID:19723702

  9. Neuron Membrane Trafficking and Protein Kinases Involved in Autism and ADHD

    Directory of Open Access Journals (Sweden)

    Yasuko Kitagishi

    2015-01-01

    Full Text Available A brain-enriched multi-domain scaffolding protein, neurobeachin has been identified as a candidate gene for autism patients. Mutations in the synaptic adhesion protein cell adhesion molecule 1 (CADM1 are also associated with autism spectrum disorder, a neurodevelopmental disorder of uncertain molecular origin. Potential roles of neurobeachin and CADM1 have been suggested to a function of vesicle transport in endosomal trafficking. It seems that protein kinase B (AKT and cyclic adenosine monophosphate (cAMP-dependent protein kinase A (PKA have key roles in the neuron membrane trafficking involved in the pathogenesis of autism. Attention deficit hyperactivity disorder (ADHD is documented to dopaminergic insufficiencies, which is attributed to synaptic dysfunction of dopamine transporter (DAT. AKT is also essential for the DAT cell-surface redistribution. In the present paper, we summarize and discuss the importance of several protein kinases that regulate the membrane trafficking involved in autism and ADHD, suggesting new targets for therapeutic intervention.

  10. Glycogen synthase kinase-3β activity and cognitive functioning in patients with bipolar I disorder

    DEFF Research Database (Denmark)

    Munkholm, Klaus; Miskowiak, Kamilla Woznica; Jacoby, Anne Sophie

    2018-01-01

    Cognitive deficits are common in patients with bipolar disorder (BD) in remission and may be associated with glycogen synthase kinase-3 (GSK-3) activity, which is inhibited by lithium. GSK-3 may be a relevant treatment target for interventions tailored at cognitive disturbances in BD...... ratio (serine-9-pGSK-3β /total GSK-3β), was negatively associated with sustained attention (p = 0.009 and p = 0.042, respectively), but not with other cognitive domains or global cognition. A crossover interaction between lithium treatment and the GSK activity was observed, indicating that lower pGSK-3β...... but the relation between GSK-3 activity, cognition and lithium treatment is unknown. We therefore investigated the possible association between GSK-3 activity and cognition and whether lithium treatment moderates this association in patients with BD. In a prospective 6-12 month follow-up study, GSK- 3β activity...

  11. Biomimetic Mussel Adhesive Inspired Clickable Anchors Applied to the Functionalization of Fe3O4 Nanoparticles

    NARCIS (Netherlands)

    Goldmann, Anja S.; Schoedel, Christine; Walther, Andreas; Yuan, Jiayin; Loos, Katja; Mueller, Axel H. E.; Müller, Axel H.E.

    2010-01-01

    The functionalization of magnetite (Fe3O4) nanoparticles with dopamine-derived clickable biomimetic anchors is reported. Herein, an alkyne-modified catechol-derivative is employed as the anchor, as i) the catechol-functional anchor groups possess irreversible covalent binding affinity to Fe3O4

  12. Facile fabrication of functional PDMS surfaces with tunable wettablity and high adhesive force via femtosecond laser textured templating

    Directory of Open Access Journals (Sweden)

    Yanlei Hu

    2014-12-01

    Full Text Available Femtosecond laser processing is emerged as a promising tool to functionalize surfaces of various materials, including metals, semiconductors, and polymers. However, the productivity of this technique is limited by the low efficiency of laser raster scanning. Here we report a facile approach for efficiently producing large-area functional polymer surfaces, by which metal is firstly textured by a femtosecond laser, and the as-prepared hierarchical structures are subsequently transferred onto polydimethylsiloxane (PDMS surfaces. Aluminum pieces covered by laser induced micro/nano-structures act as template masters and their performance of displaying diverse colors are investigated. Polymer replicas are endowed with tunable wetting properties, which are mainly attributed to the multi-scale surface structures. Furthermore, the surfaces are found to have extremely high adhesive force for water drops because of the high water penetration depth and the resultant high contact angle hysteresis. This characteristic facilitates many potential applications like loss-free tiny water droplets transportation. The reusability of metal master and easiness of soft lithography make it to be a very simple, fast and cost-efficient way for mass production of functional polymeric surfaces.

  13. Inhibition of tumor vasculogenic mimicry and prolongation of host survival in highly aggressive gallbladder cancers by norcantharidin via blocking the ephrin type a receptor 2/focal adhesion kinase/paxillin signaling pathway.

    Directory of Open Access Journals (Sweden)

    Hui Wang

    Full Text Available Vasculogenic mimicry (VM is a newly-defined tumor microcirculation pattern in highly aggressive malignant tumors. We recently reported tumor growth and VM formation of gallbladder cancers through the contribution of the ephrin type a receptor 2 (EphA2/focal adhesion kinase (FAK/Paxillin signaling pathways. In this study, we further investigated the anti-VM activity of norcantharidin (NCTD as a VM inhibitor for gallbladder cancers and the underlying mechanisms. In vivo and in vitro experiments to determine the effects of NCTD on tumor growth, host survival, VM formation of GBC-SD nude mouse xenografts, and vasculogenic-like networks, malignant phenotypes i.e., proliferation, apoptosis, invasion and migration of GBC-SD cells. Expression of VM signaling-related markers EphA2, FAK and Paxillin in vivo and in vitro were examined by immunofluorescence, western blotting and real-time polymerase chain reaction (RT-PCR, respectively. The results showed that after treatment with NCTD, GBC-SD cells were unable to form VM structures when injecting into nude mouse, growth of the xenograft was inhibited and these observations were confirmed by facts that VM formation by three-dimensional (3-D matrix, proliferation, apoptosis, invasion, migration of GBC-SD cells were affected; and survival time of the xenograft mice was prolonged. Furthermore, expression of EphA2, FAK and Paxillin proteins/mRNAs of the xenografts was downregulated. Thus, we concluded that NCTD has potential anti-VM activity against human gallbladder cancers; one of the underlying mechanisms may be via blocking the EphA2/FAK/Paxillin signaling pathway.

  14. Corticosteroid Injections Accelerate Pain Relief and Recovery of Function Compared With Oral NSAIDs in Patients With Adhesive Capsulitis: A Randomized Controlled Trial.

    Science.gov (United States)

    Ranalletta, Maximiliano; Rossi, Luciano A; Bongiovanni, Santiago L; Tanoira, Ignacio; Elizondo, Cristina M; Maignon, Gastón D

    2016-02-01

    Intra-articular corticosteroid injection is a common therapy for adhesive capsulitis, but there is a lack of prospective randomized controlled studies analyzing the efficacy of single injections applied blindly to accelerate improvement in pain and function. In patients with adhesive capsulitis, a single intra-articular corticosteroid injection without image control applied before the beginning of a physical therapy program will accelerate pain relief and recovery of function compared with oral nonsteroidal anti-inflammatory drugs (NSAIDs) and physical therapy. Randomized controlled trial; Level of evidence, 1. A total of 74 patients with primary adhesive capsulitis in the freezing stage were randomized to receive either intra-articular injections with betamethasone or oral NSAIDs. Clinical outcome was documented at baseline and after 2, 4, 8, and 12 weeks and comprised a visual analog scale (VAS) for pain, the American Shoulder and Elbow Surgeons (ASES) Shoulder Score, the abbreviated Constant-Murley score, and the abbreviated Disabilities of the Arm, Shoulder and Hand (QuickDASH) score for function. Passive range of motion was measured with a goniometer. Patients treated with corticosteroid injections achieved faster pain relief compared with control patients during the first 8 weeks after treatment (P adhesive capsulitis, a single corticosteroid injection applied without image control provides faster pain relief and earlier improvement of shoulder function and motion compared with oral NSAIDs. © 2015 The Author(s).

  15. Effect of various surface conditioning methods on the adhesion of dual-cure resin cement with MDP functional monomer to zirconia after thermal aging

    NARCIS (Netherlands)

    Ozcan, Mutlu; Nijhuis, Henk; Felipe Valandro, Luiz

    This study evaluated the effect of chairside and laboratory types of surface conditioning methods on the adhesion of dual-cure resin cement with MDP functional monomer to zirconia ceramic after thermocycling. Disk-shaped (diameter: 10 mm, thickness: 2 mm) Y-TZP ceramics (Lava(TM), 3M ESPE) were used

  16. Switchable bio-inspired adhesives

    Science.gov (United States)

    Kroner, Elmar

    2015-03-01

    Geckos have astonishing climbing abilities. They can adhere to almost any surface and can run on walls and even stick to ceilings. The extraordinary adhesion performance is caused by a combination of a complex surface pattern on their toes and the biomechanics of its movement. These biological dry adhesives have been intensely investigated during recent years because of the unique combination of adhesive properties. They provide high adhesion, allow for easy detachment, can be removed residue-free, and have self-cleaning properties. Many aspects have been successfully mimicked, leading to artificial, bio-inspired, patterned dry adhesives, and were addressed and in some aspects they even outperform the adhesion capabilities of geckos. However, designing artificial patterned adhesion systems with switchable adhesion remains a big challenge; the gecko's adhesion system is based on a complex hierarchical surface structure and on advanced biomechanics, which are both difficult to mimic. In this paper, two approaches are presented to achieve switchable adhesion. The first approach is based on a patterned polydimethylsiloxane (PDMS) polymer, where adhesion can be switched on and off by applying a low and a high compressive preload. The switch in adhesion is caused by a reversible mechanical instability of the adhesive silicone structures. The second approach is based on a composite material consisting of a Nickel- Titanium (NiTi) shape memory alloy and a patterned adhesive PDMS layer. The NiTi alloy is trained to change its surface topography as a function of temperature, which results in a change of the contact area and of alignment of the adhesive pattern towards a substrate, leading to switchable adhesion. These examples show that the unique properties of bio-inspired adhesives can be greatly improved by new concepts such as mechanical instability or by the use of active materials which react to external stimuli.

  17. Inducible activation of ERK5 MAP kinase enhances adult neurogenesis in the olfactory bulb and improves olfactory function.

    Science.gov (United States)

    Wang, Wenbin; Lu, Song; Li, Tan; Pan, Yung-Wei; Zou, Junhui; Abel, Glen M; Xu, Lihong; Storm, Daniel R; Xia, Zhengui

    2015-05-20

    Recent discoveries have suggested that adult neurogenesis in the subventricular zone (SVZ) and olfactory bulb (OB) may be required for at least some forms of olfactory behavior in mice. However, it is unclear whether conditional and selective enhancement of adult neurogenesis by genetic approaches is sufficient to improve olfactory function under physiological conditions or after injury. Furthermore, specific signaling mechanisms regulating adult neurogenesis in the SVZ/OB are not fully defined. We previously reported that ERK5, a MAP kinase selectively expressed in the neurogenic regions of the adult brain, plays a critical role in adult neurogenesis in the SVZ/OB. Using a site-specific knock-in mouse model, we report here that inducible and targeted activation of the endogenous ERK5 in adult neural stem/progenitor cells enhances adult neurogenesis in the OB by increasing cell survival and neuronal differentiation. This conditional ERK5 activation also improves short-term olfactory memory and odor-cued associative olfactory learning under normal physiological conditions. Furthermore, these mice show enhanced recovery of olfactory function and have more adult-born neurons after a zinc sulfate-induced lesion of the main olfactory epithelium. We conclude that ERK5 MAP kinase is an important endogenous signaling pathway regulating adult neurogenesis in the SVZ/OB, and that conditional activation of endogenous ERK5 is sufficient to enhance adult neurogenesis in the OB thereby improving olfactory function both under normal conditions and after injury. Copyright © 2015 the authors 0270-6474/15/357833-17$15.00/0.

  18. Preparation, Characterization and Mechanical Properties of Bio-Based Polyurethane Adhesives from Isocyanate-Functionalized Cellulose Acetate and Castor Oil for Bonding Wood

    Directory of Open Access Journals (Sweden)

    Adrián Tenorio-Alfonso

    2017-04-01

    Full Text Available Nowadays, different types of natural carbohydrates such as sugars, starch, cellulose and their derivatives are widely used as renewable raw materials. Vegetable oils are also considered as promising raw materials to be used in the synthesis of high quality products in different applications, including in the adhesive field. According to this, several bio-based formulations with adhesion properties were synthesized first by inducing the functionalization of cellulose acetate with 1,6-hexamethylene diisocyanate and then mixing the resulting biopolymer with a variable amount of castor oil, from 20% to 70% (wt. These bio-based adhesives were mechanically characterized by means of small-amplitude oscillatory torsion measurements, at different temperatures, and standardized tests to evaluate tension loading (ASTM-D906 and peel strength (ASTM-D903. In addition, thermal properties and stability of the synthesized bio-polyurethane formulations were also analyzed through differential scanning calorimetry and thermal gravimetric analysis. As a result, the performance of these bio-polyurethane products as wood adhesives were compared and analyzed. Bio-polyurethane formulations exhibited a simple thermo-rheological behavior below a critical temperature of around 80–100 °C depending on the castor oil/cellulose acetate weight ratio. Formulation with medium castor oil/biopolymer weight ratio (50:50 % wt showed the most suitable mechanical properties and adhesion performance for bonding wood.

  19. Structural and Functional Analysis of the Escherichia coli Acid-Sensing Histidine Kinase EvgS.

    Science.gov (United States)

    Sen, Hrishiraj; Aggarwal, Nikhil; Ishionwu, Chibueze; Hussain, Nosheen; Parmar, Chandni; Jamshad, Mohammed; Bavro, Vassiliy N; Lund, Peter A

    2017-09-15

    The EvgS/EvgA two-component system of Escherichia coli is activated in response to low pH and alkali metals and regulates many genes, including those for the glutamate-dependent acid resistance system and a number of efflux pumps. EvgS, the sensor kinase, is one of five unconventional histidine kinases (HKs) in E. coli and has a large periplasmic domain and a cytoplasmic PAS domain in addition to phospho-acceptor, HK and dimerization, internal receiver, and phosphotransfer domains. Mutations that constitutively activate the protein at pH 7 map to the PAS domain. Here, we built a homology model of the periplasmic region of EvgS, based on the structure of the equivalent region of the BvgS homologue, to guide mutagenesis of potential key residues in this region. We show that histidine 226 is required for induction and that it is structurally colocated with a proline residue (P522) at the top of the predicted transmembrane helix that is expected to play a key role in passing information to the cytoplasmic domains. We also show that the constitutive mutations in the PAS domain can be further activated by low external pH. Expression of the cytoplasmic part of the protein alone also gives constitutive activation, which is lost if the constitutive PAS mutations are present. These findings are consistent with a model in which EvgS senses both external and internal pH and is activated by a shift from a tight inactive to a weak active dimer, and we present an analysis of the purified cytoplasmic portion of EvgS that supports this. IMPORTANCE One of the ways bacteria sense their environment is through two-component systems, which have one membrane-bound protein to do the sensing and another inside the cell to turn genes on or off in response to what the membrane-bound protein has detected. The membrane-bound protein must thus be able to detect the stress and signal this detection event to the protein inside the cell. To understand this process, we studied a protein that helps

  20. Spatiotemporal distribution and function of N-cadherin in postnatal Schwann cells: A matter of adhesion?

    DEFF Research Database (Denmark)

    Corell, Mikael; Wicher, Grzegorz; Limbach, Christoph

    2010-01-01

    ), and myenteric plexi. In the sciatic nerve, N-cadherin decreases with age and progress of myelination. In adult animals, N-cadherin was found exclusively in nonmyelinating Schwann cells. The distribution of N-cadherin in developing E17 DRG primary cultures is similar to what was observed in vivo. Functional...

  1. Spatiotemporal and functional characterisation of the Plasmodium falciparum cGMP-dependent protein kinase.

    Directory of Open Access Journals (Sweden)

    Christine S Hopp

    Full Text Available Signalling by 3'-5'-cyclic guanosine monophosphate (cGMP exists in virtually all eukaryotes. In the apicomplexan parasite Plasmodium, the cGMP-dependent protein kinase (PKG has previously been reported to play a critical role in four key stages of the life cycle. The Plasmodium falciparum isoform (PfPKG is essential for the initiation of gametogenesis and for blood stage schizont rupture and work on the orthologue from the rodent malaria parasite P. berghei (PbPKG has shown additional roles in ookinete differentiation and motility as well as liver stage schizont development. In the present study, PfPKG expression and subcellular location in asexual blood stages was investigated using transgenic epitope-tagged PfPKG-expressing P. falciparum parasites. In Western blotting experiments and immunofluorescence analysis (IFA, maximal PfPKG expression was detected at the late schizont stage. While IFA suggested a cytosolic location, a degree of overlap with markers of the endoplasmic reticulum (ER was found and subcellular fractionation showed some association with the peripheral membrane fraction. This broad localisation is consistent with the notion that PfPKG, as with the mammalian orthologue, has numerous cellular substrates. This idea is further supported by the global protein phosphorylation pattern of schizonts which was substantially changed following PfPKG inhibition, suggesting a complex role for PfPKG during schizogony.

  2. Optimization of ATP synthase function in mitochondria and chloroplasts via the adenylate kinase equilibrium

    Directory of Open Access Journals (Sweden)

    Abir U Igamberdiev

    2015-01-01

    Full Text Available The bulk of ATP synthesis in plants is performed by ATP synthase, the main bioenergetics engine of cells, operating both in mitochondria and in chloroplasts. The reaction mechanism of ATP synthase has been studied in detail for over half a century; however, its optimal performance depends also on the steady delivery of ATP synthase substrates and the removal of its products. For mitochondrial ATP synthase, we analyze here the provision of stable conditions for (i the supply of ADP and Mg2+, supported by adenylate kinase (AK equilibrium in the intermembrane space, (ii the supply of phosphate via membrane transporter in symport with H+, and (iii the conditions of outflow of ATP by adenylate transporter carrying out the exchange of free adenylates. We also show that, in chloroplasts, AK equilibrates adenylates and governs Mg2+ contents in the stroma, optimizing ATP synthase and Calvin cycle operation, and affecting the import of inorganic phosphate in exchange with triose phosphates. It is argued that chemiosmosis is not the sole component of ATP synthase performance, which also depends on AK-mediated equilibrium of adenylates and Mg2+, adenylate transport and phosphate release and supply.

  3. Rho-associated kinase (ROCK) function is essential for cell cycle progression, senescence and tumorigenesis.

    Science.gov (United States)

    Kümper, Sandra; Mardakheh, Faraz K; McCarthy, Afshan; Yeo, Maggie; Stamp, Gordon W; Paul, Angela; Worboys, Jonathan; Sadok, Amine; Jørgensen, Claus; Guichard, Sabrina; Marshall, Christopher J

    2016-01-14

    Rho-associated kinases 1 and 2 (ROCK1/2) are Rho-GTPase effectors that control key aspects of the actin cytoskeleton, but their role in proliferation and cancer initiation or progression is not known. Here, we provide evidence that ROCK1 and ROCK2 act redundantly to maintain actomyosin contractility and cell proliferation and that their loss leads to cell-cycle arrest and cellular senescence. This phenotype arises from down-regulation of the essential cell-cycle proteins CyclinA, CKS1 and CDK1. Accordingly, while the loss of either Rock1 or Rock2 had no negative impact on tumorigenesis in mouse models of non-small cell lung cancer and melanoma, loss of both blocked tumor formation, as no tumors arise in which both Rock1 and Rock2 have been genetically deleted. Our results reveal an indispensable role for ROCK, yet redundant role for isoforms 1 and 2, in cell cycle progression and tumorigenesis, possibly through the maintenance of cellular contractility.

  4. In Vitro Functional Study of Rice Adenosine 5’-Phosphosulfate Kinase

    Directory of Open Access Journals (Sweden)

    Wang De-zhen

    2016-05-01

    Full Text Available Sulfate can be activated by ATP sulfurylase and adenosine 5’-phosphosulfate kinase (APSK in vivo. Recent studies suggested that APSK in Arabidopsis thaliana regulated the partition between APS reduction and phosphorylation and its activity can be modulated by cellular redox status. In order to study regulation of APSK in rice (OsAPSK, OsAPSK1 gene was cloned and its activity was analyzed. OsAPSK1 C36 and C69 were found to be the conserved counterparts of C86 and C119, which involved in disulfide formation in AtAPSK. C36A/C69A OsAPSK1 double mutation was made by site directed mutagenesis. OsAPSK1 and its mutant were prokaryotically over-expressed and purified, and then assayed for APS phosphorylation activity. OsAPSK1 activity was depressed by oxidized glutathione, while the activity of its mutant was not. Further studies in the case that oxidative stress will fluctuate in vivo 3’-phosphoadenosine-5’-phosphosulfate content, and all APSK isoenzymes have similar regulation patterns are necessary to be performed.

  5. Regulation of Discrete Functional Responses by Syk and Src Family Tyrosine Kinases in Human Neutrophils

    Directory of Open Access Journals (Sweden)

    Thornin Ear

    2017-01-01

    Full Text Available Neutrophils play a critical role in innate immunity and also influence adaptive immune responses. This occurs in good part through their production of inflammatory and immunomodulatory cytokines, in conjunction with their prolonged survival at inflamed foci. While a picture of the signaling machinery underlying these neutrophil responses is now emerging, much remains to be uncovered. In this study, we report that neutrophils constitutively express various Src family isoforms (STKs, as well as Syk, and that inhibition of these protein tyrosine kinases selectively hinders inflammatory cytokine generation by acting posttranscriptionally. Accordingly, STK or Syk inhibition decreases the phosphorylation of signaling intermediates (e.g., eIF-4E, S6K, and MNK1 involved in translational control. By contrast, delayed apoptosis appears to be independent of either STKs or Syk. Our data therefore significantly extend our understanding of which neutrophil responses are governed by STKs and Syk and pinpoint some signaling intermediates that are likely involved. In view of the foremost role of neutrophils in several chronic inflammatory conditions, our findings identify potential molecular targets that could be exploited for future therapeutic intervention.

  6. TEC family kinases in health and disease--loss-of-function of BTK and ITK and the gain-of-function fusions ITK-SYK and BTK-SYK.

    Science.gov (United States)

    Hussain, Alamdar; Yu, Liang; Faryal, Rani; Mohammad, Dara K; Mohamed, Abdalla J; Smith, C I Edvard

    2011-06-01

    The TEC family is ancient and constitutes the second largest family of cytoplasmic tyrosine kinases. In 1993, loss-of-function mutations in the BTK gene were reported as the cause of X-linked agammaglobulinemia. Of all the existing 90 tyrosine kinases in humans, Bruton's tyrosine kinase (BTK) is the kinase for which most mutations have been identified. These experiments of nature collectively provide a form of mutation scanning with direct implications for the several hundred endogenous signaling proteins carrying domains also found in BTK. In 2009, an inactivating mutation in the ITK gene was shown to cause susceptibility to lethal Epstein-Barr virus infection. Both kinases represent interesting targets for inhibition: in the case of BTK, as an immunosuppressant, whereas there is evidence that the inhibition of inducible T-cell kinase (ITK) could influence the infectivity of HIV and also have anti-inflammatory activity. Since 2006, several patients carrying a fusion protein, originating from a translocation joining genes encoding the kinases ITK and spleen tyrosine kinase (SYK), have been shown to develop T-cell lymphoma. We review these disease processes and also describe the role of the N-terminal pleckstrin homology-Tec homology (PH-TH) domain doublet of BTK and ITK in the downstream intracellular signaling of such fusion proteins. © 2011 The Authors Journal compilation © 2011 FEBS.

  7. Temporal quantitation of mutant Kit tyrosine kinase signaling attenuated by a novel thiophene kinase inhibitor OSI-930.

    Science.gov (United States)

    Petti, Filippo; Thelemann, April; Kahler, Jen; McCormack, Siobhan; Castaldo, Linda; Hunt, Tony; Nuwaysir, Lydia; Zeiske, Lynn; Haack, Herbert; Sullivan, Laura; Garton, Andrew; Haley, John D

    2005-08-01

    OSI-930, a potent thiophene inhibitor of the Kit, KDR, and platelet-derived growth factor receptor tyrosine kinases, was used to selectively inhibit tyrosine phosphorylation downstream of juxtamembrane mutant Kit in the mast cell leukemia line HMC-1. Inhibition of Kit kinase activity resulted in a rapid dephosphorylation of Kit and inhibition of the downstream signaling pathways. Attenuation of Ras-Raf-Erk (phospho-Erk, phospho-p38), phosphatidyl inositol-3' kinase (phospho-p85, phospho-Akt, phospho-S6), and signal transducers and activators of transcription signaling pathways (phospho-STAT3/5/6) were measured by affinity liquid chromatography tandem mass spectrometry, by immunoblot, and by tissue microarrays of fixed cell pellets. To more globally define additional components of Kit signaling temporally altered by kinase inhibition, a novel multiplex quantitative isobaric peptide labeling approach was used. This approach allowed clustering of proteins by temporal expression patterns. Kit kinase, which dephosphorylates rapidly upon kinase inhibition, was shown to regulate both Shp-1 and BDP-1 tyrosine phosphatases and the phosphatase-interacting protein PSTPIP2. Interactions with SH2 domain adapters [growth factor receptor binding protein 2 (Grb2), Cbl, Slp-76] and SH3 domain adapters (HS1, cortactin, CD2BP3) were attenuated by inhibition of Kit kinase activity. Functional crosstalk between Kit and the non-receptor tyrosine kinases Fes/Fps, Fer, Btk, and Syk was observed. Inhibition of Kit modulated phosphorylation-dependent interactions with pathways controlling focal adhesion (paxillin, leupaxin, p130CAS, FAK1, the Src family kinase Lyn, Wasp, Fhl-3, G25K, Ack-1, Nap1, SH3P12/ponsin) and septin-actin complexes (NEDD5, cdc11, actin). The combined use of isobaric protein quantitation and expression clustering, immunoblot, and tissue microarray strategies allowed temporal measurement signaling pathways modulated by mutant Kit inhibition in a model of mast cell

  8. Multi-functional electrospun antibacterial core-shell nanofibrous membranes for prolonged prevention of post-surgical tendon adhesion and inflammation.

    Science.gov (United States)

    Shalumon, K T; Sheu, Chialin; Chen, Chih-Hao; Chen, Shih-Heng; Jose, Gils; Kuo, Chang-Yi; Chen, Jyh-Ping

    2018-04-05

    The possibility of endowing an electrospun anti-adhesive barrier membrane with multi-functionality, such as lubrication, prevention of fibroblast attachment and anti-infection and anti-inflammation properties, is highly desirable for the management of post-surgical tendon adhesion. To this end, we fabricated core-shell nanofibrous membranes (CSNMs) with embedded silver nanoparticles (Ag NPs) in the poly(ethylene glycol) (PEG)/poly(caprolactone) (PCL) shell and hyaluronic acid (HA)/ibuprofen in the core. HA imparted a lubrication effect for smooth tendon gliding and reduced fibroblast attachment, while Ag NPs and ibuprofen functioned as anti-infection and anti-inflammation agents, respectively. CSNMs with a PEG/PCL/Ag shell (PPA) and HA core containing 0% (H/PPA), 10% (HI10/PPA), 30% (HI30/PPA) and 50% (HI50/PPA) ibuprofen were fabricated through co-axial electrospinning and assessed through microscopic, spectroscopic, thermal, mechanical and drug release analyses. Considering nutrient passage through the barrier, the microporous CSNMs exerted the same barrier effect but drastically increased the mass transfer coefficients of bovine serum albumin compared with the commercial anti-adhesive membrane SurgiWrap®. Cell attachment/focal adhesion formation of fibroblasts revealed effective reduction of initial cell attachment on the CSNM surface with minimum cytotoxicity (except HI50/PPA). The anti-bacterial effect against both Gram-negative and Gram-positive bacteria was verified to be due to the Ag NPs in the membranes. In vivo studies using H/PPA and HI30/PPA CSNMs and SurgiWrap® in a rabbit flexor tendon rupture model demonstrated the improved efficacy of HI30/PPA CSNMs in reducing inflammation and tendon adhesion formation based on gross observation, histological analysis and functional assays. We conclude that HI30/PPA CSNMs can act as a multifunctional barrier membrane to prevent peritendinous adhesion after tendon surgery. A multi-functional anti-adhesion barrier

  9. Genomic dissection and expression profiling revealed functional divergence in Triticum aestivum leucine rich repeat receptor like kinases (TaLRRKs

    Directory of Open Access Journals (Sweden)

    Shumayla .

    2016-09-01

    Full Text Available The leucine rich repeat receptor like kinases (LRRK constitute the largest subfamily of receptor like kinases (RLK, which play critical roles in plant development and stress responses. Herein, we identified 531 TaLRRK genes in Triticum aestivum (bread wheat, which were distributed throughout the A, B, and D sub-genomes and chromosomes. These were clustered into 233 homologous groups, which were mostly located on either homeologous chromosomes from various sub-genomes or in proximity on the same chromosome. A total of 255 paralogous genes were predicted which depicted the role of duplication events in expansion of this gene family. Majority of TaLRRKs consisted of trans-membrane region and localized on plasma-membrane. The TaLRRKs were further categorized into eight phylogenetic groups with numerous subgroups on the basis of sequence homology. The gene and protein structure in terms of exon/intron ratio, domains and motifs organization were found to be variably conserved across the different phylogenetic groups/subgroups, which indicated a potential divergence and neofunctionalization during evolution. High-throughput transcriptome data and quantitative real time PCR analyses in various developmental stages, and biotic and abiotic (heat, drought and salt stresses provided insight into modus operandi of TaLRRKs during these conditions. Distinct expression of majority of stress responsive TaLRRKs homologous genes suggested their specified role in a particular condition. These results provided a comprehensive analysis of various characteristic features including functional divergence, which may provide the way for future functional characterization of this important gene family in bread wheat.

  10. Function of AURKA protein kinase in the formation of vasculogenic mimicry in triple-negative breast cancer stem cells

    Directory of Open Access Journals (Sweden)

    Liu Y

    2016-06-01

    Full Text Available Ying Liu,1,2,* Baocun Sun,1–3,* Tieju Liu,1,2,* Xiulan Zhao,1,2 Xudong Wang,3 Yanlei Li,1,2 Jie Meng,2 Qiang Gu,1,2 Fang Liu,1,2 Xueyi Dong,1,2 Peimei Liu,2 Ran Sun,2 Nan Zhao1 1Department of Pathology, General Hospital of Tianjin Medical University, 2Department of Pathology, Tianjin Medical University, 3Department of Pathology, Cancer Hospital of Tianjin Medical University, Tianjin, People’s Republic of China *These authors contributed equally to this work Abstract: Tumor cell vasculogenic mimicry (VM, a newly defined pattern of tumor blood supply, signifies the functional plasticity of aggressive cancer cells forming vascular networks. VM and cancer stem cells (CSCs have been shown to be associated with tumor growth, local invasion, and distant metastasis. In our previous study, CSCs in triple-negative breast cancer were potential to participate in VM formation. In this study, breast CSCs were isolated from the triple-negative breast cancer cell line MDA-MB-231 by using mammosphere culture. Western blotting and reverse transcription polymerase chain reaction showed that mammosphere cells displayed an increased expression of AURKA protein kinase and stem cell marker c-myc and sox2. The VM formation by mammosphere cells was inhibited by AURKA knockdown or the addition of AURKA inhibitor MLN8237. In the meantime, MLN8237 induced the increased E-cadherin and decreased c-myc, sox2, and β-catenin expressions. The function of AURKA in VM formation was further confirmed using a xenograft-murine model. The results suggested that AURKA protein kinase is involved in VM formation of CSCs and may become a new treatment target in suppressing VM and metastasis of breast cancer. Keywords: AURKA, cancer stem cells, vasculogenic mimicry, breast cancer

  11. Integrin-linked kinase mediates force transduction in cardiomyocytes by modulating SERCA2a/PLN function.

    Science.gov (United States)

    Traister, Alexandra; Li, Mark; Aafaqi, Shabana; Lu, Mingliang; Arab, Sara; Radisic, Milica; Gross, Gil; Guido, Fiorella; Sherret, John; Verma, Subodh; Slorach, Cameron; Mertens, Luc; Hui, Wei; Roy, Anna; Delgado-Olguín, Paul; Hannigan, Gregory; Maynes, Jason T; Coles, John G

    2014-09-11

    Human dilated cardiomyopathy (DCM) manifests as a profound reduction in biventricular cardiac function that typically progresses to death or cardiac transplantation. There is no effective mechanism-based therapy currently available for DCM, in part because the transduction of mechanical load into dynamic changes in cardiac contractility (termed mechanotransduction) remains an incompletely understood process during both normal cardiac function and in disease states. Here we show that the mechanoreceptor protein integrin-linked kinase (ILK) mediates cardiomyocyte force transduction through regulation of the key calcium regulatory protein sarcoplasmic/endoplasmic reticulum Ca(2+)ATPase isoform 2a (SERCA-2a) and phosphorylation of phospholamban (PLN) in the human heart. A non-oncogenic ILK mutation with a synthetic point mutation in the pleckstrin homology-like domain (ILK(R211A)) is shown to enhance global cardiac function through SERCA-2a/PLN. Thus, ILK serves to link mechanoreception to the dynamic modulation of cardiac contractility through a previously undiscovered interaction with the functional SERCA-2a/PLN module that can be exploited to rescue impaired mechanotransduction in DCM.

  12. Metabolic functions of atypical protein kinase C: "good" and "bad" as defined by nutritional status.

    Science.gov (United States)

    Farese, Robert V; Sajan, Mini P

    2010-03-01

    Atypical protein kinase C (aPKC) isoforms mediate insulin effects on glucose transport in muscle and adipose tissues and lipid synthesis in liver and support other metabolic processes, expression of enzymes needed for islet insulin secretion and hepatic glucose production/release, CNS appetite suppression, and inflammatory responses. In muscle, selective aPKC deficiency impairs glucose uptake and produces insulin resistance and hyperinsulinemia, which, by activating hepatic aPKC, provokes inordinate increases in lipid synthesis and produces typical "metabolic syndrome" features. In contrast, hepatic aPKC deficiency diminishes lipid synthesis and protects against metabolic syndrome features. Unfortunately, aPKC is deficient in muscle but paradoxically conserved in liver in obesity and type 2 diabetes mellitus; this combination is particularly problematic because it promotes lipid and carbohydrate abnormalities. Accordingly, metabolic effects of aPKCs can be "good" or "bad," depending upon nutritional status; thus, muscle glucose uptake, islet insulin secretion, hepatic glucose and lipid production/release, and adipose fat synthesis/storage would be important for survival during periods of limited food availability and therefore be "good." However, during times of food surfeit, excessive activation of hepatic aPKC, whether caused by overnutrition or impairments in extrahepatic effects of insulin, would lead to inordinate increases in hepatic lipid synthesis and metabolic syndrome features and therefore be "bad." In keeping with these ideas, the inhibition of hepatic aPKC markedly ameliorates lipid and carbohydrate abnormalities in experimental models of obesity and type 2 diabetes. We postulate that a similar approach may be useful for treating humans.

  13. Targeting polo-like kinase 1 suppresses essential functions of alloreactive T cells.

    Science.gov (United States)

    Berges, Carsten; Chatterjee, Manik; Topp, Max S; Einsele, Hermann

    2016-06-01

    Acute graft-versus-host disease (aGvHD) is still a major cause of transplant-related mortality after allogeneic stem cell transplantation (ASCT). It requires immunosuppressive treatments that broadly abrogate T cell responses including beneficial ones directed against tumor cells or infective pathogens. Polo-like kinase 1 (PLK1) is overexpressed in many cancer types including leukemia, and clinical studies demonstrated that targeting PLK1 using selective PLK1 inhibitors resulted in inhibition of proliferation and induction of apoptosis predominantly in tumor cells, supporting the feasibility of PLK1 as target for anticancer therapy. Here, we show that activation of alloreactive T cells (Tallo) up-regulate expression of PLK1, suggesting that PLK1 is a potential new candidate for dual therapy of aGvHD and leukemia after ASCT. Inhibition of PLK1, using PLK1-specific inhibitor GSK461364A selectively depletes Tallo by preventing activation and by inducing apoptosis in already activated Tallo, while memory T cells are preserved. Activated Tallo cells which survive exposure to PLK1 undergo inhibition of proliferation by induction of G2/M cell cycle arrest, which is accompanied by accumulation of cell cycle regulator proteins p21(WAF/CIP1), p27(Kip1), p53 and cyclin B1, whereas abundance of CDK4 decreased. We also show that suppressive effects of PLK1 inhibition on Tallo were synergistically enhanced by concomitant inhibition of molecular chaperone Hsp90. Taken together, our data suggest that PLK1 inhibition represents a reasonable dual strategy to suppress residual tumor growth and efficiently deplete Tallo, and thus provide a rationale to selectively prevent and treat aGvHD.

  14. Adhesion molecule expression and function of primary endothelial cells in benign and malignant tissues correlates with proliferation.

    Directory of Open Access Journals (Sweden)

    Wolfgang Sievert

    Full Text Available BACKGROUND: Comparative analysis of the cellular biology of the microvasculature in different tissues requires the availability of viable primary endothelial cells (ECs. This study describes a novel method to isolate primary ECs from healthy organs, repair blastemas and tumors as examples of non-proliferating and proliferating benign and malignant tissues and their functional characterization. METHODOLOGY/PRINCIPAL FINDINGS: Single cell suspensions from hearts, lungs, repair blastemas and tumors were incubated consecutively with an anti-CD31 antibody and magnetic micro-beads, coupled to a derivative of biotin and streptavidin, respectively. Following magnetic bead separation, CD31-positive ECs were released by biotin-streptavidin competition. In the absence of micro-beads, ECs became adherent to plastic surfaces. ECs from proliferating repair blastemas and tumors were larger and exhibited higher expression densities of CD31, CD105 and CD102 compared to those from non-proliferating normal tissues such as heart and lung. The expression density of CD34 was particularly high in tumor-derived ECs, and that of CD54 and CD144 in ECs of repair blastemas. Functionally, ECs of non-proliferating and proliferating tissues differed in their capacity to form tubes in matrigel and to align under flow conditions. CONCLUSIONS/SIGNIFICANCE: This method provides a powerful tool to generate high yields of viable, primary ECs of different origins. The results suggest that an altered expression of adhesion molecules on ECs in proliferating tissues contribute to loss of EC function that might cause a chaotic tumor vasculature.

  15. Bacterial adhesion

    NARCIS (Netherlands)

    Loosdrecht, van M.C.M.

    1988-01-01

    As mentioned in the introduction of this thesis bacterial adhesion has been studied from a variety of (mostly practice oriented) starting points. This has resulted in a range of widely divergent approaches. In order to elucidate general principles in bacterial adhesion phenomena, we felt it

  16. Adhesive Categories

    DEFF Research Database (Denmark)

    Lack, Stephen; Sobocinski, Pawel

    2004-01-01

    We introduce adhesive categories, which are categories with structure ensuring that pushouts along monomorphisms are well-behaved. Many types of graphical structures used in computer science are shown to be examples of adhesive categories. Double-pushout graph rewriting generalises well...

  17. Identification of Human Junctional Adhesion Molecule 1 as a Functional Receptor for the Hom-1 Calicivirus on Human Cells

    Directory of Open Access Journals (Sweden)

    Stanislav V. Sosnovtsev

    2017-02-01

    Full Text Available The Hom-1 vesivirus was reported in 1998 following the inadvertent transmission of the animal calicivirus San Miguel sea lion virus to a human host in a laboratory. We characterized the Hom-1 strain and investigated the mechanism by which human cells could be infected. An expression library of 3,559 human plasma membrane proteins was screened for reactivity with Hom-1 virus-like particles, and a single interacting protein, human junctional adhesion molecule 1 (hJAM1, was identified. Transient expression of hJAM1 conferred susceptibility to Hom-1 infection on nonpermissive Chinese hamster ovary (CHO cells. Virus infection was markedly inhibited when CHO cells stably expressing hJAM were pretreated with anti-hJAM1 monoclonal antibodies. Cell lines of human origin were tested for growth of Hom-1, and efficient replication was observed in HepG2, HuH7, and SK-CO15 cells. The three cell lines (of hepatic or intestinal origin were confirmed to express hJAM1 on their surface, and clustered regularly interspaced short palindromic repeats/Cas9-mediated knockout of the hJAM1 gene in each line abolished Hom-1 propagation. Taken together, our data indicate that entry of the Hom-1 vesivirus into these permissive human cell lines is mediated by the plasma membrane protein hJAM1 as a functional receptor.

  18. Ouabain modulates cell contacts as well as functions that depend on cell adhesion.

    Science.gov (United States)

    Larre, Isabel; Contreras, Ruben G; Cereijido, Marcelino

    2011-01-01

    Ouabain, a toxic of vegetal origin used for centuries to treat heart failure, has recently been demonstrated to have an endogenous counterpart, most probably ouabain itself, which behaves as a hormone. Therefore, the challenge now is to discover the physiological role of hormone ouabain. We have recently shown that it modulates cell contacts such as gap junctions, which communicate neighboring cells, as well as tight junctions (TJs), which are one of the two differentiated features of epithelial cells, the other being apical/basolateral polarity. The importance of cell contacts can be hardly overestimated, since the most complex object in the universe, the brain, assembles itself depending on what cells contacts what other(s) how, when, and how is the molecular composition and special arrangement of the contacts involved. In the present chapter, we detail the protocols used to demonstrate the effect of ouabain on the molecular structure and functional properties of one of those cell-cell contacts: the TJ.

  19. Proteoglycans, ion channels and cell-matrix adhesion

    DEFF Research Database (Denmark)

    Mitsou, Ioli; Multhaupt, Hinke A.B.; Couchman, John R.

    2017-01-01

    , growth factors and morphogens to enzymes and extracellular matrix (ECM) glycoproteins and collagens.Extracellular interactions with other receptors, such as some integrins, are mediated by the core protein.This places syndecans at the nexus of many cellular responses to extracellular cues in development......, maintenance, repair and disease.The cytoplasmic domains of syndecans, while having no intrinsic kinase activity, can nevertheless signal through binding proteins.All syndecans appear to be connected to the actin cytoskeleton and can therefore contribute to cell adhesion, notably to the ECM and migration.......Recent data now suggest that syndecans can regulate stretchactivated ion channels.The structure and function of the syndecans and the ion channels are reviewed here, along with an analysis of ion channel functions in cell-matrix adhesion.This area sheds new light on the syndecans, not least since evidence...

  20. Neutrophil development and function critically depend on Bruton tyrosine kinase in a mouse model of X-linked agammaglobulinemia.

    Science.gov (United States)

    Fiedler, Katja; Sindrilaru, Anca; Terszowski, Grzegorz; Kokai, Enikö; Feyerabend, Thorsten B; Bullinger, Lars; Rodewald, Hans-Reimer; Brunner, Cornelia

    2011-01-27

    Bruton tyrosine kinase (Btk) is essential for B cell development and function and also appears to be important for myeloid cells. The bone marrow of Btk-deficient mice shows enhanced granulopoiesis compared with that of wild-type mice. In purified granulocyte-monocyte-progenitors (GMP) from Btk-deficient mice, the development of granulocytes is favored at the expense of monocytes. However, Btk-deficient neutrophils are impaired in maturation and function. Using bone marrow chimeras, we show that this defect is cell-intrinsic to neutrophils. In GMP and neutrophils, Btk plays a role in GM-CSF- and Toll-like receptor-induced differentiation. Molecular analyses revealed that expression of the lineage-determining transcription factors C/EBPα, C/EBPβ, and PU.1, depends on Btk. In addition, expression of several granule proteins, including myeloperoxidase, neutrophilic granule protein, gelatinase and neutrophil elastase, is Btk-dependent. In the Arthus reaction, an acute inflammatory response, neutrophil migration into tissues, edema formation, and hemorrhage are significantly reduced in Btk-deficient animals. Together, our findings implicate Btk as an important regulator of neutrophilic granulocyte maturation and function in vivo.

  1. Consequences of two naturally occurring missense mutations in the structure and function of Bruton agammaglobulinemia tyrosine kinase.

    Science.gov (United States)

    Vargas-Hernández, Alexander; López-Herrera, Gabriela; Maravillas-Montero, José L; Vences-Catalán, Felipe; Mogica-Martínez, Dolores; Rojo-Domínguez, Arturo; Espinosa-Rosales, Francisco J; Santos-Argumedo, Leopoldo

    2012-04-01

    Bruton agammaglobulinemia tyrosine kinase (BTK) is a key protein in the B-cell receptor (BCR) signaling pathway and plays an essential role in the differentiation of B lymphocytes. X-linked agammaglobulinemia (XLA) is a primary humoral immunodeficiency caused by mutations in the gene encoding BTK. Previously, we identified two novel variations, L111P and E605G, in BTK; these are localized within the pleckstrin homology and Src homology 1 domains, respectively. In the present study, we evaluated the potential effects of these variations on the structural conformation and the function of BTK. Using in silico methods, we found that the L111P and E650G variations are not located directly in protein-protein interfaces but close to them. They distorted the native structural conformation of the BTK protein, affecting not only its geometry and stability but also its ability for protein recognition and in consequence its functionality. To confirm the results of the in silico assays, WT BTK, L111P, and E650G variants were expressed in the BTK-deficient DT40 cell line. The mutant proteins exhibited an absence of catalytic activity, aberrant redistribution after BCR-crosslinking, and deficient intracellular calcium mobilization. This work demonstrates that L111 and E605 residues are fundamental for the activation and function of BTK. Copyright © 2012 Wiley Periodicals, Inc.

  2. Discovery of novel inhibitors for Leishmania nucleoside diphosphatase kinase (NDK) based on its structural and functional characterization

    Science.gov (United States)

    Mishra, Arjun K.; Singh, Nidhi; Agnihotri, Pragati; Mishra, Shikha; Singh, Saurabh P.; Kolli, Bala K.; Chang, Kwang Poo; Sahasrabuddhe, Amogh A.; Siddiqi, M. I.; Pratap, J. Venkatesh

    2017-06-01

    Nucleoside diphosphate kinases (NDKs) are ubiquitous enzymes that catalyze the transfer of the γ-phosphate moiety from an NTP donor to an NDP acceptor, crucial for maintaining the cellular level of nucleoside triphosphates (NTPs). The inability of trypanosomatids to synthesize purines de novo and their dependence on the salvage pathway makes NDK an attractive target to develop drugs for the diseases they cause. Here we report the discovery of novel inhibitors for Leishmania NDK based on the structural and functional characterization of purified recombinant NDK from Leishmania amazonensis. Recombinant LaNDK possesses auto-phosphorylation, phosphotransferase and kinase activities with Histidine 117 playing an essential role. LaNDK crystals were grown by hanging drop vapour diffusion method in a solution containing 18% PEG-MME 500, 100 mM Bis-Tris propane pH 6.0 and 50 mM MgCl2. It belongs to the hexagonal space group P6322 with unit cell parameters a = b = 115.18, c = 62.18 Å and α = β = 90°, γ = 120°. The structure solved by molecular replacement methods was refined to crystallographic R-factor and Rfree values of 22.54 and 26.52%, respectively. Molecular docking and dynamics simulation -based virtual screening identified putative binding compounds. Protein inhibition studies of selected hits identified five inhibitors effective at micromolar concentrations. One of the compounds showed 45% inhibition of Leishmania promastigotes proliferation. Analysis of inhibitor-NDK complexes reveals the mode of their binding, facilitating design of new compounds for optimization of activities as drugs against leishmaniasis.

  3. Loss of protein kinase 2 subunit alpha 2 (CK2α') effect ram sperm function after freezing and thawing process.

    Science.gov (United States)

    He, Yuxuan; Li, Hongyan; Wang, Ke; Zhang, Yong; Zhao, Xingxu

    2017-06-01

    Protein kinase 2 subunit alpha 2 (CK2α'), a serine/threonine-selective protein kinase, is associated with sperm apoptosis. However, the presence of CK2α' in ram sperm during the freezing-thawing process has not been previously reported. Thus, this study was conducted to determine the effect of cryopreservation on the association between CK2α' and ram sperm function. Sperm variables, including sperm motility, DNA damage and acrosome integrity, were measured in fresh (F), cooled (CO) and freeze-thawed (FT) sperm. Sperm proteins and total mRNA were extracted form cells of each group, and subjected to western blot and real-time PCR analysis for detection of CK2α' proteins and relative abundance of mRNA. The distribution pattern of CK2α' protein in ram sperm was also monitored in each group using an immunofluorescence technique. The results provided evidence that the freeze-thaw process has an impact on ram sperm variables, and the normalized CK2α' protein and relative abundance of CK2α' mRNA were both significantly less in FT than F sperm. The amount of CK2α' in FT extended seminal plasma was increased as compared with F samples. Furthermore, immunofluorescence revealed that CK2α' was distributed throughout the acrosome of ram sperm. The association of CK2α' with DNA damage and acrosome integrity was confirmed using Pearson's linear correlation. In conclusion, the understanding the molecular effects of cryopreservation on CK2α' in ram sperm could provide insight into methods for improving fertility associated with frozen-thawed ram sperm. Copyright © 2017 Elsevier B.V. All rights reserved.

  4. Arabidopsis cysteine-rich receptor-like kinase 45 functions in the responses to abscisic acid and abiotic stresses

    KAUST Repository

    Zhang, Xiujuan

    2013-06-01

    The phytohormone abscisic acid (ABA) regulates seed germination, plant growth and development, and response to abiotic stresses such as drought and salt stresses. Receptor-like kinases are well known signaling components that mediate plant responses to developmental and environmental stimuli. Here, we characterized the biological function of an ABA and stress-inducible cysteine-rich receptor-like protein kinase, CRK45, in ABA signaling in Arabidopsis thaliana. The crk45 mutant was less sensitive to ABA than the wild type during seed germination and early seedling development, whereas CRK45 overexpression plants were more sensitive to ABA compared to the wild type. Furthermore, overexpression of CRK45 led to hypersensitivity to salt and glucose inhibition of seed germination, whereas the crk45 mutant showed the opposite phenotypes. In addition, CRK45 overexpression plants had enhanced tolerance to drought. Gene expression analyses revealed that the expression of representative stress-responsive genes was significantly enhanced in CRK45 overexpression plants in response to salt stress. ABA biosynthetic genes such as NCED3,. 22NCED3, 9-Cis-Epoxycarotenoid Dioxygenase 3.NCED5,. 33NCED5, 9-Cis-Epoxycarotenoid Dioxygenase 5.ABA2,. 44ABA2, Abscisic Acid Deficient 2. and AAO355AAO3, Abscisic Aldehyde Oxidase 3. were also constitutively elevated in the CRK45 overexpression plants. We concluded that CRK45 plays an important role in ABA signaling that regulates Arabidopsis seeds germination, early seedling development and abiotic stresses response, by positively regulating ABA responses in these processes. © 2013 Elsevier Masson SAS.

  5. Genetic analysis of beta1 integrin function: confirmed, new and revised roles for a crucial family of cell adhesion molecules

    DEFF Research Database (Denmark)

    Brakebusch, C; Hirsch, E; Potocnik, A

    1997-01-01

    Integrins are heterodimeric cell adhesion proteins connecting the extracellular matrix to the cytoskeleton and transmitting signals in both directions. These integrins are suggested to be involved in many different biological processes such as growth, differentiation, migration, and cell death...

  6. The Adhesion Molecule-Characteristic HNK-1 Carbohydrate Contributes to Functional Recovery After Spinal Cord Injury in Adult Zebrafish.

    Science.gov (United States)

    Ma, Liping; Shen, Hui-Fan; Shen, Yan-Qin; Schachner, Melitta

    2017-07-01

    The human natural killer cell antigen-1 (HNK-1) is functionally important in development, synaptic activity, and regeneration after injury in the nervous system of several mammalian species. It contains a sulfated glucuronic acid which is carried by neural adhesion molecules and expressed in nonmammalian species, including zebrafish, which, as opposed to mammals, spontaneously regenerate after injury in the adult. To evaluate HNK-1's role in recovery of function after spinal cord injury (SCI) of adult zebrafish, we assessed the effects of the two HNK-1 synthesizing enzymes, glucuronyl transferase and HNK-1 sulfotransferase. Expression of these two enzymes was increased at the messenger RNA (mRNA) level 11 days after injury in the brainstem nuclei that are capable of regrowth of severed axons, namely, the nucleus of medial longitudinal fascicle and intermediate reticular formation, but not at earlier time points after SCI. mRNA levels of glucuronyl transferase and sulfotransferase were increased in neurons, not only of these nuclei but also in the spinal cord caudal to the injury site at 11 days. Mauthner neurons which are not capable of regeneration did not show increased levels of enzyme mRNAs after injury. Reducing protein levels of the enzymes by application of anti-sense morpholinos resulted in reduction of locomotor recovery for glucuronyl transferase, but not for HNK-1 sulfotransferase. The combined results indicate that HNK-1 is upregulated in expression only in those neurons that are intrinsically capable of regeneration and contributes to regeneration after spinal cord injury in adult zebrafish in the absence of its sulfate moiety.

  7. Expression pattern and function of tyrosine receptor kinase B isoforms in rat mesenteric arterial smooth muscle cells

    International Nuclear Information System (INIS)

    Otani, Kosuke; Okada, Muneyoshi; Yamawaki, Hideyuki

    2015-01-01

    Tyrosine receptor kinaseB (TrkB) is a high affinity receptor for brain-derived neurotrophic factor (BDNF). TrkB isoforms involve full length TrkB (TrkB FL) and truncated TrkB type1 (TrkB T1) and type 2 (TrkB T2) in rats. The aim of present study was to explore their expression pattern and function in mesenteric arterial smooth muscle cells (MASMCs). The expression of TrkB isoform protein and mRNA was examined by Western blotting, immunofluorescence and quantitative RT-PCR analyses. Cell proliferation was measured by a bromodeoxyuridine (BrdU) incorporation assay. Cell migration was measured by a Boyden chamber assay. Cell morphology was observed with a phase-contrast microscope. Protein and mRNA expression of BDNF and TrkB isoforms was confirmed in MASMCs. Expression level of TrkB FL was less, while that of TrkB T1 was the highest in MASMCs. Although BDNF increased phosphorylation of ERK, it had no influence on migration and proliferation of MASMCs. TrkB T1 gene knockdown by a RNA interference induced morphological changes and reduced expression level of α-smooth muscle actin (α-SMA) in MASMCs. Similar morphological changes and reduced α-SMA expression were induced in MASMCs by a Rho kinase inhibitor, Y-27632. In conclusion, we for the first time demonstrate that TrkB T1 expressed highly in MASMCs contributes to maintain normal cell morphology possibly via regulation of Rho activity. This study firstly defined expression level of TrkB isoforms and partly revealed their functions in peripheral vascular cells. - Highlights: • BDNF-TrkB axis mediates neurogenesis, growth, differentiation and survival. • Expression pattern and function of TrkB in vascular smooth muscle remain unclear. • Expression of TrkB FL is low, while that of TrkB T1 is the highest. • TrkB T1 contributes to maintain normal morphology possibly via activating Rho.

  8. Expression pattern and function of tyrosine receptor kinase B isoforms in rat mesenteric arterial smooth muscle cells

    Energy Technology Data Exchange (ETDEWEB)

    Otani, Kosuke; Okada, Muneyoshi; Yamawaki, Hideyuki, E-mail: yamawaki@vmas.kitasato-u.ac.jp

    2015-11-27

    Tyrosine receptor kinaseB (TrkB) is a high affinity receptor for brain-derived neurotrophic factor (BDNF). TrkB isoforms involve full length TrkB (TrkB FL) and truncated TrkB type1 (TrkB T1) and type 2 (TrkB T2) in rats. The aim of present study was to explore their expression pattern and function in mesenteric arterial smooth muscle cells (MASMCs). The expression of TrkB isoform protein and mRNA was examined by Western blotting, immunofluorescence and quantitative RT-PCR analyses. Cell proliferation was measured by a bromodeoxyuridine (BrdU) incorporation assay. Cell migration was measured by a Boyden chamber assay. Cell morphology was observed with a phase-contrast microscope. Protein and mRNA expression of BDNF and TrkB isoforms was confirmed in MASMCs. Expression level of TrkB FL was less, while that of TrkB T1 was the highest in MASMCs. Although BDNF increased phosphorylation of ERK, it had no influence on migration and proliferation of MASMCs. TrkB T1 gene knockdown by a RNA interference induced morphological changes and reduced expression level of α-smooth muscle actin (α-SMA) in MASMCs. Similar morphological changes and reduced α-SMA expression were induced in MASMCs by a Rho kinase inhibitor, Y-27632. In conclusion, we for the first time demonstrate that TrkB T1 expressed highly in MASMCs contributes to maintain normal cell morphology possibly via regulation of Rho activity. This study firstly defined expression level of TrkB isoforms and partly revealed their functions in peripheral vascular cells. - Highlights: • BDNF-TrkB axis mediates neurogenesis, growth, differentiation and survival. • Expression pattern and function of TrkB in vascular smooth muscle remain unclear. • Expression of TrkB FL is low, while that of TrkB T1 is the highest. • TrkB T1 contributes to maintain normal morphology possibly via activating Rho.

  9. Functional intersection of ATM and DNA-dependent protein kinase catalytic subunit in coding end joining during V(D)J recombination

    DEFF Research Database (Denmark)

    Lee, Baeck-Seung; Gapud, Eric J; Zhang, Shichuan

    2013-01-01

    V(D)J recombination is initiated by the RAG endonuclease, which introduces DNA double-strand breaks (DSBs) at the border between two recombining gene segments, generating two hairpin-sealed coding ends and two blunt signal ends. ATM and DNA-dependent protein kinase catalytic subunit (DNA......-PKcs. Mutation of these threonine residues to alanine (DNA-PKcs(3A)) renders DNA-PKcs dependent on its intrinsic kinase activity during coding end joining, at a step downstream of opening hairpin-sealed coding ends. Thus, DNA-PKcs has critical functions in coding end joining beyond promoting Artemis endonuclease...

  10. Enhancement of growth and osteogenic differentiation of MC3T3-E1 cells via facile surface functionalization of polylactide membrane with chitooligosaccharide based on polydopamine adhesive coating

    Energy Technology Data Exchange (ETDEWEB)

    Li, Huihua [Biomaterial Research Laboratory, Department of Material Science and Engineering, College of Science and Engineering, Jinan University, Guangzhou 510632 (China); Luo, Chuang [Engineering Research Center of Artificial Organs and Materials, Ministry of Education, Guangzhou 510632 (China); Luo, Binghong, E-mail: tluobh@jnu.edu.cn [Biomaterial Research Laboratory, Department of Material Science and Engineering, College of Science and Engineering, Jinan University, Guangzhou 510632 (China); Engineering Research Center of Artificial Organs and Materials, Ministry of Education, Guangzhou 510632 (China); Wen, Wei [Biomaterial Research Laboratory, Department of Material Science and Engineering, College of Science and Engineering, Jinan University, Guangzhou 510632 (China); Engineering Research Center of Artificial Organs and Materials, Ministry of Education, Guangzhou 510632 (China); Wang, Xiaoying [Department of Biomedical Engineering, College of Life Science and Technology, Jinan University, Guangzhou 510632 (China); Ding, Shan [Biomaterial Research Laboratory, Department of Material Science and Engineering, College of Science and Engineering, Jinan University, Guangzhou 510632 (China); Engineering Research Center of Artificial Organs and Materials, Ministry of Education, Guangzhou 510632 (China); Zhou, Changren, E-mail: tcrz9@jnu.edu.cn [Biomaterial Research Laboratory, Department of Material Science and Engineering, College of Science and Engineering, Jinan University, Guangzhou 510632 (China); Engineering Research Center of Artificial Organs and Materials, Ministry of Education, Guangzhou 510632 (China)

    2016-01-01

    Graphical abstract: - Highlights: • COS was conveniently immobilized on PDLLA membrane based on PDOPA adhesive layer. • The hydrophilicity of PDLLA membrane was improved by modified with PDOPA and COS. • COS-functionalized PDLLA membrane is favorable to cell adhesion and proliferation. • COS-coated PDLLA membrane notably promote osteogenic differentiation of MC3T3-E1. - Abstract: To develop a chitooligosaccharide(COS)-functionalized poly(D,L-lactide) (PDLLA) membrane to enhance growth and osteogenic differentiation of MC3T3-E1 cells, firstly a thin polydopamine (PDOPA) layer was adhered to the PDLLA membrane via the self-polymerization and strong adhesion behavior of dopamine. Subsequently, COS was immobilized covalently on the resultant PDLLA/PDOPA composite membrane by coupling with PDOPA active coating. The successful immobilization of the PDOPA and COS was confirmed by attenuated total reflectance Fourier transform infrared (ATR-FTIR) spectroscopy and X-ray photoelectron spectroscopy (XPS). Scanning electronic microscopy (SEM) and atomic force microscopy (AFM) results indicated that the surface topography and roughness of the membranes were changed, and the root mean square increased from 0.613 nm to 6.96 and 7.12 nm, respectively after coating PDOPA and COS. Water contact angle and surface energy measurements revealed that the membrane hydrophilicity was remarkably improved by surface modification. In vitro cells culture results revealed that the PDOPA- and COS-functionalized surfaces showed a significant increase in MC3T3-E1 cells adhesion, proliferation, osteogenic differentiation and alkaline phosphate activity compared to the pristine PDLLA substrate. Furthermore the COS-functionalized PDLLA membrane was more effectively at enhancing osteoblast activity than the PDOPA-functionalized PDLLA membrane.

  11. Myosin light chain kinase knockout improves gut barrier function and confers a survival advantage in polymicrobial sepsis.

    Science.gov (United States)

    Lorentz, C Adam; Liang, Zhe; Meng, Mei; Chen, Ching-Wen; Yoseph, Benyam P; Breed, Elise R; Mittal, Rohit; Klingensmith, Nathan J; Farris, Alton B; Burd, Eileen M; Koval, Michael; Ford, Mandy L; Coopersmith, Craig M

    2017-06-07

    Sepsis-induced intestinal hyperpermeability is mediated by disruption of the epithelial tight junction, which is closely associated with the peri-junctional actin-myosin ring. Myosin light chain kinase (MLCK) phosphorylates the myosin regulatory light chain, resulting in increased permeability. The purpose of this study was to determine whether genetic deletion of MLCK would alter gut barrier function and survival from sepsis. MLCK -/- and wild type (WT) mice were subjected to cecal ligation and puncture and assayed for both survival and mechanistic studies. Survival was significantly increased in MLCK -/- mice (95% vs. 24%, p<0.0001). Intestinal permeability increased in septic WT mice compared to unmanipulated mice. In contrast, permeability in septic MLCK -/- mice was similar to that seen in unmanipulated animals. Improved gut barrier function in MLCK -/- mice was associated with increases in the tight junction mediators ZO-1 and claudin 15 without alterations in claudin 1, 2, 3, 4, 5, 7, 8, 13, occludin or JAM-A. Other components of intestinal integrity (apoptosis, proliferation and villus length) were unaffected by MLCK deletion as were local peritoneal inflammation and distant lung injury. Systemic IL-10 was decreased greater than 10-fold in MLCK -/- mice; however, survival was similar between septic MLCK -/- mice given exogenous IL-10 or vehicle. These data demonstrate that deletion of MLCK improves survival following sepsis, associated with normalization of intestinal permeability and selected tight junction proteins.

  12. Glucagon Receptor Antagonism Improves Glucose Metabolism and Cardiac Function by Promoting AMP-Mediated Protein Kinase in Diabetic Mice

    Directory of Open Access Journals (Sweden)

    Ankit X. Sharma

    2018-02-01

    Full Text Available The antidiabetic potential of glucagon receptor antagonism presents an opportunity for use in an insulin-centric clinical environment. To investigate the metabolic effects of glucagon receptor antagonism in type 2 diabetes, we treated Leprdb/db and Lepob/ob mice with REMD 2.59, a human monoclonal antibody and competitive antagonist of the glucagon receptor. As expected, REMD 2.59 suppresses hepatic glucose production and improves glycemia. Surprisingly, it also enhances insulin action in both liver and skeletal muscle, coinciding with an increase in AMP-activated protein kinase (AMPK-mediated lipid oxidation. Furthermore, weekly REMD 2.59 treatment over a period of months protects against diabetic cardiomyopathy. These functional improvements are not derived simply from correcting the systemic milieu; nondiabetic mice with cardiac-specific overexpression of lipoprotein lipase also show improvements in contractile function after REMD 2.59 treatment. These observations suggest that hyperglucagonemia enables lipotoxic conditions, allowing the development of insulin resistance and cardiac dysfunction during disease progression.

  13. Protein Kinase C-theta (PKC-theta in Natural Killer (NK cell function and anti-tumor immunity

    Directory of Open Access Journals (Sweden)

    Alberto eAnel

    2012-07-01

    Full Text Available The protein kinase C-theta (PKCtheta, which is essential for T cell function and survival, is also required for efficient anti-tumor immune surveillance. Natural killer (NK cells, which express PKCtheta, play a prominent role in this process, mainly by elimination of tumor cells with reduced or absent major histocompatibility complex class-I (MHC-I expression. This justifies the increased interest of the use of activated NK cells in anti-tumor immunotherapy in the clinic. The in vivo development of MHC-I-deficient tumors is much favored in PKCtheta-/- mice compared with wild-type mice. Recent data offer some clues on the mechanism that could explain the important role of PKCtheta in NK cell-mediated anti-tumor immune surveillance: some studies show that PKCtheta is implicated in signal transduction and anti-tumoral activity of NK cells elicited by interleukin (IL-12 or IL-15, while others show that it is implicated in NK cell functional activation mediated by certain killer activating receptors (KAR. Alternatively, the possibility that PKCtheta is involved in NK cell degranulation is discussed, since recent data indicate that it is implicated in microtubule-organizing center (MTOC polarization to the immune synapse in CD4+ T cells. The implication of PKC isoforms in degranulation has been more extensively studied in CTL, and these studies will be also summarized.

  14. Comparative evaluation of bone marrow cells morpho-functional activity in chronic myeloid leukemia patients treated with tyrosine kinase inhibitors of the first and second generation

    Directory of Open Access Journals (Sweden)

    I. O. Zhaleyko

    2014-07-01

    Full Text Available The efficiency of using the culture techniques of research for monitoring the patient’s response to the treatment by tyrosine kinase inhibitors of the first and second generation is shown. Thus, the functional activity of bone marrow cells in patients having the optimal treatment response to inhibitors of tyrosine kinases was significantly lower compared with patients with the acquired resistance to the drug, and patients who had CML diagnosed for first time. Furthermore, for patients with the optimal response to the nilotinib therapy, numbers of colonies in semi-solid agar in vitro was lower, than in patients with the optimal response to imatinib. When the leukaemic cell clone becomes resistant to tyrosine kinase inhibitors, the prevalence of early cells of granulocyte-macrophage hematopoietic stem cells is observed in CFU culture which can be an important prognostic factor for choosing the appropriate treatment strategy.

  15. Signaling transduction pathways involved in basophil adhesion and histamine release

    DEFF Research Database (Denmark)

    Sha, Quan; Poulsen, Lars K.; Gerwien, Jens

    2006-01-01

    Little is known about basophil with respect to the different signaling transduction pathways involved in spontaneous, cytokine or anti-IgE induced adhesion and how this compares to IgE-dependent and IgE-independent mediator secretion. The purpose of the present study was to investigate the roles ...... of beta1 and beta2 integrins in basophil adhesion as well as hosphatidylinositol 3-kinase (PI3K), src-kinases and extracellular signal regulated kinase (ERK) 1/2 in basophil adhesion and histamine release (HR)....

  16. Hepatocyte growth factor regulated tyrosine kinase substrate in the peripheral development and function of B-cells

    International Nuclear Information System (INIS)

    Nagata, Takayuki; Murata, Kazuko; Murata, Ryo; Sun, Shu-lan; Saito, Yutaro; Yamaga, Shuhei; Tanaka, Nobuyuki; Tamai, Keiichi; Moriya, Kunihiko; Kasai, Noriyuki; Sugamura, Kazuo; Ishii, Naoto

    2014-01-01

    Highlights: •ESCRT-0 protein regulates the development of peripheral B-cells. •BCR expression on cell surface should be controlled by the endosomal-sorting system. •Hrs plays important roles in responsiveness to Ag stimulation in B lymphocytes. -- Abstract: Hepatocyte growth factor (HGF)-regulated tyrosine kinase substrate (Hrs) is a vesicular sorting protein that functions as one of the endosomal-sorting proteins required for transport (ESCRT). Hrs, which binds to ubiquitinated proteins through its ubiquitin-interacting motif (UIM), contributes to the lysosomal transport and degradation of ubiquitinated membrane proteins. However, little is known about the relationship between B-cell functions and ESCRT proteins in vivo. Here we examined the immunological roles of Hrs in B-cell development and functions using B-cell-specific Hrs-deficient (Hrs flox/flox ;mb1 cre/+ :Hrs-cKO) mice, which were generated using a cre-LoxP recombination system. Hrs deficiency in B-cells significantly reduced T-cell-dependent antibody production in vivo and impaired the proliferation of B-cells treated in vitro with an anti-IgM monoclonal antibody but not with LPS. Although early development of B-cells in the bone marrow was normal in Hrs-cKO mice, there was a significant decrease in the number of the peripheral transitional B-cells and marginal zone B-cells in the spleen of Hrs-cKO mice. These results indicate that Hrs plays important roles during peripheral development and physiological functions of B lymphocytes

  17. Hepatocyte growth factor regulated tyrosine kinase substrate in the peripheral development and function of B-cells

    Energy Technology Data Exchange (ETDEWEB)

    Nagata, Takayuki [Department of Pharmacy, Iwaki Meisei University, 5-5-1 Chuodai Iino, Iwaki, Fukushima 970-8551 (Japan); Department of Microbiology and Immunology, Tohoku University Graduate School of Medicine, 2-1 Seiryo-machi, Aoba-ku, Sendai 980-8575 (Japan); Murata, Kazuko, E-mail: murata-k@iwakimu.ac.jp [Department of Pharmacy, Iwaki Meisei University, 5-5-1 Chuodai Iino, Iwaki, Fukushima 970-8551 (Japan); Murata, Ryo [Department of Pharmacy, Iwaki Meisei University, 5-5-1 Chuodai Iino, Iwaki, Fukushima 970-8551 (Japan); Sun, Shu-lan [Department of Microbiology and Immunology, Tohoku University Graduate School of Medicine, 2-1 Seiryo-machi, Aoba-ku, Sendai 980-8575 (Japan); Saito, Yutaro; Yamaga, Shuhei [Department of Pharmacy, Iwaki Meisei University, 5-5-1 Chuodai Iino, Iwaki, Fukushima 970-8551 (Japan); Tanaka, Nobuyuki; Tamai, Keiichi [Division of Immunology, Miyagi Cancer Research Institute, 47-1 Nodayama, Medeshima-Shiode, Natori 981-1293 (Japan); Moriya, Kunihiko [Department of Pediatrics, Tohoku University School of Medicine, 1-1 Seiryo-machi, Aoba-ku, Sendai 980-8575 (Japan); Kasai, Noriyuki [Institute for Animal Experimentation, Tohoku University Graduate School of Medicine, 2-1 Seiryo-machi, Aoba-ku, Sendai 980-8575 (Japan); Sugamura, Kazuo [Division of Immunology, Miyagi Cancer Research Institute, 47-1 Nodayama, Medeshima-Shiode, Natori 981-1293 (Japan); Ishii, Naoto [Department of Microbiology and Immunology, Tohoku University Graduate School of Medicine, 2-1 Seiryo-machi, Aoba-ku, Sendai 980-8575 (Japan)

    2014-01-10

    Highlights: •ESCRT-0 protein regulates the development of peripheral B-cells. •BCR expression on cell surface should be controlled by the endosomal-sorting system. •Hrs plays important roles in responsiveness to Ag stimulation in B lymphocytes. -- Abstract: Hepatocyte growth factor (HGF)-regulated tyrosine kinase substrate (Hrs) is a vesicular sorting protein that functions as one of the endosomal-sorting proteins required for transport (ESCRT). Hrs, which binds to ubiquitinated proteins through its ubiquitin-interacting motif (UIM), contributes to the lysosomal transport and degradation of ubiquitinated membrane proteins. However, little is known about the relationship between B-cell functions and ESCRT proteins in vivo. Here we examined the immunological roles of Hrs in B-cell development and functions using B-cell-specific Hrs-deficient (Hrs{sup flox/flox};mb1{sup cre/+}:Hrs-cKO) mice, which were generated using a cre-LoxP recombination system. Hrs deficiency in B-cells significantly reduced T-cell-dependent antibody production in vivo and impaired the proliferation of B-cells treated in vitro with an anti-IgM monoclonal antibody but not with LPS. Although early development of B-cells in the bone marrow was normal in Hrs-cKO mice, there was a significant decrease in the number of the peripheral transitional B-cells and marginal zone B-cells in the spleen of Hrs-cKO mice. These results indicate that Hrs plays important roles during peripheral development and physiological functions of B lymphocytes.

  18. Expression and functional analysis of genes encoding cytokinin receptor-like histidine kinase in maize (Zea mays L.).

    Science.gov (United States)

    Wang, Bo; Chen, Yanhong; Guo, Baojian; Kabir, Muhammad Rezaul; Yao, Yingyin; Peng, Huiru; Xie, Chaojie; Zhang, Yirong; Sun, Qixin; Ni, Zhongfu

    2014-08-01

    Cytokinin signaling is vital for plant growth and development which function via the two-component system (TCS). As one of the key component of TCS, transmembrane histidine kinases (HK) are encoded by a small gene family in plants. In this study, we focused on expression and functional analysis of cytokinin receptor-like HK genes (ZmHK) in maize. Firstly, bioinformatics analysis revealed that seven cloned ZmHK genes have different expression patterns during maize development. Secondly, ectopic expression by CaMV35S promoter in Arabidopsis further revealed that functional differentiation exists among these seven members. Among them, the ZmHK1a2-OX transgenic line has the lowest germination rate in the dark, ZmHK1-OX and ZmHK2a2-OX can delay leaf senescence, and seed size of ZmHK1-OX, ZmHK1a2-OX, ZmHK2-OX, ZmHK3b-OX and ZmHK2a2-OX was obviously reduced as compared to wild type. Additionally, ZmHK genes play opposite roles in shoot and root development; all ZmHK-OX transgenic lines display obvious shorter root length and reduced number of lateral roots, but enhanced shoot development compared with the wild type. Most notably, Arabidopsis response regulator ARR5 gene was up-regulated in ZmHK1-OX, ZmHK1a2-OX, ZmHK2-OX, ZmHK3b-OX and ZmHK2a2-OX as compared to wild type. Although the causal link between ZmHK genes and cytokinin signaling pathway is still an area to be further elucidated, these findings reflected that the diversification of ZmHK genes expression patterns and functions occurred in the course of maize evolution, indicating that some ZmHK genes might play different roles during maize development.

  19. Denture Adhesives

    Science.gov (United States)

    ... prevent overuse if zinc is an ingredient. (Some companies include graphics of the amount of adhesive to ... and adequate directions for use or a clear definition of an unsafe dosage or methods or duration ...

  20. Thermoresponsive cell culture substrates based on PNIPAM brushes functionalized with adhesion peptides: theoretical considerations of mechanism and design.

    Science.gov (United States)

    Halperin, Avraham; Kröger, Martin

    2012-12-04

    Thermoresponsive tissue culture substrates based on PNIPAM brushes are used to harvest confluent cell sheets for tissue engineering. The prospect of clinical use imposes the utilization of culture medium free of bovine serum, thus suggesting conjugation with adhesion peptides containing the RGD minimal recognition sequence. The optimum position of the RGD along the chain should ensure both cell adhesion at 37 °C and cell detachment at T(L) below the lower critical solution temperature of PNIPAM. Design guidelines are formulated from considerations of brush confinement by the cells: (i) Cell adhesion at 37 °C is controlled by the RGDs accessible without brush compression. (ii) Cell detachment at T(L) is driven by a disjoining force due to confinement of the swollen brush by cells retaining integrin-RGD bonds formed at 37 °C. These suggest placing the RGDs at the grafting surface or its vicinity. Randomly placed RGDs do not enable efficient detachment because a large fraction of the integrin-RGD bonds are not sufficiently tensioned at T(L), in line with experimental observations (Ebara, M.; Yamato, M.; Aoyagi, T.; Kikuchi, A.; Sakai, K.; Okano, T. Immobilization of celladhesive peptides to temperature-responsive surfaces facilitates both serum-free cell adhesion and noninvasive cell harvest. Tissue Eng. 2004, 10, 1125-1135). The theory framework enables analysis of culture media based on polymer brushes conjugated with adhesion peptides in general.

  1. Double-Layer Surface Modification of Polyamide Denture Base Material by Functionalized Sol-Gel Based Silica for Adhesion Improvement.

    Science.gov (United States)

    Hafezeqoran, Ali; Koodaryan, Roodabeh

    2017-09-21

    Limited surface treatments have been proposed to improve the bond strength between autopolymerizing resin and polyamide denture base materials. Still, the bond strength of autopolymerizing resins to nylon polymer is not strong enough to repair the fractured denture effectively. This study aimed to introduce a novel method to improve the adhesion of autopolymerizing resin to polyamide polymer by a double layer deposition of sol-gel silica and N-2-(aminoethyl)-3-aminopropyltrimethoxysilane (AE-APTMS). The silica sol was synthesized by acid-catalyzed hydrolysis of tetraethylorthosilicate (TEOS) as silica precursors. Polyamide specimens were dipped in TEOS-derived sol (TS group, n = 28), and exposed to ultraviolet (UV) light under O 2 flow for 30 minutes. UV-treated specimens were immersed in AE-APTMS solution and left for 24 hours at room temperature. The other specimens were either immersed in AE-APTMS solution (AP group, n = 28) or left untreated (NT group, n = 28). Surface characterization was investigated by fourier transform infrared spectroscopy (FTIR) and atomic force microscopy (AFM). Two autopolymerizing resins (subgroups G and T, n = 14) were bonded to the specimens, thermocycled, and then tested for shear bond strength with a universal testing machine. Data were analyzed with one-way ANOVA followed by Tukey's HSD (α = 0.05). FTIR spectra of treated surfaces confirmed the chemical modification and appearance of functional groups on the polymer. One-way ANOVA revealed significant differences in shear bond strength among the study groups. Tukey's HSD showed that TS T and TS G groups had significantly higher shear bond strength than control groups (p = 0.001 and p < 0.001, respectively). Moreover, bond strength values of AP T were statistically significant compared to controls (p = 0.017). Amino functionalized TEOS-derived silica coating is a simple and cost-effective method for improving the bond strength between the autopolymerizing resin and polyamide

  2. Increased Rho-kinase expression and activity and pulmonary endothelial dysfunction in smokers with normal lung function.

    Science.gov (United States)

    Duong-Quy, S; Dao, P; Hua-Huy, T; Guilluy, C; Pacaud, P; Dinh-Xuan, A T

    2011-02-01

    Endothelial dysfunction is one of the main consequences of the toxic effects of cigarette smoke on the vascular system. Increasing evidence suggests that the small G-protein RhoA and its downstream effectors, the Rho-kinases (ROCKs), are involved in systemic endothelial dysfunction induced by cigarette smoke. This study aimed to evaluate the role of the RhoA/ROCKs pathway in pulmonary artery endothelial function in current smokers with normal lung function. Lung tissues were obtained from nonsmokers and smokers who underwent lobectomy for lung carcinoma. Arterial relaxation in response to acetylcholine (ACh) was assessed in isolated pulmonary arterial rings. Protein expressions and activities of endothelial nitric oxide synthase (eNOS), ROCKs and the myosin phosphatase subunit 1 (MYPT-1) were sought. Relaxation in response to ACh was significantly lower in smokers as compared with nonsmokers (n = 8 in each group), consistent with reduced eNOS activity in the former compared with the latter. eNOS protein expression remained, however, the same in both groups. Expression of ROCKs, guanosine triphosphate-RhoA and phosphorylated MYPT-1 were significantly increased in smokers compared with controls. Pulmonary endothelial dysfunction is present in smokers whose lung function has not yet been impaired. Reduced activity of eNOS accounts at least in part for this endothelial dysfunction. Increased expression and activity of ROCKs accounts for another part through direct or indirect inhibition of the Rho-A/ROCKs pathway on nitric oxide synthesis and sustained pulmonary vasoconstriction through inhibition of myosin phosphatase.

  3. Cellular function and adhesion mechanisms of human bone marrow mesenchymal stem cells on multi-walled carbon nanotubes.

    Science.gov (United States)

    Kroustalli, Anthoula A; Kourkouli, Souzana N; Deligianni, Despina D

    2013-12-01

    Multiwalled carbon nanotubes (MWCNTs) are considered to be excellent reinforcements for biorelated applications, but, before being incorporated into biomedical devices, their biocompatibility need to be investigated thoroughly. We investigated the ability of films of pristine MWCNTs to influence human mesenchymal stem cells' proliferation, morphology, and differentiation into osteoblasts. Moreover, the selective integrin subunit expression and the adhesion mechanism to the substrate were evaluated on the basis of adherent cell number and adhesion strength, following the treatment of cells with blocking antibodies to a series of integrin subunits. Results indicated that MWCNTs accelerated cell differentiation to a higher extent than tissue culture plastic, even in the absence of additional biochemical inducing agents. The pre-treatment with anti-integrin antibodies decreased number of adherent cells and adhesion strength at 4-60%, depending on integrin subunit. These findings suggest that pristine MWCNTs represent a suitable reinforcement for bone tissue engineering scaffolds.

  4. TOR1 and TOR2 are structurally and functionally similar but not identical phosphatidylinositol kinase homologues in yeast

    OpenAIRE

    Helliwell, S. B.; Wagner, P.; Kunz, J.; Deuter-Reinhard, M.; Henriquez, R.; Hall, M. N.

    1994-01-01

    The Saccharomyces cerevisiae genes TOR1 and TOR2 were originally identified by mutations that confer resistance to the immunosuppressant rapamycin. TOR2 was previously shown to encode an essential 282-kDa phosphatidylinositol kinase (PI kinase) homologue. The TOR1 gene product is also a large (281 kDa) PI kinase homologue, with 67% identity to TOR2. TOR1 is not essential, but a TOR1 TOR2 double disruption uniquely confers a cell cycle (G1) arrest as does exposure to rapamycin; disruption of T...

  5. Rac function is crucial for cell migration but is not required for spreading and focal adhesion formation

    DEFF Research Database (Denmark)

    Steffen, Anika; Ladwein, Markus; Dimchev, Georgi A

    2013-01-01

    can be potently stimulated by Rho GTPases of the Rac subfamily, but also by RhoG or Cdc42. Here we describe viable fibroblast cell lines genetically deficient for Rac1 that lack detectable levels of Rac2 and Rac3. Rac-deficient cells were devoid of apparent lamellipodia, but these structures were...... filopodia and established focal adhesions. Spreading in these cells was achieved by the extension of filopodia followed by the advancement of cytoplasmic veils between them. The number and size of focal adhesions as well as their intensity were largely unaffected by genetic removal of Rac1. However, Rac...

  6. Identification of calcium-dependent protein kinase (CDPK): A multi-functional gene family in Rafflesia cantleyi

    Science.gov (United States)

    Amini, Safoora; Goh, Hoe-Han; Wan, Kiew-Lian

    2016-11-01

    Rafflesia, a parasitic plant that belongs to the Rafflesiaceae family, is notable for producing the largest flowers in the world. This study focused on identification of Calcium-dependent protein kinases (CDPKs) due to their vital roles in plant growth and development, biotic and abiotic stress responses, and hormone signaling. RNA-seq data generated from three bud stages of Rafflesia cantleyi ie BS1, BS2, and BS3 and were assembled. Based on the BLAST searches of Rafflesia unique transcripts (UTs) to Arabidopsis TAIR database, a total of 14 unique transcripts (UTs) were identified as CDPK1 to CDPK5, CDPK7 to CDPK11, CDPK16, CDPK18, CDPK19, and CDPK28. These genes are expressed at all three bud stages of R. cantleyi with up-regulation pattern at BS1 vs. BS2 and BS2 vs. BS3. This result shows that the expression of CDPK gene family increases by developmental progress in Rafflesia in order to regulate biochemical and molecular changes at the cellular level in response to exposure to environmental changes. However, CDPKs functions in plants growth and defense process still need more experimental evidence to deeply understand their biological roles in R. cantleyi.

  7. Eukaryotic initiation factor 5A and Ca2+/calmodulin-dependent protein kinase 1D modulate trophoblast cell function.

    Science.gov (United States)

    Qin, Xiaoli; Liang, Yan; Guo, Yuna; Liu, Xiaorui; Zeng, Weihong; Wu, Fan; Lin, Yi; Zhang, Yan

    2018-03-13

    Trophoblast cells regulate embryo implantation and placental development. Eukaryotic initiation factor 5A (eIF5A) is an initiator of translation involved in cellular processes, such as migration, proliferation, and apoptosis. However, the function of eIF5A in trophoblast cells is unknown. We inhibited eIF5A and Ca 2+ /calmodulin-dependent protein kinase 1D (CAMK1D) expression in HTR8 cells using RNA interference. The effects of eIF5A and CAMK1D on HTR8 cells were investigated using real-time polymerase chain reaction, Western blotting, flow cytometry, cell transfection assays, cell migration assays, and terminal deoxynucleotidyl transferase dUTP nick-end labeling. eIF5A inhibition decreased CAMK1D expression, proliferation, migration, and invasion, but upregulated apoptosis, in HTR8 cells. Cross-talk between eIF5A and CAMK1D enhances proliferation, migration, and invasion, but inhibits apoptosis, in trophoblasts. © 2018 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

  8. I kappa B kinase alpha (IKKα) activity is required for functional maturation of dendritic cells and acquired immunity to infection.

    Science.gov (United States)

    Mancino, Alessandra; Habbeddine, Mohamed; Johnson, Ella; Luron, Lionel; Bebien, Magali; Memet, Sylvie; Fong, Carol; Bajenoff, Marc; Wu, Xuefeng; Karin, Michael; Caamano, Jorge; Chi, Hongbo; Seed, Michael; Lawrence, Toby

    2013-03-20

    Dendritic cells (DC) are required for priming antigen-specific T cells and acquired immunity to many important human pathogens, including Mycobacteriuim tuberculosis (TB) and influenza. However, inappropriate priming of auto-reactive T cells is linked with autoimmune disease. Understanding the molecular mechanisms that regulate the priming and activation of naïve T cells is critical for development of new improved vaccines and understanding the pathogenesis of autoimmune diseases. The serine/threonine kinase IKKα (CHUK) has previously been shown to have anti-inflammatory activity and inhibit innate immunity. Here, we show that IKKα is required in DC for priming antigen-specific T cells and acquired immunity to the human pathogen Listeria monocytogenes. We describe a new role for IKKα in regulation of IRF3 activity and the functional maturation of DC. This presents a unique role for IKKα in dampening inflammation while simultaneously promoting adaptive immunity that could have important implications for the development of new vaccine adjuvants and treatment of autoimmune diseases.

  9. Sphingosine kinase 1 and sphingosine 1-phosphate receptor 3 are functionally upregulated on astrocytes under pro-inflammatory conditions.

    Directory of Open Access Journals (Sweden)

    Iris Fischer

    Full Text Available BACKGROUND: Reactive astrocytes are implicated in the development and maintenance of neuroinflammation in the demyelinating disease multiple sclerosis (MS. The sphingosine kinase 1 (SphK1/sphingosine1-phosphate (S1P receptor signaling pathway is involved in modulation of the inflammatory response in many cell types, but the role of S1P receptor subtype 3 (S1P(3 signaling and SphK1 in activated rat astrocytes has not been defined. METHODOLOGY/PRINCIPAL FINDINGS: Using immunohistochemistry we observed the upregulation of S1P(3 and SphK1 expression on reactive astrocytes and SphK1 on macrophages in MS lesions. Increased mRNA and protein expression of S1P(3 and SphK1, as measured by qPCR and Western blotting respectively, was observed after treatment of rat primary astrocyte cultures with the pro-inflammatory stimulus lipopolysaccharide (LPS. Activation of SphK by LPS stimulation was confirmed by SphK activity assay and was blocked by the use of the SphK inhibitor SKI (2-(p-hydroxyanilino-4-(p-chlorphenyl thiazole. Treatment of astrocytes with a selective S1P(3 agonist led to increased phosphorylation of extracellular signal-regulated kinase (ERK-1/2, which was further elevated with a LPS pre-challenge, suggesting that S1P(3 upregulation can lead to increased functionality. Moreover, astrocyte migration in a scratch assay was induced by S1P and LPS and this LPS-induced migration was sensitive to inhibition of SphK1, and independent of cell proliferation. In addition, S1P induced secretion of the potentially neuroprotective chemokine CXCL1, which was increased when astrocytes were pre-challenged with LPS. A more prominent role of S1P(3 signaling compared to S1P(1 signaling was demonstrated by the use of selective S1P(3 or S1P(1 agonists. CONCLUSION/SIGNIFICANCE: In summary, our data demonstrate that the SphK1/S1P(3 signaling axis is upregulated when astrocytes are activated by LPS. This signaling pathway appears to play a role in the establishment

  10. Beneficial Effect of Intermedin 1-53 in Septic Shock Rats: Contributions of Rho Kinase and BKCA Pathway-Mediated Improvement in Cardiac Function.

    Science.gov (United States)

    Zhu, Yu; Wu, Huiling; Wu, Yue; Zhang, Jie; Peng, Xiaoyong; Zang, Jiatao; Xiang, Xinming; Liu, Liangming; Li, Tao

    2016-11-01

    Intermedin (IMD) is a calcitonin gene-related peptide shown to have a protective effect on myocardial function in ischemia-reperfusion injury. Whether IMD has beneficial effect in severe sepsis and septic shock (and its underlying mechanisms) is not known. We induced septic shock using cecal ligation and puncture (CLP). We focused on the potential beneficial effect of IMD1-53 on cardiac papillary muscle and cardiomyocytes against septic shock and its relationship with the protection of cardiac function. Early (immediately after CLP) and late (12 h after CLP) administration of IMD1-53 (0.5 μg/kg) improved animal survival significantly, increased cardiac contractility and function, and improved tissue perfusion and oxygen delivery. The effect of early administration of IMD1-53 was better than that of late administration. The Rho kinase/TnI and BKCa pathways participated in the protective effect of IMD1-53 on cardiac function in septic rats. An inhibitor of Rho kinase (Y-27632) or a BKCa opener (NS1619) abolished the protective effect of IMD1-53 on cardiac function. IMD1-53 increased expression of Rho kinase in cardiac muscle and inhibited TnI phosphorylation. IMD1-53 inhibited currents in BKCa channels and intracellular calcium concentration in cardiomyocytes. IMD1-53 is beneficial against severe sepsis/septic shock. IMD1-53 can improve cardiac contractility and cardiac function significantly, and then improve tissue perfusion and oxygen delivery. Rho kinase and the BKCa pathways have important roles in these effects. These findings provide a new treatment strategy for severe sepsis with cardiac dysfunction.

  11. Transfected HEK293 Cells Expressing Functional Recombinant Intercellular Adhesion Molecule 1 (ICAM-1) - A Receptor Associated with Severe Plasmodium falciparum Malaria

    DEFF Research Database (Denmark)

    Bengtsson, Anja; Joergensen, Louise; Barbati, Zachary R

    2013-01-01

    Intercellular adhesion molecule 1 (ICAM-1) is a membrane-bound glycoprotein expressed on endothelial cells and cells of the immune system. Human ICAM-1 mediates adhesion and migration of leucocytes, and is implicated in inflammatory pathologies, autoimmune diseases and in many cancer processes....... Additionally, ICAM-1 acts as receptor for pathogens like human rhinovirus and Plasmodium falciparum malaria parasites. A group of related P. falciparum erythrocyte membrane protein 1 (PfEMP1) domains, the DBLβ, mediates ICAM-1 binding of P. falciparum-infected erythrocytes. This ICAM‑1-binding phenotype has...... as vaccine candidates and go into clinical trials. Such studies require availability of functional recombinant ICAM-1 in large quantities. In this study, we compared recombinant ICAM-1 expressed in HEK293 and COS-7 cells with mouse myeloma NS0 ICAM-1 purchased from a commercial vendor in terms of protein...

  12. The neural cell adhesion molecule

    DEFF Research Database (Denmark)

    Berezin, V; Bock, E; Poulsen, F M

    2000-01-01

    During the past year, the understanding of the structure and function of neural cell adhesion has advanced considerably. The three-dimensional structures of several of the individual modules of the neural cell adhesion molecule (NCAM) have been determined, as well as the structure of the complex...... between two identical fragments of the NCAM. Also during the past year, a link between homophilic cell adhesion and several signal transduction pathways has been proposed, connecting the event of cell surface adhesion to cellular responses such as neurite outgrowth. Finally, the stimulation of neurite...

  13. Structure and Function of the Hypertension Variant A486V of G Protein-coupled Receptor Kinase 4

    Energy Technology Data Exchange (ETDEWEB)

    Allen, Samantha J.; Parthasarathy, Gopal; Darke, Paul L.; Diehl, Ronald E.; Ford, Rachael E.; Hall, Dawn L.; Johnson, Scott A.; Reid, John C.; Rickert, Keith W.; Shipman, Jennifer M.; Soisson, Stephen M.; Zuck, Paul; Munshi, Sanjeev K.; Lumb, Kevin J. (Merck)

    2015-07-01

    G-protein-coupled receptor (GPCR) kinases (GRKs) bind to and phosphorylate GPCRs, initiating the process of GPCR desensitization and internalization. GRK4 is implicated in the regulation of blood pressure, and three GRK4 polymorphisms (R65L, A142V, and A486V) are associated with hypertension. Here, we describe the 2.6 Å structure of human GRK4α A486V crystallized in the presence of 5'-adenylyl β,γ-imidodiphosphate. The structure of GRK4α is similar to other GRKs, although slight differences exist within the RGS homology (RH) bundle subdomain, substrate-binding site, and kinase C-tail. The RH bundle subdomain and kinase C-terminal lobe form a strikingly acidic surface, whereas the kinase N-terminal lobe and RH terminal subdomain surfaces are much more basic. In this respect, GRK4α is more similar to GRK2 than GRK6. A fully ordered kinase C-tail reveals interactions linking the C-tail with important determinants of kinase activity, including the αB helix, αD helix, and the P-loop. Autophosphorylation of wild-type GRK4α is required for full kinase activity, as indicated by a lag in phosphorylation of a peptide from the dopamine D1 receptor without ATP preincubation. In contrast, this lag is not observed in GRK4α A486V. Phosphopeptide mapping by mass spectrometry indicates an increased rate of autophosphorylation of a number of residues in GRK4α A486V relative to wild-type GRK4α, including Ser-485 in the kinase C-tail.

  14. LIM kinase1 modulates function of membrane type matrix metalloproteinase 1: implication in invasion of prostate cancer cells

    Directory of Open Access Journals (Sweden)

    Chakrabarti Ratna

    2011-01-01

    Full Text Available Abstract Background LIM kinase 1 (LIMK1 is an actin and microtubule cytoskeleton modulatory protein that is overexpressed in a number of cancerous tissues and cells and also promotes invasion and metastasis of prostate and breast cancer cells. Membrane type matrix metalloproteinase 1 (MT1-MMP is a critical modulator of extracellular matrix (ECM turnover through pericellular proteolysis and thus plays crucial roles in neoplastic cell invasion and metastasis. MT1-MMP and its substrates pro-MMP-2 and pro-MMP-9 are often overexpressed in a variety of cancers including prostate cancer and the expression levels correlate with the grade of malignancy in prostate cancer cells. The purpose of this study is to determine any functional relation between LIMK1 and MT1-MMP and its implication in cell invasion. Results Our results showed that treatment with the hydroxamate inhibitor of MT1-MMP, MMP-2 and MMP-9 ilomastat inhibited LIMK1-induced invasion of benign prostate epithelial cells. Over expression of LIMK1 resulted in increased collagenolytic activity of MMP-2, and secretion of pro-MMP2 and pro-MMP-9. Cells over expressing LIMK1 also exhibited increased expression of MT1-MMP, transcriptional activation and its localization to the plasma membrane. LIMK1 physically associates with MT1-MMP and is colocalized with it to the Golgi vesicles. We also noted increased expression of both MT1-MMP and LIMK1 in prostate tumor tissues. Conclusion Our results provide new information on regulation of MT1-MMP function by LIMK1 and showed for the first time, involvement of MMPs in LIMK1 induced cell invasion.

  15. Impaired vascular function in sepsis-surviving rats mediated by oxidative stress and Rho-Kinase pathway

    Directory of Open Access Journals (Sweden)

    Priscila de Souza

    2016-12-01

    Full Text Available We investigated long-lasting changes in endothelial and vascular function in adult rat survivors of severe sepsis induced by cecal ligation and puncture (CLP model. For this, male Wistar rats (200–350 g had their cecum punctured once (non-transfixing hole with a 14-gauge needle. Performed in this way, a mortality rate around 30% was achieved in the first 72 h. The survivors, together with age-matched control rats (not subjected to CLP, were maintained in our holding room for 60 days (S60 group and had the descending thoracic aorta processed for functional, histological, biochemical or molecular analyses. Endothelium-intact aortic rings obtained from sepsis-surviving S60 group displayed increased angiotensin II-induced contraction, accompanied by decreased activity of the endogenous superoxide dismutase, augmented reactive oxygen species generation, and increased levels of tyrosine nitration compared with vessels from control group. The superoxide scavengers superoxide dismutase and tempol, and the antioxidant apocynin, were able to avoid this enhanced contractility to angiotensin II in aortic rings from the S60 group. In addition, aortic rings from the S60 group presented reduced sensitivity to Y-27632, a Rho-kinase (ROCK inhibitor. Immunoblot analyses revealed augmented RhoA and ROCK II, and high levels of phosphorylation of myosin phosphatase target subunit 1 in vessels from S60 rats. In conclusion, aortic rings from sepsis-surviving rats display endothelial dysfunction mediated by the increased production of reactive oxygen species, which in turn reduces the bioavailability of nitric oxide and increases the formation of peroxynitrite, and enhances RhoA-ROCK-mediated calcium sensitization, leading to augmented contractile responses to angiotensin II. Notably, this is the first study demonstrating long-term dysfunction in the vasculature of sepsis-surviving rats, which take place or remain beyond the acute septic insult.

  16. AMP-activated kinase in human spermatozoa: identification, intracellular localization, and key function in the regulation of sperm motility.

    Science.gov (United States)

    Calle-Guisado, Violeta; de Llera, Ana Hurtado; Martin-Hidalgo, David; Mijares, Jose; Gil, Maria C; Alvarez, Ignacio S; Bragado, Maria J; Garcia-Marin, Luis J

    2017-01-01

    AMP-activated kinase (AMPK), a protein that regulates energy balance and metabolism, has recently been identified in boar spermatozoa where regulates key functional sperm processes essential for fertilization. This work's aims are AMPK identification, intracellular localization, and their role in human spermatozoa function. Semen was obtained from healthy human donors. Sperm AMPK and phospho-Thr172-AMPK were analyzed by Western blotting and indirect immunofluorescence. High- and low-quality sperm populations were separated by a 40%-80% density gradient. Human spermatozoa motility was evaluated by an Integrated Semen Analysis System (ISAS) in the presence or absence of the AMPK inhibitor compound C (CC). AMPK is localized along the human spermatozoa, at the entire acrosome, midpiece and tail with variable intensity, whereas its active form, phospho-Thr172-AMPK, shows a prominent staining at the acrosome and sperm tail with a weaker staining in the midpiece and the postacrosomal region. Interestingly, spermatozoa bearing an excess residual cytoplasm show strong AMPK staining in this subcellular compartment. Both AMPK and phospho-Thr172-AMPK human spermatozoa contents exhibit important individual variations. Moreover, active AMPK is predominant in the high motility sperm population, where shows a stronger intensity compared with the low motility sperm population. Inhibition of AMPK activity in human spermatozoa by CC treatment leads to a significant reduction in any sperm motility parameter analyzed: percent of motile sperm, sperm velocities, progressivity, and other motility coefficients. This work identifies and points out AMPK as a new molecular mechanism involved in human spermatozoa motility. Further AMPK implications in the clinical efficiency of assisted reproduction and in other reproductive areas need to be studied.

  17. AMP-activated kinase in human spermatozoa: identification, intracellular localization, and key function in the regulation of sperm motility

    Science.gov (United States)

    Calle-Guisado, Violeta; de Llera, Ana Hurtado; Martin-Hidalgo, David; Mijares, Jose; Gil, Maria C; Alvarez, Ignacio S; Bragado, Maria J; Garcia-Marin, Luis J

    2017-01-01

    AMP-activated kinase (AMPK), a protein that regulates energy balance and metabolism, has recently been identified in boar spermatozoa where regulates key functional sperm processes essential for fertilization. This work's aims are AMPK identification, intracellular localization, and their role in human spermatozoa function. Semen was obtained from healthy human donors. Sperm AMPK and phospho-Thr172-AMPK were analyzed by Western blotting and indirect immunofluorescence. High- and low-quality sperm populations were separated by a 40%–80% density gradient. Human spermatozoa motility was evaluated by an Integrated Semen Analysis System (ISAS) in the presence or absence of the AMPK inhibitor compound C (CC). AMPK is localized along the human spermatozoa, at the entire acrosome, midpiece and tail with variable intensity, whereas its active form, phospho-Thr172-AMPK, shows a prominent staining at the acrosome and sperm tail with a weaker staining in the midpiece and the postacrosomal region. Interestingly, spermatozoa bearing an excess residual cytoplasm show strong AMPK staining in this subcellular compartment. Both AMPK and phospho-Thr172-AMPK human spermatozoa contents exhibit important individual variations. Moreover, active AMPK is predominant in the high motility sperm population, where shows a stronger intensity compared with the low motility sperm population. Inhibition of AMPK activity in human spermatozoa by CC treatment leads to a significant reduction in any sperm motility parameter analyzed: percent of motile sperm, sperm velocities, progressivity, and other motility coefficients. This work identifies and points out AMPK as a new molecular mechanism involved in human spermatozoa motility. Further AMPK implications in the clinical efficiency of assisted reproduction and in other reproductive areas need to be studied. PMID:27678462

  18. AMP-activated kinase in human spermatozoa: identification, intracellular localization, and key function in the regulation of sperm motility

    Directory of Open Access Journals (Sweden)

    Violeta Calle-Guisado

    2017-01-01

    Full Text Available AMP-activated kinase (AMPK, a protein that regulates energy balance and metabolism, has recently been identified in boar spermatozoa where regulates key functional sperm processes essential for fertilization. This work′s aims are AMPK identification, intracellular localization, and their role in human spermatozoa function. Semen was obtained from healthy human donors. Sperm AMPK and phospho-Thr172-AMPK were analyzed by Western blotting and indirect immunofluorescence. High- and low-quality sperm populations were separated by a 40%-80% density gradient. Human spermatozoa motility was evaluated by an Integrated Semen Analysis System (ISAS in the presence or absence of the AMPK inhibitor compound C (CC. AMPK is localized along the human spermatozoa, at the entire acrosome, midpiece and tail with variable intensity, whereas its active form, phospho-Thr172-AMPK, shows a prominent staining at the acrosome and sperm tail with a weaker staining in the midpiece and the postacrosomal region. Interestingly, spermatozoa bearing an excess residual cytoplasm show strong AMPK staining in this subcellular compartment. Both AMPK and phospho-Thr172-AMPK human spermatozoa contents exhibit important individual variations. Moreover, active AMPK is predominant in the high motility sperm population, where shows a stronger intensity compared with the low motility sperm population. Inhibition of AMPK activity in human spermatozoa by CC treatment leads to a significant reduction in any sperm motility parameter analyzed: percent of motile sperm, sperm velocities, progressivity, and other motility coefficients. This work identifies and points out AMPK as a new molecular mechanism involved in human spermatozoa motility. Further AMPK implications in the clinical efficiency of assisted reproduction and in other reproductive areas need to be studied.

  19. Integrin-linked kinase regulates interphase and mitotic microtubule dynamics.

    Directory of Open Access Journals (Sweden)

    Simin Lim

    Full Text Available Integrin-linked kinase (ILK localizes to both focal adhesions and centrosomes in distinct multiprotein complexes. Its dual function as a kinase and scaffolding protein has been well characterized at focal adhesions, where it regulates integrin-mediated cell adhesion, spreading, migration and signaling. At the centrosomes, ILK regulates mitotic spindle organization and centrosome clustering. Our previous study showed various spindle defects after ILK knockdown or inhibition that suggested alteration in microtubule dynamics. Since ILK expression is frequently elevated in many cancer types, we investigated the effects of ILK overexpression on microtubule dynamics. We show here that overexpressing ILK in HeLa cells was associated with a shorter duration of mitosis and decreased sensitivity to paclitaxel, a chemotherapeutic agent that suppresses microtubule dynamics. Measurement of interphase microtubule dynamics revealed that ILK overexpression favored microtubule depolymerization, suggesting that microtubule destabilization could be the mechanism behind the decreased sensitivity to paclitaxel, which is known to stabilize microtubules. Conversely, the use of a small molecule inhibitor selective against ILK, QLT-0267, resulted in suppressed microtubule dynamics, demonstrating a new mechanism of action for this compound. We further show that treatment of HeLa cells with QLT-0267 resulted in higher inter-centromere tension in aligned chromosomes during mitosis, slower microtubule regrowth after cold depolymerization and the presence of a more stable population of spindle microtubules. These results demonstrate that ILK regulates microtubule dynamics in both interphase and mitotic cells.

  20. Functional Analysis of Lactobacillus rhamnosus GG Pili in Relation to Adhesion and Immunomodulatory Interactions with Intestinal Epithelial Cells

    NARCIS (Netherlands)

    Lebeer, S.; Claes, I.J.; Tytgat, H.L.P.; Verhoeven, T.L.A.; Marien, E.; Ossowski, von I.; Reunanen, J.; Palva, A.; Vos, de W.M.; Keersmaecker, de S.C.; Vanderleyden, J.

    2012-01-01

    Lactobacillus rhamnosus GG, a probiotic with good survival capacity in the human gut, has well-documented adhesion properties and health effects. Recently, spaCBA-encoded pili that bind to human intestinal mucus were identified on its cell surface. Here, we report on the phenotypic analysis of a

  1. Two functional reticulocyte binding-like (RBL) invasion ligands of zoonotic Plasmodium knowlesi exhibit differential adhesion to monkey and human erythrocytes.

    Science.gov (United States)

    Semenya, Amma A; Tran, Tuan M; Meyer, Esmeralda Vs; Barnwell, John W; Galinski, Mary R

    2012-07-06

    Plasmodium knowlesi is a monkey malaria species that is becoming a serious public health concern infecting hundreds and perhaps thousands of humans in Southeast Asia. Invasion of erythrocytes by merozoites entails a cascade of molecular interactions. One step involves the adhesion of Plasmodium reticulocyte binding-like (RBL) proteins. Plasmodium knowlesi merozoites express only two RBL invasion ligands, known as Normocyte Binding Proteins (PkNBPXa and PkNBPXb). Overlapping N-terminal regions of PkNBPXa and PkNBPXb were expressed in COS7 cells and tested for surface expression and adhesion to rhesus monkey erythrocytes. Subsequent tests to study specific receptor ligand interactions included adhesion to a panel of human and non-human primate erythrocytes, enzymatic treatment, and site directed mutagenesis. An N-terminal cysteine-rich region of PkNBPXb (PkNBPXb-II) exhibited specific adhesion to rhesus monkey erythrocytes. Mutation of four of five cysteines in PkNBPXb-II interfered with its surface expression on COS7 cells, suggesting disulphide bond conformation is critical for intracellular trafficking. Binding of PkNBPXb-II was abolished when rhesus erythrocytes were pre-treated with chymotrypsin, but not trypsin or neuraminidase. PkNBPXb-II also bound other Old World monkey species and gibbon erythrocytes. However, erythrocytes from other primate species including humans did not bind to PkNBPXb-II or native PkNBPXb. Importantly, unlike PkNBPXb, PkNBPXa bound human erythrocytes, and this binding was independent of the Duffy blood group determinant. The data reported here begins to clarify the functional domains of the P. knowlesi RBLs. A binding domain has been identified and characterized in PkNBPXb. Notably, this study demonstrates that unlike PkNBPXb, PkNBPXa can bind to human erythrocytes, suggesting that PkNBPXa may function as a ligand to enable the invasion of P. knowlesi merozoites into human cells.

  2. Two functional reticulocyte binding-like (RBL invasion ligands of zoonotic Plasmodium knowlesi exhibit differential adhesion to monkey and human erythrocytes

    Directory of Open Access Journals (Sweden)

    Semenya Amma A

    2012-07-01

    Full Text Available Abstract Background Plasmodium knowlesi is a monkey malaria species that is becoming a serious public health concern infecting hundreds and perhaps thousands of humans in Southeast Asia. Invasion of erythrocytes by merozoites entails a cascade of molecular interactions. One step involves the adhesion of Plasmodium reticulocyte binding-like (RBL proteins. Plasmodium knowlesi merozoites express only two RBL invasion ligands, known as Normocyte Binding Proteins (PkNBPXa and PkNBPXb. Methods Overlapping N-terminal regions of PkNBPXa and PkNBPXb were expressed in COS7 cells and tested for surface expression and adhesion to rhesus monkey erythrocytes. Subsequent tests to study specific receptor ligand interactions included adhesion to a panel of human and non-human primate erythrocytes, enzymatic treatment, and site directed mutagenesis. Results An N-terminal cysteine-rich region of PkNBPXb (PkNBPXb-II exhibited specific adhesion to rhesus monkey erythrocytes. Mutation of four of five cysteines in PkNBPXb-II interfered with its surface expression on COS7 cells, suggesting disulphide bond conformation is critical for intracellular trafficking. Binding of PkNBPXb-II was abolished when rhesus erythrocytes were pre-treated with chymotrypsin, but not trypsin or neuraminidase. PkNBPXb-II also bound other Old World monkey species and gibbon erythrocytes. However, erythrocytes from other primate species including humans did not bind to PkNBPXb-II or native PkNBPXb. Importantly, unlike PkNBPXb, PkNBPXa bound human erythrocytes, and this binding was independent of the Duffy blood group determinant. Conclusions The data reported here begins to clarify the functional domains of the P. knowlesi RBLs. A binding domain has been identified and characterized in PkNBPXb. Notably, this study demonstrates that unlike PkNBPXb, PkNBPXa can bind to human erythrocytes, suggesting that PkNBPXa may function as a ligand to enable the invasion of P. knowlesi merozoites into

  3. The effects of cannabinoids and cannabispiro compounds on Escherichia coli adhesion to tissue culture cells and on leukocyte functions in vitro.

    Science.gov (United States)

    Molnár, J; Petri, I; Berek, I; Shoyama, Y; Nishioka, I

    1987-01-01

    delta 9-Tetrahydrocannabinol, cannabidiol, cannabidiolic acid, tetrahydrocannabidiolic acid, cannabispirol, acetylcannabispirol, cannabispirone, and cannabispirenone in a low concentration did not affect the adhesion of Escherichia coli on cultured HEp-2 cells. Cannabinoids at 10(-6) M increased the chemiluminescence of human polymorphonuclear leukocytes, while the cannabispiro compounds failed to enhance the oxidative burst of leukocytes. In lymphocyte and granulocyte function tests (E- and EA-rosette formation, blast transformation of T-lymphocytes in the presence of phytohaemagglutinin and concanavalin-A, ADCC and phagocytosis) all compounds displayed immunosuppressive effect at 1.5 X 10(-5) M. Tetrahydrocannabidiolic acid exerted the weakest immunosuppression on human leukocyte functions.

  4. The neural cell adhesion molecule

    DEFF Research Database (Denmark)

    Berezin, V; Bock, E; Poulsen, F M

    2000-01-01

    During the past year, the understanding of the structure and function of neural cell adhesion has advanced considerably. The three-dimensional structures of several of the individual modules of the neural cell adhesion molecule (NCAM) have been determined, as well as the structure of the complex...

  5. Multi-functional and redundant roles of Borrelia burgdorferi outer surface proteins in tissue adhesion, colonization, and complement evasion.

    Directory of Open Access Journals (Sweden)

    Jennifer A Caine

    2016-10-01

    Full Text Available Borrelia burgdorferi is the causative agent of Lyme disease in the U.S., with at least 25,000 cases reported to the CDC each year. B. burgdorferi is thought to enter and exit the bloodstream to achieve rapid dissemination to distal tissue sites during infection. Travel through the bloodstream requires evasion of immune surveillance and pathogen clearance in the host, a process at which B. burgdorferi is adept. B. burgdorferi encodes greater than 19 adhesive outer surface proteins many of which have been found to bind to host cells or components of the extracellular matrix. Several others bind to host complement regulatory factors, in vitro. Production of many of these adhesive proteins is tightly regulated by environmental cues, and some have been shown to aid in vascular interactions and tissue colonization, as well as survival in the blood, in vivo. Recent work has described multifaceted and redundant roles of B. burgdorferi outer surface proteins in complement component interactions and tissue targeted adhesion and colonization, distinct from their previously identified in vitro binding capabilities. Recent insights into the multifunctional roles of previously well characterized outer surface proteins such as BBK32, DbpA, CspA, and OspC have changed the way we think about the surface proteome of these organisms during the tick-mammal lifecycle. With the combination of new and old in vivo models and in vitro techniques, the field has identified distinct ligand binding domains on BBK32 and DbpA that afford tissue colonization or blood survival to B. burgdorferi. In this review we describe the multifunctional and redundant roles of many adhesive outer surface proteins of B. burgdorferi in tissue adhesion, colonization, and bloodstream survival that, together, promote the survival of Borrelia spp. throughout maintenance in their multi-host lifestyle.

  6. Inhibitory Effects of KP-A159, a Thiazolopyridine Derivative, on Osteoclast Differentiation, Function, and Inflammatory Bone Loss via Suppression of RANKL-Induced MAP Kinase Signaling Pathway.

    Directory of Open Access Journals (Sweden)

    Hye Jung Ihn

    Full Text Available Abnormally elevated formation and activation of osteoclasts are primary causes for a majority of skeletal diseases. In this study, we found that KP-A159, a newly synthesized thiazolopyridine derivative, inhibited osteoclast differentiation and function in vitro, and inflammatory bone loss in vivo. KP-A159 did not cause a cytotoxic response in bone marrow macrophages (BMMs, but significantly inhibited the formation of multinucleated tartrate-resistant acid phosphatase (TRAP-positive osteoclasts induced by macrophage colony-stimulating factor (M-CSF and receptor activator of nuclear factor-κB ligand (RANKL. KP-A159 also dramatically inhibited the expression of marker genes related to osteoclast differentiation, including TRAP (Acp5, cathepsin K (Ctsk, dendritic cell-specific transmembrane protein (Dcstamp, matrix metallopeptidase 9 (Mmp9, and nuclear factor of activated T-cells, cytoplasmic 1 (Nfatc1. Moreover, actin ring and resorption pit formation were inhibited by KP-A159. Analysis of the signaling pathway involved showed that KP-A159 inhibited RANKL-induced activation of extracellular signal-regulated kinase (ERK, c-Jun N-terminal kinase (JNK, and mitogen-activated protein kinase kinase1/2 (MEK1/2. In a mouse inflammatory bone loss model, KP-A159 significantly rescued lipopolysaccharide (LPS-induced bone loss by suppressing osteoclast numbers. Therefore, KP-A159 targets osteoclasts, and may be a potential candidate compound for prevention and/or treatment of inflammatory bone loss.

  7. an Adhesive Patch

    Directory of Open Access Journals (Sweden)

    S. Mojtaba Taghizadeh

    2013-01-01

    Full Text Available Drug-in-adhesive transdermal drug delivery systems  TDDSs containing stimulants, termed as energetic substances, such as caffeine and pantothenic acid, were studied. Caffeine is a white crystalline substance and a stimulant to central nervous system. In humans, caffeine acts as a central nervous system stimulant, temporarily warding off drowsiness and restoring alertness. Pantothenic acid, also recognized as vitamin B5, is a water-soluble vitamin. For many animals, pantothenic acid is an essential nutrient. Animals require pantothenic acid to synthesize and metabolize proteins, carbohydrates and fats. For this purpose caffeine and pantothenic acid were  used  as  drug  components with  6.32%  and  1.12%  loadings,  in  different functional and non-functional acrylic pressure sensitive adhesives (PSAs of 52.89%, respectively. Ethylene glycol as a chemical enhancer was used in all TDDSs with 39.67%. The effect of PSAs  type on  in vitro  release and adhesion properties  (peel strength and tack values from drug delivery devices were evaluated. It was found that TDDS containing -COOH functional PSA showed  the  lowest steady state fux. The adhesion properties of the samples were improved by addition of functional acrylic PSA in formulations.

  8. Functional Coexpression of HSV-1 Thymidine Kinase and Green Fluorescent Protein: Implications for Noninvasive Imaging of Transgene Expression

    Directory of Open Access Journals (Sweden)

    Andreas Jacobs

    1999-06-01

    Full Text Available Current gene therapy technology is limited by the paucity of methodology for determining the location and magnitude of therapeutic transgene expression in vivo. We describe and validate a paradigm for monitoring therapeutic transgene expression by noninvasive imaging of the herpes simplex virus type 1 thymidine kinase (HSV-1-tk marker gene expression. To test proportional coexpression of therapeutic and marker genes, a model fusion gene comprising green fluorescent protein (gfp and HSV-1-tk genes was generated (tkgfp gene and assessed for the functional coexpression of the gene product, TKGFP fusion protein, in rat 9L gliosarcoma, RG2 glioma, and W256 carcinoma cells. Analysis of the TKGFP protein demonstrated that it can serve as a therapeutic gene by rendering tkgfp transduced cells sensitive to ganciclovir or as a screening marker useful for identifying transduced cells by fluorescence microscopy or fluorescence-activated cell sorting (FACS. TK and GFP activities in the TKGFP fusion protein were similar to corresponding wild-type proteins and accumulation of the HSV-1-tk-specific radiolabeled substrate, 2′-fluoro-2′-deoxy-1β-D-arabino-furanosyl-5-iodo-uracil (FIAU, in stability transduced clones correlated with gfp-fluorescence intensity over a wide range of expression levels. The tkgfp fusion gene itself may be useful in developing novel cancer gene therapy approaches. Valuable information about the efficiency of gene transfer and expression could be obtained by non-invasive imaging of tkgfp expression with FIAU and clinical imaging devices (gamma camera, positron-emission tomography [PET], single photon emission computed tomography [SPECT], and/or direct visualization of gfp expression in situ by fluorescence microscopy or endoscopy.

  9. Overexpression of MERTK Receptor Tyrosine Kinase in Epithelial Cancer Cells Drives Efferocytosis in a Gain-of-Function Capacity*

    Science.gov (United States)

    Nguyen, Khanh-Quynh N.; Tsou, Wen-I; Calarese, Daniel A.; Kimani, Stanley G.; Singh, Sukhwinder; Hsieh, Shelly; Liu, Yongzhang; Lu, Bin; Wu, Yi; Garforth, Scott J.; Almo, Steve C.; Kotenko, Sergei V.; Birge, Raymond B.

    2014-01-01

    MERTK, a member of the TAM (TYRO3, AXL, and MERTK) receptor tyrosine kinases, has complex and diverse roles in cell biology. On the one hand, knock-out of MERTK results in age-dependent autoimmunity characterized by failure of apoptotic cell clearance, while on the other, MERTK overexpression in cancer drives classical oncogene pathways leading to cell transformation. To better understand the interplay between cell transformation and efferocytosis, we stably expressed MERTK in human MCF10A cells, a non-tumorigenic breast epithelial cell line devoid of endogenous MERTK. While stable expression of MERTK in MCF10A resulted in enhanced motility and AKT-mediated chemoprotection, MERTK-10A cells did not form stable colonies in soft agar, or enhance proliferation compared with parental MCF10A cells. Concomitant to chemoresistance, MERTK also stimulated efferocytosis in a gain-of-function capacity. However, unlike AXL, MERTK activation was highly dependent on apoptotic cells, suggesting MERTK may preferentially interface with phosphatidylserine. Consistent with this idea, knockdown of MERTK in breast cancer cells MDA-MB 231 reduced efferocytosis, while transient or stable expression of MERTK stimulated apoptotic cell clearance in all cell lines tested. Moreover, human breast cancer cells with higher endogenous MERTK showed higher levels of efferocytosis that could be blocked by soluble TAM receptors. Finally, through MERTK, apoptotic cells induced PD-L1 expression, an immune checkpoint blockade, suggesting that cancer cells may adopt MERTK-driven efferocytosis as an immune suppression mechanism for their advantage. These data collectively identify MERTK as a significant link between cancer progression and efferocytosis, and a potentially unrealized tumor-promoting event when MERTK is overexpressed in epithelial cells. PMID:25074939

  10. Determination of creatine kinase kinetic parameters in rat brain by NMR magnetization transfer. Correlation with brain function.

    Science.gov (United States)

    Sauter, A; Rudin, M

    1993-06-25

    The pseudo first-order rate constant kf of the creatine kinase (CK) forward reaction as well as the CK forward flux FCK,f have been shown to correlate better with cardiac performance than the steady-state levels of ATP and PCr (Bittl, J. A., and Ingwall, J. S. (1985) J. Biol. Chem. 260, 3512-3517). In order to elucidate the relationship between the CK kinetic parameters and brain activity, we have determined, using the non-invasive NMR technique of magnetization transfer, kf and FCK,f in rats, in which brain activity was experimentally varied by administration of either thiopental sodium or bicuculline to decrease or increase electro-encephalogram (EEG) intensity, respectively. The steady-state levels of ATP and PCr, as well as the accumulation of deoxyglucose 6-phosphate (DG-6P) in brain following intraperitoneal administration of deoxyglucose, were determined simultaneously by the NMR technique, whereas the cortical EEG was recorded in a separate experiment. The EEG intensity (range, 1-20 Hz), taken as a measure for brain performance, as well as the amount of DG-6P formed in brain, reflecting the synthesis rate of high energy phosphates (ATP and PCr), linearly correlated with kf. Despite large changes in both EEG intensity (50-250%) and kf (0.12-0.69 s-1) between thiopental sodium- and bicuculline-treated rats, the ATP levels remained constant, whereas the PCr levels decreased with high EEG activity. In contrast to the expectation based on model calculations of CK kinetics, the PCr levels did not increase above control values at reduced EEG intensity (50% of controls). At EEG intensities exceeding control values (bicuculline-treated rats) FCK,f increased as predicted by CK equilibrium. In conclusion, we have shown that in the rat brain, like in the heart, the CK forward rate constant kf, in contrast to ATP and PCr levels, is a sensitive reliable indicator of both increased and reduced function.

  11. Endotoxemia Engages the RhoA Kinase Pathway to Impair Cardiac Function By Altering Cytoskeleton, Mitochondrial Fission, and Autophagy.

    Science.gov (United States)

    Preau, Sebastien; Delguste, Florian; Yu, Yichi; Remy-Jouet, Isabelle; Richard, Vincent; Saulnier, Fabienne; Boulanger, Eric; Neviere, Remi

    2016-04-01

    The RhoA/ROCK pathway controls crucial biological processes involved in cardiovascular pathophysiology, such as cytoskeleton dynamics, vascular smooth muscle contraction, and inflammation. In this work, we tested whether Rho kinase inhibition would beneficially impact cardiac cytoskeleton organization, bioenergetics, and autophagy in experimental endotoxemia induced by lipopolysaccharides (LPSs) in mice. Fasudil, a potent ROCK inhibitor, prevented LPS-induced cardiac inflammation, oxidative stress, cytoskeleton disarray, and mitochondrial injury. ROCK inhibition prevented phosphorylation of cofilin and dynamin-related protein-1, which promotes stabilization-polymerization of F-actin and mediates mitochondrial fission, respectively. Pyr1, which exclusively alters actin dynamics, prevented LPS-induced myocardial dysfunction, suggesting that beneficial impact of ROCK inhibition was not mainly related to pleiotropic effects of fasudil on cardiac inflammation and oxidative stress. Fasudil reduced mitochondrial fragmentation, stimulated initiation of autophagy, and elicited cardioprotection in LPS heart. Mdivi-1, a potent mitochondria fission inhibitor, converted cardioprotective autophagy to an inefficient form due to cargo loading failure in which autophagic vacuoles fail to trap cytosolic cargo, despite their formation at enhanced rates and lysosomal elimination. In experimental endotoxemia, cardioprotection by RhoA/ROCK inhibition may be related to changes in actin cytoskeleton reorganization and mitochondrial homeostasis. Improvement of LPS-induced mitochondrial dysfunction by fasudil was attributed to inhibition of ROCK-dependent Drp1 phosphorylation and activation of autophagic processes that can limit mitochondrial fragmentation and enhance degradation of damaged mitochondria, respectively. Fasudil prevented LPS-induced heart oxidative stress, abnormal F-actin distribution, and oxidative phosphorylation, which concur to improve cardiac contractile and

  12. p38 signaling and receptor recycling events in a microfluidic endothelial cell adhesion assay.

    Directory of Open Access Journals (Sweden)

    Dwayne A L Vickers

    Full Text Available Adhesion-based microfluidic cell separation has proven to be very useful in applications ranging from cancer diagnostics to tissue engineering. This process involves functionalizing microchannel surfaces with a capture molecule. High specificity and purity capture can be achieved using this method. Despite these advances, little is known about the mechanisms that govern cell capture within these devices and their relationships to basic process parameters such as fluid shear stress and the presence of soluble factors. This work examines how the adhesion of human endothelial cells (ECs is influenced by a soluble tetrapeptide, Arg-Glu-Asp-Val (REDV and fluidic shear stress. The ability of these ECs to bind within microchannels coated with REDV is shown to be governed by shear- and soluble-factor mediated changes in p38 mitogen-activated protein kinase expression together with recycling of adhesion receptors from the endosome.

  13. A cell-free method for expressing and reconstituting membrane proteins enables functional characterization of the plant receptor-like protein kinase FERONIA.

    Science.gov (United States)

    Minkoff, Benjamin B; Makino, Shin-Ichi; Haruta, Miyoshi; Beebe, Emily T; Wrobel, Russell L; Fox, Brian G; Sussman, Michael R

    2017-04-07

    There are more than 600 receptor-like kinases (RLKs) in Arabidopsis , but due to challenges associated with the characterization of membrane proteins, only a few have known biological functions. The plant RLK FERONIA is a peptide receptor and has been implicated in plant growth regulation, but little is known about its molecular mechanism of action. To investigate the properties of this enzyme, we used a cell-free wheat germ-based expression system in which mRNA encoding FERONIA was co-expressed with mRNA encoding the membrane scaffold protein variant MSP1D1. With the addition of the lipid cardiolipin, assembly of these proteins into nanodiscs was initiated. FERONIA protein kinase activity in nanodiscs was higher than that of soluble protein and comparable with other heterologously expressed protein kinases. Truncation experiments revealed that the cytoplasmic juxtamembrane domain is necessary for maximal FERONIA activity, whereas the transmembrane domain is inhibitory. An ATP analogue that reacts with lysine residues inhibited catalytic activity and labeled four lysines; mutagenesis demonstrated that two of these, Lys-565 and Lys-663, coordinate ATP in the active site. Mass spectrometric phosphoproteomic measurements further identified phosphorylation sites that were examined using phosphomimetic mutagenesis. The results of these experiments are consistent with a model in which kinase-mediated phosphorylation within the C-terminal region is inhibitory and regulates catalytic activity. These data represent a step further toward understanding the molecular basis for the protein kinase catalytic activity of FERONIA and show promise for future characterization of eukaryotic membrane proteins. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

  14. Anhydride-functional silane immobilized onto titanium surfaces induces osteoblast cell differentiation and reduces bacterial adhesion and biofilm formation

    Energy Technology Data Exchange (ETDEWEB)

    Godoy-Gallardo, Maria, E-mail: maria.godoy.gallardo@upc.edu [Biomaterials, Biomechanics and Tissue Engineering Group, Department of Materials Science and Metallurgy, Technical University of Catalonia (UPC), ETSEIB, Av. Diagonal 647, 08028 Barcelona (Spain); Centre for Research in NanoEngineering (CRNE) — UPC, C/ Pascual i Vila 15, 08028 Barcelona (Spain); Guillem-Marti, Jordi, E-mail: jordi.guillem.marti@upc.edu [Biomaterials, Biomechanics and Tissue Engineering Group, Department of Materials Science and Metallurgy, Technical University of Catalonia (UPC), ETSEIB, Av. Diagonal 647, 08028 Barcelona (Spain); Centre for Research in NanoEngineering (CRNE) — UPC, C/ Pascual i Vila 15, 08028 Barcelona (Spain); Sevilla, Pablo, E-mail: psevilla@euss.es [Department of Mechanics, Escola Universitària Salesiana de Sarrià (EUSS), C/ Passeig de Sant Bosco, 42, 08017 Barcelona (Spain); Manero, José M., E-mail: jose.maria.manero@upc.edu [Biomaterials, Biomechanics and Tissue Engineering Group, Department of Materials Science and Metallurgy, Technical University of Catalonia (UPC), ETSEIB, Av. Diagonal 647, 08028 Barcelona (Spain); Centre for Research in NanoEngineering (CRNE) — UPC, C/ Pascual i Vila 15, 08028 Barcelona (Spain); Gil, Francisco J., E-mail: francesc.xavier.gil@upc.edu [Biomaterials, Biomechanics and Tissue Engineering Group, Department of Materials Science and Metallurgy, Technical University of Catalonia (UPC), ETSEIB, Av. Diagonal 647, 08028 Barcelona (Spain); Centre for Research in NanoEngineering (CRNE) — UPC, C/ Pascual i Vila 15, 08028 Barcelona (Spain); and others

    2016-02-01

    Bacterial infection in dental implants along with osseointegration failure usually leads to loss of the device. Bioactive molecules with antibacterial properties can be attached to titanium surfaces with anchoring molecules such as silanes, preventing biofilm formation and improving osseointegration. Properties of silanes as molecular binders have been thoroughly studied, but research on the biological effects of these coatings is scarce. The aim of the present study was to determine the in vitro cell response and antibacterial effects of triethoxysilypropyl succinic anhydride (TESPSA) silane anchored on titanium surfaces. X-ray photoelectron spectroscopy confirmed a successful silanization. The silanized surfaces showed no cytotoxic effects. Gene expression analyses of Sarcoma Osteogenic (SaOS-2) osteoblast-like cells cultured on TESPSA silanized surfaces reported a remarkable increase of biochemical markers related to induction of osteoblastic cell differentiation. A manifest decrease of bacterial adhesion and biofilm formation at early stages was observed on treated substrates, while favoring cell adhesion and spreading in bacteria–cell co-cultures. Surfaces treated with TESPSA could enhance a biological sealing on implant surfaces against bacteria colonization of underlying tissues. Furthermore, it can be an effective anchoring platform of biomolecules on titanium surfaces with improved osteoblastic differentiation and antibacterial properties. - Highlights: • TESPSA silane induces osteoblast differentiation. • TESPSA reduces bacterial adhesion and biofilm formation. • TESPSA is a promising anchoring platform of biomolecules onto titanium.

  15. Anhydride-functional silane immobilized onto titanium surfaces induces osteoblast cell differentiation and reduces bacterial adhesion and biofilm formation

    International Nuclear Information System (INIS)

    Godoy-Gallardo, Maria; Guillem-Marti, Jordi; Sevilla, Pablo; Manero, José M.; Gil, Francisco J.

    2016-01-01

    Bacterial infection in dental implants along with osseointegration failure usually leads to loss of the device. Bioactive molecules with antibacterial properties can be attached to titanium surfaces with anchoring molecules such as silanes, preventing biofilm formation and improving osseointegration. Properties of silanes as molecular binders have been thoroughly studied, but research on the biological effects of these coatings is scarce. The aim of the present study was to determine the in vitro cell response and antibacterial effects of triethoxysilypropyl succinic anhydride (TESPSA) silane anchored on titanium surfaces. X-ray photoelectron spectroscopy confirmed a successful silanization. The silanized surfaces showed no cytotoxic effects. Gene expression analyses of Sarcoma Osteogenic (SaOS-2) osteoblast-like cells cultured on TESPSA silanized surfaces reported a remarkable increase of biochemical markers related to induction of osteoblastic cell differentiation. A manifest decrease of bacterial adhesion and biofilm formation at early stages was observed on treated substrates, while favoring cell adhesion and spreading in bacteria–cell co-cultures. Surfaces treated with TESPSA could enhance a biological sealing on implant surfaces against bacteria colonization of underlying tissues. Furthermore, it can be an effective anchoring platform of biomolecules on titanium surfaces with improved osteoblastic differentiation and antibacterial properties. - Highlights: • TESPSA silane induces osteoblast differentiation. • TESPSA reduces bacterial adhesion and biofilm formation. • TESPSA is a promising anchoring platform of biomolecules onto titanium.

  16. Bacterial Adhesion & Blocking Bacterial Adhesion

    DEFF Research Database (Denmark)

    Vejborg, Rebecca Munk

    2008-01-01

    reduce or delay bacterial biofilm formation of a range of urinary tract infectious E.coli and Klebsiella isolates. Several other proteinaceous coatings were also found to display anti-adhesive properties, possibly providing a measure for controlling the colonization of implant materials. Several other...... components. These substances may both mediate and stabilize the bacterial biofilm. Finally, several adhesive structures were examined, and a novel physiological biofilm phenotype in E.coli biofilms was characterized, namely cell chain formation. The autotransporter protein, antigen 43, was implicated...

  17. The calcium-dependent protein kinase 3 of toxoplasma influences basal calcium levels and functions beyond egress as revealed by quantitative phosphoproteome analysis.

    Directory of Open Access Journals (Sweden)

    Moritz Treeck

    2014-06-01

    Full Text Available Calcium-dependent protein kinases (CDPKs are conserved in plants and apicomplexan parasites. In Toxoplasma gondii, TgCDPK3 regulates parasite egress from the host cell in the presence of a calcium-ionophore. The targets and the pathways that the kinase controls, however, are not known. To identify pathways regulated by TgCDPK3, we measured relative phosphorylation site usage in wild type and TgCDPK3 mutant and knock-out parasites by quantitative mass-spectrometry using stable isotope-labeling with amino acids in cell culture (SILAC. This revealed known and novel phosphorylation events on proteins predicted to play a role in host-cell egress, but also a novel function of TgCDPK3 as an upstream regulator of other calcium-dependent signaling pathways, as we also identified proteins that are differentially phosphorylated prior to egress, including proteins important for ion-homeostasis and metabolism. This observation is supported by the observation that basal calcium levels are increased in parasites where TgCDPK3 has been inactivated. Most of the differential phosphorylation observed in CDPK3 mutants is rescued by complementation of the mutants with a wild type copy of TgCDPK3. Lastly, the TgCDPK3 mutants showed hyperphosphorylation of two targets of a related calcium-dependent kinase (TgCDPK1, as well as TgCDPK1 itself, indicating that this latter kinase appears to play a role downstream of TgCDPK3 function. Overexpression of TgCDPK1 partially rescues the egress phenotype of the TgCDPK3 mutants, reinforcing this conclusion. These results show that TgCDPK3 plays a pivotal role in regulating tachyzoite functions including, but not limited to, egress.

  18. The Calcium-Dependent Protein Kinase 3 of Toxoplasma Influences Basal Calcium Levels and Functions beyond Egress as Revealed by Quantitative Phosphoproteome Analysis

    Science.gov (United States)

    Treeck, Moritz; Sanders, John L.; Gaji, Rajshekhar Y.; LaFavers, Kacie A.; Child, Matthew A.; Arrizabalaga, Gustavo; Elias, Joshua E.; Boothroyd, John C.

    2014-01-01

    Calcium-dependent protein kinases (CDPKs) are conserved in plants and apicomplexan parasites. In Toxoplasma gondii, TgCDPK3 regulates parasite egress from the host cell in the presence of a calcium-ionophore. The targets and the pathways that the kinase controls, however, are not known. To identify pathways regulated by TgCDPK3, we measured relative phosphorylation site usage in wild type and TgCDPK3 mutant and knock-out parasites by quantitative mass-spectrometry using stable isotope-labeling with amino acids in cell culture (SILAC). This revealed known and novel phosphorylation events on proteins predicted to play a role in host-cell egress, but also a novel function of TgCDPK3 as an upstream regulator of other calcium-dependent signaling pathways, as we also identified proteins that are differentially phosphorylated prior to egress, including proteins important for ion-homeostasis and metabolism. This observation is supported by the observation that basal calcium levels are increased in parasites where TgCDPK3 has been inactivated. Most of the differential phosphorylation observed in CDPK3 mutants is rescued by complementation of the mutants with a wild type copy of TgCDPK3. Lastly, the TgCDPK3 mutants showed hyperphosphorylation of two targets of a related calcium-dependent kinase (TgCDPK1), as well as TgCDPK1 itself, indicating that this latter kinase appears to play a role downstream of TgCDPK3 function. Overexpression of TgCDPK1 partially rescues the egress phenotype of the TgCDPK3 mutants, reinforcing this conclusion. These results show that TgCDPK3 plays a pivotal role in regulating tachyzoite functions including, but not limited to, egress. PMID:24945436

  19. FTY720 and two novel butterfly derivatives exert a general anti-inflammatory potential by reducing immune cell adhesion to endothelial cells through activation of S1P(3) and phosphoinositide 3-kinase.

    Science.gov (United States)

    Imeri, Faik; Blanchard, Olivier; Jenni, Aurelio; Schwalm, Stephanie; Wünsche, Christin; Zivkovic, Aleksandra; Stark, Holger; Pfeilschifter, Josef; Huwiler, Andrea

    2015-12-01

    Sphingosine-1-phosphate (S1P) is a key lipid regulator of a variety of cellular responses including cell proliferation and survival, cell migration, and inflammatory reactions. Here, we investigated the effect of S1P receptor activation on immune cell adhesion to endothelial cells under inflammatory conditions. We show that S1P reduces both tumor necrosis factor (TNF)-α- and lipopolysaccharide (LPS)-stimulated adhesion of Jurkat and U937 cells to an endothelial monolayer. The reducing effect of S1P was reversed by the S1P1+3 antagonist VPC23019 but not by the S1P1 antagonist W146. Additionally, knockdown of S1P3, but not S1P1, by short hairpin RNA (shRNA) abolished the reducing effect of S1P, suggesting the involvement of S1P3. A suppression of immune cell adhesion was also seen with the immunomodulatory drug FTY720 and two novel butterfly derivatives ST-968 and ST-1071. On the molecular level, S1P and all FTY720 derivatives reduced the mRNA expression of LPS- and TNF-α-induced adhesion molecules including ICAM-1, VCAM-1, E-selectin, and CD44 which was reversed by the PI3K inhibitor LY294002, but not by the MEK inhibitor U0126.In summary, our data demonstrate a novel molecular mechanism by which S1P, FTY720, and two novel butterfly derivatives acted anti-inflammatory that is by suppressing gene transcription of various endothelial adhesion molecules and thereby preventing adhesion of immune cells to endothelial cells and subsequent extravasation.

  20. Polymeric immunoglobulin receptor-mediated invasion of Streptococcus pneumoniae into host cells requires a coordinate signaling of SRC family of protein-tyrosine kinases, ERK, and c-Jun N-terminal kinase.

    Science.gov (United States)

    Agarwal, Vaibhav; Asmat, Tauseef M; Dierdorf, Nina I; Hauck, Christof R; Hammerschmidt, Sven

    2010-11-12

    Streptococcus pneumoniae are commensals of the human nasopharynx with the capacity to invade mucosal respiratory cells. PspC, a pneumococcal surface protein, interacts with the human polymeric immunoglobulin receptor (pIgR) to promote bacterial adherence to and invasion into epithelial cells. Internalization of pneumococci requires the coordinated action of actin cytoskeleton rearrangements and the retrograde machinery of pIgR. Here, we demonstrate the involvement of Src protein-tyrosine kinases (PTKs), focal adhesion kinase (FAK), extracellular signal-regulated kinase (ERK), and c-Jun N-terminal kinase (JNK) but not p38 mitogen-activated protein kinases (MAPK) in pneumococcal invasion via pIgR. Pharmacological inhibitors of PTKs and MAPKs and genetic interference with Src PTK and FAK functions caused a significant reduction of pIgR-mediated pneumococcal invasion but did not influence bacterial adhesion to host cells. Furthermore, pneumococcal ingestion by host cells induces activation of ERK1/2 and JNK. In agreement with activated JNK, its target molecule and DNA-binding protein c-Jun was phosphorylated. We also show that functionally active Src PTK is essential for activation of ERK1/2 upon pneumococcal infections. In conclusion, these data illustrate the importance of a coordinated signaling between Src PTKs, ERK1/2, and JNK during PspC-pIgR-mediated uptake of pneumococci by host epithelial cells.

  1. Pleiotropic functions of the yeast Greatwall-family protein kinase Rim15p: a novel target for the control of alcoholic fermentation.

    Science.gov (United States)

    Watanabe, Daisuke; Takagi, Hiroshi

    2017-06-01

    Rim15p, a Greatwall-family protein kinase in yeast Saccharomyces cerevisiae, is required for cellular nutrient responses, such as the entry into quiescence and the induction of meiosis and sporulation. In higher eukaryotes, the orthologous gene products are commonly involved in the cell cycle G 2 /M transition. How are these pleiotropic functions generated from a single family of protein kinases? Recent advances in both research fields have identified the conserved Greatwall-mediated signaling pathway and a variety of downstream target molecules. In addition, our studies of S. cerevisiae sake yeast strains revealed that Rim15p also plays a significant role in the control of alcoholic fermentation. Despite an extensive history of research on glycolysis and alcoholic fermentation, there has been no critical clue to artificial modification of fermentation performance of yeast cells. Our finding of an in vivo metabolic regulatory mechanism is expected to provide a major breakthrough in yeast breeding technologies for fermentation applications.

  2. The Syk protein tyrosine kinase can function independently of CD45 or Lck in T cell antigen receptor signaling

    NARCIS (Netherlands)

    Chu, D. H.; Spits, H.; Peyron, J. F.; Rowley, R. B.; Bolen, J. B.; Weiss, A.

    1996-01-01

    The protein tyrosine phosphatase CD45 is a critical component of the T cell antigen receptor (TCR) signaling pathway, acting as a positive regulator of Src family protein tyrosine kinases (PTKs) such as Lck. Most CD45-deficient human and murine T cell lines are unable to signal through their TCRs.

  3. Functional p53 in cells contributes to the anticancer effect of the cyclin-dependent kinase inhibitor roscovitine

    Czech Academy of Sciences Publication Activity Database

    Paprskářová, Martina; Kryštof, Vladimír; Jorda, Radek; Džubák, P.; Hajdúch, M.; Wesierska-Gadek, J.; Strnad, Miroslav

    2009-01-01

    Roč. 107, č. 3 (2009), s. 428-437 ISSN 0730-2312 R&D Projects: GA ČR GA204/08/0511 Institutional research plan: CEZ:AV0Z50380511 Keywords : APOPTOSIS * CYCLIN-DEPENDENT KINASE * OLOMOUCINE II * p53 Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 2.935, year: 2009

  4. Functional Characterization of the Canine Heme-Regulated eIF2α Kinase: Regulation of Protein Synthesis

    Directory of Open Access Journals (Sweden)

    Kimon C. Kanelakis

    2009-01-01

    Full Text Available The heme-regulated inhibitor (HRI negatively regulates protein synthesis by phosphorylating eukaryotic initiation factor-2α (eIF2α thereby inhibiting protein translation. The importance of HRI in regulating hemoglobin synthesis in erythroid cells makes it an attractive molecular target in need of further characterization. In this work, we have cloned and expressed the canine form of the HRI kinase. The canine nucleotide sequence has 86%, 82%, and 81% identity to the human, mouse, and rat HRI, respectively. It was noted that an isoleucine residue in the ATP binding site of human, rat, and mouse HRI is replaced by a valine in the canine kinase. The expression of canine HRI protein by in vitro translation using wheat germ lysate or in Sf9 cells using a baculovirus expression system was increased by the addition of hemin. Following purification, the canine protein was found to be 72 kD and showed kinase activity determined by its ability to phosphorylate a synthetic peptide substrate. Quercetin, a kinase inhibitor known to inhibit mouse and human HRI, inhibits canine HRI in a concentration-dependent manner. Additionally, quercetin is able to increase de novo protein synthesis in canine reticulocytes. We conclude that the canine is a suitable model species for studying the role of HRI in erythropoiesis.

  5. Reduced corporal fibrosis to protect erectile function by inhibiting the Rho-kinase/LIM-kinase/cofilin pathway in the aged transgenic rat harboring human tissue kallikrein 1

    Directory of Open Access Journals (Sweden)

    Kai Cui

    2017-01-01

    Full Text Available Our previous studies have demonstrated that erectile function was preserved in aged transgenic rats (TGR harboring the human tissue kallikrein 1 (hKLK1, while the molecular level of hKLK1 on corporal fibrosis to inhibit age-related erectile dysfunction (ED is poorly understood. Male wild-type Sprague-Dawley rats (WTR and TGR harboring the hKLK1 gene were fed to 4- or 18-month-old and divided into three groups: young WTR (yWTR as the control, aged WTR (aWTR, and aged TGR (aTGR. Erectile function of all rats was assessed by cavernous nerve electrostimulation method. Masson′s trichrome staining was used to evaluate corporal fibrosis in the corpus cavernosum. We found that the erectile function of rats in the aWTR group was significantly lower than that of other two groups. Masson′s trichrome staining revealed that compared with those of the yWTR and aTGR groups, the ratio of smooth muscle cell (SMC/collagen (C was significantly lower in the aWTR group. Immunohistochemistry and Western blotting analysis were performed, and results demonstrated that expression of α-SMA was lower, while expressions of transforming growth factor-β 1 (TGF-β1, RhoA, ROCK1, p-MYPT1, p-LIMK2, and p-cofilin were higher in the aWTR group compared with those in other two groups. However, LIMK2 and cofilin expressions did not differ among three groups. Taken together, these results indicated that the RhoA/ROCK1/LIMK/cofilin pathway may be involved in the corporal fibrosis caused by advanced age, and hKLK1 may reduce this corporal fibrosis by inhibiting the activation of this pathway to ameliorate age-related ED.

  6. The influence of breast milk and infant formulae hydrolysates on bacterial adhesion and Caco-2 cells functioning.

    Science.gov (United States)

    Fiedorowicz, Ewa; Markiewicz, Lidia Hanna; Sidor, Katarzyna; Świątecka, Dominika; Cieślińska, Anna; Matysiewicz, Michał; Piskorz-Ogórek, Krystyna; Sienkiewicz-Szłapka, Edyta; Teodorowicz, Małgorzata; Świątecki, Aleksander; Kostyra, Elżbieta

    2016-11-01

    The aim of the study was to determine the concentration of BCM7 in human milk and infant formulae (IF) before and after eznymatic hydrolysis, and to evaluate the effect of obtained hydrolysates on interleukin-8 (IL-8) secretion and on proliferation of enterocytes in the in vitro model (Caco-2 cells). This study evaluates also the effect of hydrolysates on the adhesion of intestinal microbiota isolated from faeces of both healthy (H) and allergic (A) infants. In the study we investigated breast milk delivered by mothers of healthy ('healthy milk'; HM) and allergic ('allergic milk'; AM) infants. Three infant formulae were investigated: from hydrolysed cow casein (IF1), from hydrolysed cow whey (IF2) and from whole cow milk (IF3). Intestinal bacteria: Bifidobacterium, lactic acid bacteria, Enterobacteriaceae, Clostridium and Enterococcus were isolated from faeces of five healthy and five allergic infants. Mixtures of bacterial isolates and bacteria adhering to Caco-2 cells were characterised qualitatively with PCR-DGGE, and quantitavely with FISH. Concentration of BCM7 in breast milk and infant formulae was 1.6 to 8.9 times higher after enzymatic hydrolysis in comparison to undigested samples. The presence of this peptide resulted in alteration of intestinal epithelial proliferation and increase in secretion of IL-8. The quantitative profile of adherred bacteria applied as a mix of all isolates from healthy infants (H-MIX) was unchanged in the presence of HM hydrolysate and was modulated (increased number of beneficial Bifidobacterium and reduced commensal Enterobacteriaceae) in the presence of all IF hydrolysates. The presence of IF hydrolysates affected the profile of adhering isolates obtained from allergic infants (A-MIX) and reduced the adhesion of Enterobacteriaceae; the IF2 and IF3 hydrolysates decreased also the total number of adhering bacteria (TBN). However, a stimulating effect of AM hydrolysate on A-MIX adhesion (increased TBN) was observed. Copyright

  7. Focal adhesions and assessment of cytotoxicity

    NARCIS (Netherlands)

    van Kooten, TG; Klein, CL; Wagner, M; Kirkpatrick, CJ

    Focal adhesions are highly ordered assemblies of transmembrane receptors, extracellular matrix proteins, and a large number of cytoplasmic proteins, including structural proteins, as well as tyrosine kinases, phosphatases, and their substrates. They are now accepted as a prime component of signal

  8. Diagnostic Implementation of Fast and Selective Integrin-Mediated Adhesion of Cancer Cells on Functionalized Zeolite L Monolayers.

    Science.gov (United States)

    Greco, Arianna; Maggini, Laura; De Cola, Luisa; De Marco, Rossella; Gentilucci, Luca

    2015-09-16

    The rapid and exact identification and quantification of specific biomarkers is a key technology for always achieving more efficient diagnostic methodologies. We present the first application of a nanostructured device constituted of patterned self-assembled monolayers of disk-shaped zeolite L coated with the cyclic integrin ligand c[RGDfK] via isocyanate linker, to the rapid detection of cancer cells. With its high specificity toward HeLa and Glioma cells and fast adhesion ability, this biocompatible monolayer is a promising platform for implementation in diagnostics and personalized therapy formulation devices.

  9. Adhesive plasters

    Science.gov (United States)

    Holcombe, Jr., Cressie E.; Swain, Ronald L.; Banker, John G.; Edwards, Charlene C.

    1978-01-01

    Adhesive plaster compositions are provided by treating particles of Y.sub.2 O.sub.3, Eu.sub.2 O.sub.3, Gd.sub.2 O.sub.3 or Nd.sub.2 O.sub.3 with dilute acid solutions. The resulting compositions have been found to spontaneously harden into rigid reticulated masses resembling plaster of Paris. Upon heating, the hardened material is decomposed into the oxide, yet retains the reticulated rigid structure.

  10. Electrically Conductive Epoxy Adhesives

    Directory of Open Access Journals (Sweden)

    Lan Bai

    2011-02-01

    Full Text Available Conductive adhesives are widely used in electronic packaging applications such as die attachment and solderless interconnections, component repair, display interconnections, and heat dissipation. The effects of film thickness as functions of filler volume fraction, conductive filler size, shape, as well as uncured adhesive matrix viscosity on the electrical conduction behavior of epoxy-based adhesives are presented in this work. For this purpose, epoxy-based adhesives were prepared using conductive fillers of different size, shape, and types, including Ni powder, flakes, and filaments, Ag powder, and Cu powder. The filaments were 20 μm in diameter, and 160 or 260 μm in length. HCl and H3PO4 acid solutions were used to etch and remove the surface oxide layers from the fillers. The plane resistance of filled adhesive films was measured using the four-point method. In all cases of conductive filler addition, the planar resistivity levels for the composite adhesive films increased when the film thickness was reduced. The shape of resistivity-thickness curves was negative exponential decaying type and was modeled using a mathematical relation. The relationships between the conductive film resistivities and the filler volume fractions were also derived mathematically based on the experimental data. Thus, the effects of surface treatment of filler particles, the type, size, shape of fillers, and the uncured epoxy viscosity could be included empirically by using these mathematical relations based on the experimental data. By utilizing the relations we proposed to model thickness-dependent and volume fraction-dependent conduction behaviors separately, we were able to describe the combined and coupled volume fraction-film thickness relationship mathematically based on our experimental data.

  11. Adhesive capsulitis of the shoulder.

    Science.gov (United States)

    Neviaser, Andrew S; Neviaser, Robert J

    2011-09-01

    Adhesive capsulitis is characterized by painful, gradual loss of active and passive shoulder motion resulting from fibrosis and contracture of the joint capsule. Other shoulder pathology can produce a similar clinical picture, however, and must be considered. Management is based on the underlying cause of pain and stiffness, and determination of the etiology is essential. Subtle clues in the history and physical examination can help differentiate adhesive capsulitis from other conditions that cause a stiff, painful shoulder. The natural history of adhesive capsulitis is a matter of controversy. Management of true capsular restriction of motion (ie, true adhesive capsulitis) begins with gentle, progressive stretching exercises. Most patients improve with nonsurgical treatment. Indications for surgery should be individualized. Failure to obtain symptomatic improvement and continued functional disability following ≥6 months of physical therapy is a general guideline for surgical intervention. Diligent postoperative therapy to maintain motion is required to minimize recurrence of adhesive capsulitis.

  12. Solvent-free functionalization of silicone rubber and efficacy of PAAm brushes grafted from an amino-PPX layer against bacterial adhesion.

    Science.gov (United States)

    Fundeanu, Irina; Klee, Doris; Schouten, Arend J; Busscher, Henk J; van der Mei, Henny C

    2010-11-01

    Silicone rubber is a frequently employed biomaterial that is prone to bacterial adhesion and biofilm formation. In this study, the surface of silicone rubber was solvent-free functionalized by chemical vapor deposition (CVD) of poly(o-amino-p-xylylene-co-p-xylylene (amino-PPX). Subsequently, the amino groups of the amino-PPX layer were used to introduce the initiator from a vapor phase for atom transfer radical polymerization of acrylamide to form polyacrylamide (PAAm) brushes. The modification steps were verified by means of X-ray photoelectron spectroscopy and attenuated total reflection-Fourier transform infrared spectroscopy. Adhesion of Staphylococcus aureus ATCC 12600 and Escherichia coli 3.14 to an amino-PPX-PAAm brush coating in a parallel plate flow chamber was strongly reduced with respect to non-coated silicone rubber - by 93% and 99%, respectively. For E. coli 3.14, this reduction is larger than that obtained for solvent functionalization of γ-aminopropyltriethoxysilane-PAAm brushes due to the higher density of amino groups introduced by the CVD of amino-PPX. Copyright © 2010 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

  13. Functional characterization of a StyS sensor kinase reveals distinct domains associated with intracellular and extracellular sensing of styrene in P. putida CA-3

    Science.gov (United States)

    O'Leary, Niall D; Mooney, Aisling; O'Mahony, Mark; Dobson, Alan DW

    2014-01-01

    Bacterial two-component systems (TCSs) are of vital importance in the translation of rapidly changing environmental conditions into appropriate cellular regulatory responses enabling adaptation, growth, and survival. The diverse range of environmental signals that TCSs can process, coupled with discrete modular domains within TCS proteins, offers considerable potential for the rational design of bio-sensor and/or bio-reporter strains. In this study we functionally characterize the multi-domain StyS sensor kinase associated with sensing of the aromatic pollutant styrene by Pseudomonas putida CA-3. Deletion analysis of discrete domains was performed and the ability of the truncated StyS sensor proteins to activate a cognate reporter system in an E. coli host assessed. The essential histidine kinase and PAS input domains were identified for StyS dependent activation of the reporter system. However, co-expression of an ABC-transporter protein StyE, previously linked to styrene transport in P. putida CA-3, enabled activation of the reporter system with a StyS construct containing a non-essential PAS input domain, suggesting a novel role for intracellular detection and/or activation. Site directed mutagenesis and amino acid deletions were employed to further characterize the PAS sensing domains of both input regions. The potential implications of these findings in the use of multi-domain sensor kinases in rational design strategies and the potential link between transport and intracellular sensing are discussed. PMID:24637704

  14. The arabidopsis wall associated kinase-like 10 gene encodes a functional guanylyl cyclase and is co-expressed with pathogen defense related genes

    KAUST Repository

    Meier, Stuart

    2010-01-26

    Background: Second messengers have a key role in linking environmental stimuli to physiological responses. One such messenger, guanosine 3?,5?-cyclic monophosphate (cGMP), has long been known to be an essential signaling molecule in many different physiological processes in higher plants, including biotic stress responses. To date, however, the guanylyl cyclase (GC) enzymes that catalyze the formation of cGMP from GTP have largely remained elusive in higher plants. Principal Findings: We have identified an Arabidopsis receptor type wall associated kinase-like molecule (AtWAKL10) as a candidate GC and provide experimental evidence to show that the intracellular domain of AtWAKL10431-700 can generate cGMP in vitro. Further, we also demonstrate that the molecule has kinase activity indicating that AtWAKL10 is a twin-domain catalytic protein. A co-expression and stimulus-specific expression analysis revealed that AtWAKL10 is consistently coexpressed with well characterized pathogen defense related genes and along with these genes is induced early and sharply in response to a range of pathogens and their elicitors. Conclusions: We demonstrate that AtWAKL10 is a twin-domain, kinase-GC signaling molecule that may function in biotic stress responses that are critically dependent on the second messenger cGMP. © 2010 Meier et al.

  15. TRIM15 is a focal adhesion protein that regulates focal adhesion disassembly

    Science.gov (United States)

    Uchil, Pradeep D.; Pawliczek, Tobias; Reynolds, Tracy D.; Ding, Siyuan; Hinz, Angelika; Munro, James B.; Huang, Fang; Floyd, Robert W.; Yang, Haitao; Hamilton, William L.; Bewersdorf, Joerg; Xiong, Yong; Calderwood, David A.; Mothes, Walther

    2014-01-01

    ABSTRACT Focal adhesions are macromolecular complexes that connect the actin cytoskeleton to the extracellular matrix. Dynamic turnover of focal adhesions is crucial for cell migration. Paxillin is a multi-adaptor protein that plays an important role in regulating focal adhesion dynamics. Here, we identify TRIM15, a member of the tripartite motif protein family, as a paxillin-interacting factor and a component of focal adhesions. TRIM15 localizes to focal contacts in a myosin-II-independent manner by an interaction between its coiled-coil domain and the LD2 motif of paxillin. Unlike other focal adhesion proteins, TRIM15 is a stable focal adhesion component with restricted mobility due to its ability to form oligomers. TRIM15-depleted cells display impaired cell migration and reduced focal adhesion disassembly rates, in addition to enlarged focal adhesions. Thus, our studies demonstrate a cellular function for TRIM15 as a regulatory component of focal adhesion turnover and cell migration. PMID:25015296

  16. Functional Coexpression of HSV-1 Thymidine Kinase and Green Fluorescent Protein: Implications for Noninvasive Imaging of Transgene Expression

    OpenAIRE

    Jacobs, Andreas; Dubrovin, Michael; Hewett, Jeff; Sena-Esteves, Miguel; Tan, Cui-Wen; Slack, Mark; Sadelain, Michele; Breakefield, Xandra O; Tjuvajev, Juri G

    1999-01-01

    Current gene therapy technology is limited by the paucity of methodology for determining the location and magnitude of therapeutic transgene expression in vivo. We describe and validate a paradigm for monitoring therapeutic transgene expression by noninvasive imaging of the herpes simplex virus type 1 thymidine kinase (HSV-1-tk) marker gene expression. To test proportional coexpression of therapeutic and marker genes, a model fusion gene comprising green fluorescent protein (gfp) and HSV-1-tk...

  17. Structural and functional characterization of the Mycobacterium tuberculosis uridine monophosphate kinase: insights into the allosteric regulation †

    OpenAIRE

    Labesse, Gilles; Benkali, Khaled; Salard-Arnaud, Isabelle; Gilles, Anne-Marie; Munier-Lehmann, Hélène

    2010-01-01

    Nucleoside Monophosphate Kinases (NMPKs) family are key enzymes in nucleotide metabolism. Bacterial UMPKs depart from the main superfamily of NMPKs. Having no eukaryotic counterparts they represent attractive therapeutic targets. They are regulated by GTP and UTP, while showing different mechanisms in Gram(+), Gram(-) and archaeal bacteria. In this work, we have characterized the mycobacterial UMPK (UMPKmt) combining enzymatic and structural investigations with site-directed mutagenesis. UMPK...

  18. Deletion of mammalian sterile 20-like kinase 1 attenuates neuronal loss and improves locomotor function in a mouse model of spinal cord trauma.

    Science.gov (United States)

    Wang, Pan-Feng; Xu, Da-Yuan; Zhang, Yuntong; Liu, Xiao-Bin; Xia, Yan; Zhou, Pan-Yu; Fu, Qing-Ge; Xu, Shuo-Gui

    2017-07-01

    Neuronal cell death following spinal cord injury (SCI) is an important contributor to neurological deficits. The purpose of our work was to delineate the function of mammalian sterile 20-like kinase 1 (Mst1), a pro-apoptotic kinase and key mediator of apoptotic signaling, in the pathogenesis of an experimental mouse model of SCI. Male mice received a mid-thoracic spinal contusion injury, and it was found that phosphorylation of Mst1 at the injured site was enhanced significantly following SCI. Furthermore, when compared to the wild-type controls, Mst1-deficient mice displayed improved locomotor function by increased Basso mouse scale score. Deletion of Mst1 in mice attenuated loss of motor neurons and suppressed microglial and glial activation following SCI. Deletion of Mst1 in mice reduced apoptosis via suppressing cytochrome c release and caspase-3 activation following SCI. Deletion of Mst1 attenuated mitochondrial dysfunction and increased ATP formation following SCI. Deletion of Mst1 in mice inhibited local inflammation following SCI, evidenced by reduced activities of myeloperoxidase and protein levels of TNF-α, IL-1β, and IL-6. In conclusion, the present study demonstrated that deletion of Mst1 attenuated neuronal loss and improved locomotor function in a mouse model of SCI, via preserving mitochondrial function, attenuating mitochondria-mediated apoptotic pathway, and suppressing inflammation, at least in part.

  19. Constraint-induced movement therapy promotes motor function recovery and downregulates phosphorylated extracellular regulated protein kinase expression in ischemic brain tissue of rats

    Directory of Open Access Journals (Sweden)

    Bei Zhang

    2015-01-01

    Full Text Available Motor function impairment is a common outcome of stroke. Constraint-induced movement therapy (CIMT involving intensive use of the impaired limb while restraining the unaffected limb is widely used to overcome the effects of ′learned non-use′ and improve limb function after stroke. However, the underlying mechanism of CIMT remains unclear. In the present study, rats were randomly divided into a middle cerebral artery occlusion (model group, a CIMT + model (CIMT group, or a sham group. Restriction of the affected limb by plaster cast was performed in the CIMT and sham groups. Compared with the model group, CIMT significantly improved the forelimb functional performance in rats. By western blot assay, the expression of phosphorylated extracellular regulated protein kinase in the bilateral cortex and hippocampi of cerebral ischemic rats in the CIMT group was significantly lower than that in the model group, and was similar to sham group levels. These data suggest that functional recovery after CIMT may be related to decreased expression of phosphorylated extracellular regulated protein kinase in the bilateral cortex and hippocampi.

  20. Modes of Action and Functions of ERECTA-family Receptor-like Kinases in Plant Organ Growth and Development

    Energy Technology Data Exchange (ETDEWEB)

    TORII, Keiko U.

    2012-05-01

    Higher plants constitute the central resource for renewable lignocellulose biomass that can supplement for the world's depleting stores of fossil fuels. As such, understanding the molecular and genetic mechanisms of plant organ growth will provide key knowledge and genetic resources that enables manipulation of plant biomass feedstock for better growth and productivity. The goal of this proposal is to understand how cell proliferation and growth are coordinated during aboveground organ morphogenesis, and how cell-cell signaling mediated by a family of receptor kinases coordinates plant organogenesis. The well-established model plant Arabidopsis thaliana is used for our research to facilitate rapid progress. Specifically, we focus on how ERECTA-family leucine-rich repeat receptor kinases (LRR-RLKs) interact in a synergistic manner to promote organogenesis and pattern formation in Arabidopsis. This project was highly successful, resulted in fourteen publications including nine peer-reviewed original research articles. One provisional US patent has been filed through this DOE funding. We have addressed the critical roles for a family of receptor kinases in coordinating proliferation and differentiation of plants, and we successfully elucidated the downstream targets of this signaling pathway in specifying stomatal patterning.

  1. Adhesion rings surround invadopodia and promote maturation

    Directory of Open Access Journals (Sweden)

    Kevin M. Branch

    2012-06-01

    Invasion and metastasis are aggressive cancer phenotypes that are highly related to the ability of cancer cells to degrade extracellular matrix (ECM. At the cellular level, specialized actin-rich structures called invadopodia mediate focal matrix degradation by serving as exocytic sites for ECM-degrading proteinases. Adhesion signaling is likely to be a critical regulatory input to invadopodia, but the mechanism and location of such adhesion signaling events are poorly understood. Here, we report that adhesion rings surround invadopodia shortly after formation and correlate strongly with invadopodium activity on a cell-by-cell basis. By contrast, there was little correlation of focal adhesion number or size with cellular invadopodium activity. Prevention of adhesion ring formation by inhibition of RGD-binding integrins or knockdown (KD of integrin-linked kinase (ILK reduced the number of ECM-degrading invadopodia and reduced recruitment of IQGAP to invadopodium actin puncta. Furthermore, live cell imaging revealed that the rate of extracellular MT1-MMP accumulation at invadopodia was greatly reduced in both integrin-inhibited and ILK-KD cells. Conversely, KD of MT1-MMP reduced invadopodium activity and dynamics but not the number of adhesion-ringed invadopodia. These results suggest a model in which adhesion rings are recruited to invadopodia shortly after formation and promote invadopodium maturation by enhancing proteinase secretion. Since adhesion rings are a defining characteristic of podosomes, similar structures formed by normal cells, our data also suggest further similarities between invadopodia and podosomes.

  2. Quality of life and functional capacity of patients with adhesive capsulitis: identifying risk factors associated to better outcomes after treatment with nerve blocking

    Directory of Open Access Journals (Sweden)

    Marcos Rassi Fernandes

    Full Text Available Abstract Introduction: The objectives of this study were to assess the quality of life and functional capacity of adhesive capsulitis patients at the beginning and end of procedure and to identify risk factors associated to better outcomes after treatment with nerve blocking. Methods: A prospective cohort study was performed. Inclusion criteria were clinical signs of adhesive capsulitis and disease changes on shoulder imaging exams. The short form of World Health Organization Quality of life and Disabilities of the Arm, Shoulder and Hand questionnaires were administered at the beginning and end of treatment. A score of 55 points or more on the Constant index was used for discontinuation of treatment. We used the Wilcoxon test for paired samples. Multiple regression analysis of Poisson was carried out using exposure variables with p < 0.20 in the univariate analysis and the satisfactory quality of life and better functional capability as outcomes. The significance level was 5%. Results: 43 patients were evaluated. For the comparison between medians values at the beginning and end of treatment (physical domain: 46.43-67.86; psychologic domain: 66.67-79.17; social domain: 66.67-75; environment domain: 62.5-68.75; DASH: 64.16-38.33, p was <0.05. Aging (physical/psychologic/DASH, higher educational level (physical/environment/DASH, less severity (only physical and fewer nerve blocking (only psychologic were these independent risk factors. Conclusions: Quality of life and functional capacity of the patients improve at the end of procedure. Older patients and higher education levels are the risk factors most associated to satisfactory quality of life and better functional capacity after treatment with nerve blocking.

  3. Quality of life and functional capacity of patients with adhesive capsulitis: identifying risk factors associated to better outcomes after treatment with nerve blocking.

    Science.gov (United States)

    Fernandes, Marcos Rassi; Barbosa, Maria Alves; Faria, Ruth Minamisawa

    The objectives of this study were to assess the quality of life and functional capacity of adhesive capsulitis patients at the beginning and end of procedure and to identify risk factors associated to better outcomes after treatment with nerve blocking. A prospective cohort study was performed. Inclusion criteria were clinical signs of adhesive capsulitis and disease changes on shoulder imaging exams. The short form of World Health Organization Quality of life and Disabilities of the Arm, Shoulder and Hand questionnaires were administered at the beginning and end of treatment. A score of 55 points or more on the Constant index was used for discontinuation of treatment. We used the Wilcoxon test for paired samples. Multiple regression analysis of Poisson was carried out using exposure variables with p<0.20 in the univariate analysis and the satisfactory quality of life and better functional capability as outcomes. The significance level was 5%. 43 patients were evaluated. For the comparison between medians values at the beginning and end of treatment (physical domain: 46.43-67.86; psychologic domain: 66.67-79.17; social domain: 66.67-75; environment domain: 62.5-68.75; DASH: 64.16-38.33), p was <0.05. Aging (physical/psychologic/DASH), higher educational level (physical/environment/DASH), less severity (only physical) and fewer nerve blocking (only psychologic) were these independent risk factors. Quality of life and functional capacity of the patients improve at the end of procedure. Older patients and higher education levels are the risk factors most associated to satisfactory quality of life and better functional capacity after treatment with nerve blocking. Copyright © 2017. Published by Elsevier Editora Ltda.

  4. Adhesion and Cohesion

    Directory of Open Access Journals (Sweden)

    J. Anthony von Fraunhofer

    2012-01-01

    Full Text Available The phenomena of adhesion and cohesion are reviewed and discussed with particular reference to dentistry. This review considers the forces involved in cohesion and adhesion together with the mechanisms of adhesion and the underlying molecular processes involved in bonding of dissimilar materials. The forces involved in surface tension, surface wetting, chemical adhesion, dispersive adhesion, diffusive adhesion, and mechanical adhesion are reviewed in detail and examples relevant to adhesive dentistry and bonding are given. Substrate surface chemistry and its influence on adhesion, together with the properties of adhesive materials, are evaluated. The underlying mechanisms involved in adhesion failure are covered. The relevance of the adhesion zone and its importance with regard to adhesive dentistry and bonding to enamel and dentin is discussed.

  5. Syndecans and cell adhesion

    DEFF Research Database (Denmark)

    Couchman, J R; Chen, L; Woods, A

    2001-01-01

    Now that transmembrane signaling through primary cell-matrix receptors, integrins, is being elucidated, attention is turning to how integrin-ligand interactions can be modulated. Syndecans are transmembrane proteoglycans implicated as coreceptors in a variety of physiological processes, including...... cell adhesion, migration, response to growth factors, development, and tumorigenesis. This review will describe this family of proteoglycans in terms of their structures and functions and their signaling in conjunction with integrins, and indicate areas for future research....

  6. Kinases and Cancer.

    Science.gov (United States)

    Cicenas, Jonas; Zalyte, Egle; Bairoch, Amos; Gaudet, Pascale

    2018-03-01

    Protein kinases are a large family of enzymes catalyzing protein phosphorylation. The human genome contains 518 protein kinase genes, 478 of which belong to the classical protein kinase family and 40 are atypical protein kinases [...].

  7. Kinases and Cancer

    OpenAIRE

    Jonas Cicenas; Egle Zalyte; Amos Bairoch; Pascale Gaudet

    2018-01-01

    Protein kinases are a large family of enzymes catalyzing protein phosphorylation. The human genome contains 518 protein kinase genes, 478 of which belong to the classical protein kinase family and 40 are atypical protein kinases [...

  8. Proteomic dataset of the sea urchin Paracentrotus lividus adhesive organs and secreted adhesive

    Directory of Open Access Journals (Sweden)

    Nicolas Lebesgue

    2016-06-01

    Full Text Available Sea urchins have specialized adhesive organs called tube feet, which mediate strong but reversible adhesion. Tube feet are composed by a disc, producing adhesive and de-adhesive secretions for substratum attachment, and a stem for movement. After detachment the secreted adhesive remains bound to the substratum as a footprint. Recently, a label-free quantitative proteomic approach coupled with the latest mass-spectrometry technology was used to analyze the differential proteome of Paracentrotus lividus adhesive organ, comparing protein expression levels in the tube feet adhesive part (the disc versus the non-adhesive part (the stem, and also to profile the proteome of the secreted adhesive (glue. This data article contains complementary figures and results related to the research article “Deciphering the molecular mechanisms underlying sea urchin reversible adhesion: a quantitative proteomics approach” (Lebesgue et al., 2016 [1]. Here we provide a dataset of 1384 non-redundant proteins, their fragmented peptides and expression levels, resultant from the analysis of the tube feet differential proteome. Of these, 163 highly over-expressed tube feet disc proteins (>3-fold, likely representing the most relevant proteins for sea urchin reversible adhesion, were further annotated in order to determine the potential functions. In addition, we provide a dataset of 611 non-redundant proteins identified in the secreted adhesive proteome, as well as their functional annotation and grouping in 5 major protein groups related with adhesive exocytosis, and microbial protection. This list was further analyzed to identify the most abundant protein groups and pinpoint putative adhesive proteins, such as Nectin, the most abundant adhesive protein in sea urchin glue. The obtained data uncover the key proteins involved in sea urchins reversible adhesion, representing a step forward to the development of new wet-effective bio-inspired adhesives.

  9. Proteomic dataset of the sea urchin Paracentrotus lividus adhesive organs and secreted adhesive.

    Science.gov (United States)

    Lebesgue, Nicolas; da Costa, Gonçalo; Ribeiro, Raquel Mesquita; Ribeiro-Silva, Cristina; Martins, Gabriel G; Matranga, Valeria; Scholten, Arjen; Cordeiro, Carlos; Heck, Albert J R; Santos, Romana

    2016-06-01

    Sea urchins have specialized adhesive organs called tube feet, which mediate strong but reversible adhesion. Tube feet are composed by a disc, producing adhesive and de-adhesive secretions for substratum attachment, and a stem for movement. After detachment the secreted adhesive remains bound to the substratum as a footprint. Recently, a label-free quantitative proteomic approach coupled with the latest mass-spectrometry technology was used to analyze the differential proteome of Paracentrotus lividus adhesive organ, comparing protein expression levels in the tube feet adhesive part (the disc) versus the non-adhesive part (the stem), and also to profile the proteome of the secreted adhesive (glue). This data article contains complementary figures and results related to the research article "Deciphering the molecular mechanisms underlying sea urchin reversible adhesion: a quantitative proteomics approach" (Lebesgue et al., 2016) [1]. Here we provide a dataset of 1384 non-redundant proteins, their fragmented peptides and expression levels, resultant from the analysis of the tube feet differential proteome. Of these, 163 highly over-expressed tube feet disc proteins (>3-fold), likely representing the most relevant proteins for sea urchin reversible adhesion, were further annotated in order to determine the potential functions. In addition, we provide a dataset of 611 non-redundant proteins identified in the secreted adhesive proteome, as well as their functional annotation and grouping in 5 major protein groups related with adhesive exocytosis, and microbial protection. This list was further analyzed to identify the most abundant protein groups and pinpoint putative adhesive proteins, such as Nectin, the most abundant adhesive protein in sea urchin glue. The obtained data uncover the key proteins involved in sea urchins reversible adhesion, representing a step forward to the development of new wet-effective bio-inspired adhesives.

  10. Marine Bioinspired Underwater Contact Adhesion.

    Science.gov (United States)

    Clancy, Sean K; Sodano, Antonio; Cunningham, Dylan J; Huang, Sharon S; Zalicki, Piotr J; Shin, Seunghan; Ahn, B Kollbe

    2016-05-09

    Marine mussels and barnacles are sessile biofouling organisms that adhere to a number of surfaces in wet environments and maintain remarkably strong bonds. Previous synthetic approaches to mimic biological wet adhesive properties have focused mainly on the catechol moiety, present in mussel foot proteins (mfps), and especially rich in the interfacial mfps, for example, mfp-3 and -5, found at the interface between the mussel plaque and substrate. Barnacles, however, do not use Dopa for their wet adhesion, but are instead rich in noncatecholic aromatic residues. Due to this anomaly, we were intrigued to study the initial contact adhesion properties of copolymerized acrylate films containing the key functionalities of barnacle cement proteins and interfacial mfps, for example, aromatic (catecholic or noncatecholic), cationic, anionic, and nonpolar residues. The initial wet contact adhesion of the copolymers was measured using a probe tack testing apparatus with a flat-punch contact geometry. The wet contact adhesion of an optimized, bioinspired copolymer film was ∼15.0 N/cm(2) in deionized water and ∼9.0 N/cm(2) in artificial seawater, up to 150 times greater than commercial pressure-sensitive adhesive (PSA) tapes (∼0.1 N/cm(2)). Furthermore, maximum wet contact adhesion was obtained at ∼pH 7, suggesting viability for biomedical applications.

  11. A Novel Role for Keratin 17 in Coordinating Oncogenic Transformation and Cellular Adhesion in Ewing Sarcoma

    Science.gov (United States)

    Sankar, Savita; Tanner, Jason M.; Bell, Russell; Chaturvedi, Aashi; Randall, R. Lor; Beckerle, Mary C.

    2013-01-01

    Oncogenic transformation in Ewing sarcoma is caused by EWS/FLI, an aberrant transcription factor fusion oncogene. Glioma-associated oncogene homolog 1 (GLI1) is a critical target gene activated by EWS/FLI, but the mechanism by which GLI1 contributes to the transformed phenotype of Ewing sarcoma was unknown. In this work, we identify keratin 17 (KRT17) as a direct downstream target gene upregulated by GLI1. We demonstrate that KRT17 regulates cellular adhesion by activating AKT/PKB (protein kinase B) signaling. In addition, KRT17 is necessary for oncogenic transformation in Ewing sarcoma and accounts for much of the GLI1-mediated transformation function but via a mechanism independent of AKT signaling. Taken together, our data reveal previously unknown molecular functions for a cytoplasmic intermediate filament protein, KRT17, in coordinating EWS/FLI- and GLI1-mediated oncogenic transformation and cellular adhesion in Ewing sarcoma. PMID:24043308

  12. Regulation of the Tumor-Suppressor Function of the Class III Phosphatidylinositol 3-Kinase Complex by Ubiquitin and SUMO

    International Nuclear Information System (INIS)

    Reidick, Christina; El Magraoui, Fouzi; Meyer, Helmut E.; Stenmark, Harald; Platta, Harald W.

    2014-01-01

    The occurrence of cancer is often associated with a dysfunction in one of the three central membrane-involution processes—autophagy, endocytosis or cytokinesis. Interestingly, all three pathways are controlled by the same central signaling module: the class III phosphatidylinositol 3-kinase (PI3K-III) complex and its catalytic product, the phosphorylated lipid phosphatidylinositol 3-phosphate (PtdIns3P). The activity of the catalytic subunit of the PI3K-III complex, the lipid-kinase VPS34, requires the presence of the membrane-targeting factor VPS15 as well as the adaptor protein Beclin 1. Furthermore, a growing list of regulatory proteins associates with VPS34 via Beclin 1. These accessory factors define distinct subunit compositions and thereby guide the PI3K-III complex to its different cellular and physiological roles. Here we discuss the regulation of the PI3K-III complex components by ubiquitination and SUMOylation. Especially Beclin 1 has emerged as a highly regulated protein, which can be modified with Lys11-, Lys48- or Lys63-linked polyubiquitin chains catalyzed by distinct E3 ligases from the RING-, HECT-, RBR- or Cullin-type. We also point out other cross-links of these ligases with autophagy in order to discuss how these data might be merged into a general concept

  13. Regulation of the Tumor-Suppressor Function of the Class III Phosphatidylinositol 3-Kinase Complex by Ubiquitin and SUMO

    Energy Technology Data Exchange (ETDEWEB)

    Reidick, Christina [Biochemie Intrazellulärer Transportprozesse, Ruhr-Universität Bochum, Bochum 44801 (Germany); El Magraoui, Fouzi; Meyer, Helmut E. [Biomedical Research, Human Brain Proteomics II, Leibniz-Institut für Analytische Wissenschaften-ISAS, Dortmund 44139 (Germany); Stenmark, Harald [Department of Biochemistry, Institute for Cancer Research, Oslo University Hospital, Montebello, Oslo 0310 (Norway); Platta, Harald W., E-mail: harald.platta@rub.de [Biochemie Intrazellulärer Transportprozesse, Ruhr-Universität Bochum, Bochum 44801 (Germany)

    2014-12-23

    The occurrence of cancer is often associated with a dysfunction in one of the three central membrane-involution processes—autophagy, endocytosis or cytokinesis. Interestingly, all three pathways are controlled by the same central signaling module: the class III phosphatidylinositol 3-kinase (PI3K-III) complex and its catalytic product, the phosphorylated lipid phosphatidylinositol 3-phosphate (PtdIns3P). The activity of the catalytic subunit of the PI3K-III complex, the lipid-kinase VPS34, requires the presence of the membrane-targeting factor VPS15 as well as the adaptor protein Beclin 1. Furthermore, a growing list of regulatory proteins associates with VPS34 via Beclin 1. These accessory factors define distinct subunit compositions and thereby guide the PI3K-III complex to its different cellular and physiological roles. Here we discuss the regulation of the PI3K-III complex components by ubiquitination and SUMOylation. Especially Beclin 1 has emerged as a highly regulated protein, which can be modified with Lys11-, Lys48- or Lys63-linked polyubiquitin chains catalyzed by distinct E3 ligases from the RING-, HECT-, RBR- or Cullin-type. We also point out other cross-links of these ligases with autophagy in order to discuss how these data might be merged into a general concept.

  14. AMP-activated protein kinase regulates lymphocyte responses to metabolic stress but is largely dispensable for immune cell development and function.

    Science.gov (United States)

    Mayer, Alice; Denanglaire, Sébastien; Viollet, Benoit; Leo, Oberdan; Andris, Fabienne

    2008-04-01

    AMP-activated protein kinase (AMPK), a phylogenetically conserved serine/threonine protein kinase, represents an energy sensor able to adapt cellular metabolism in response to nutritional environmental variations. TCR stimulation activates AMPK, a regulatory event that is known to stimulate ATP-producing processes, possibly in anticipation of the increased energetic needs associated with cell division and expression of effector function. Taking advantage of the selective expression of the AMPKalpha1 catalytic subunit in lymphoid cells, we have analyzed the in vitro and in vivo capacity of lymphocytes lacking AMPK activity (AMPKalpha1-KO cells) to respond to metabolic stress and to initiate and sustain an immune response. AMPKalpha1-KO cells displayed increasing sensitivity to energetic stress in vitro, and were found unable to maintain adequate ATP levels in response to ATP synthase inhibition. These cells were, however, able to respond to antigen stimulation in vitro, as shown by optimal proliferation and cytokine production. Similarly, AMPKalpha1-KO mice were fully immunocompetent in vivo and displayed normal cell proliferation, humoral, cytotoxic and delayed-type hypersensitivity (DTH) responses following antigen injection. In conclusion, AMPK represents an important enzyme allowing lymphocytes to resist a mild energy crisis in vitro, but is largely dispensable for activation and expression of effector function in response to antigen stimulation.

  15. Central functions of bicarbonate in S-type anion channel activation and OST1 protein kinase in CO 2 signal transduction in guard cell

    KAUST Repository

    Xue, Shaowu

    2011-03-18

    Plants respond to elevated CO(2) via carbonic anhydrases that mediate stomatal closing, but little is known about the early signalling mechanisms following the initial CO(2) response. It remains unclear whether CO(2), HCO(3)(-) or a combination activates downstream signalling. Here, we demonstrate that bicarbonate functions as a small-molecule activator of SLAC1 anion channels in guard cells. Elevated intracellular [HCO(3)(-)](i) with low [CO(2)] and [H(+)] activated S-type anion currents, whereas low [HCO(3)(-)](i) at high [CO(2)] and [H(+)] did not. Bicarbonate enhanced the intracellular Ca(2+) sensitivity of S-type anion channel activation in wild-type and ht1-2 kinase mutant guard cells. ht1-2 mutant guard cells exhibited enhanced bicarbonate sensitivity of S-type anion channel activation. The OST1 protein kinase has been reported not to affect CO(2) signalling. Unexpectedly, OST1 loss-of-function alleles showed strongly impaired CO(2)-induced stomatal closing and HCO(3)(-) activation of anion channels. Moreover, PYR/RCAR abscisic acid (ABA) receptor mutants slowed but did not abolish CO(2)/HCO(3)(-) signalling, redefining the convergence point of CO(2) and ABA signalling. A new working model of the sequence of CO(2) signalling events in gas exchange regulation is presented.

  16. Furosin, an ellagitannin, suppresses RANKL-induced osteoclast differentiation and function through inhibition of MAP kinase activation and actin ring formation

    International Nuclear Information System (INIS)

    Park, Eui Kyun; Kim, Myung Sunny; Lee, Seung Ho; Kim, Kyung Hee; Park, Ju-Young; Kim, Tae-Ho; Lee, In-Seon; Woo, Je-Tae; Jung, Jae-Chang; Shin, Hong-In; Choi, Je-Yong; Kim, Shin-Yoon

    2004-01-01

    Phenolic compounds including tannins and flavonoids have been implicated in suppression of osteoclast differentiation/function and prevention of bone diseases. However, the effects of hydrolysable tannins on bone metabolism remain to be elucidated. In this study, we found that furosin, a hydrolysable tannin, markedly decreased the differentiation of both murine bone marrow mononuclear cells and Raw264.7 cells into osteoclasts, as revealed by the reduced number of tartrate resistant acid phosphatase (TRAP)-positive multinucleated cells and decreased TRAP activity. Furosin appears to target at the early stage of osteoclastic differentiation while having no cytotoxic effect on osteoclast precursors. Analysis of the inhibitory mechanisms of furosin revealed that it inhibited the receptor activator of nuclear factor-κB ligand (RANKL)-induced activation of p38 mitogen-activated protein kinase (p38MAPK) and c-Jun N-terminal kinase (JNK)/activating protein-1 (AP-1). Furthermore, furosin reduced resorption pit formation in osteoclasts, which was accompanied by disruption of the actin rings. Taken together, these results demonstrate that naturally occurring furosin has an inhibitory activity on both osteoclast differentiation and function through mechanisms involving inhibition of the RANKL-induced p38MAPK and JNK/AP-1 activation as well as actin ring formation

  17. Platelet adhesion onto artificial red blood cells.

    Science.gov (United States)

    Muramatsu, N; Kondo, T

    1980-05-01

    Several kinds of polyamide microcapsules containing mammalian hemolysate were prepared by making use of the interfacial polycondensation reaction between diamines and terephthaloyl dichloride and their blood compatibility in terms of platelet adhesion was examined aiming at their ultimate clinical use as artificial red blood cells. It was found that rabbit platelets adhere onto the hemolysate-loaded microcapsules in the presence of the plasma, while no platelet adhesion takes place in the absence of the plasma. This was interpreted as indicating an important role of plasma components in platelet adhesion. Moreover, platelet adhesion was observed to be facilitated by negative charges on the surface of the hemolysate-loaded microcapsules; the more negatively the surface was charge, the more easily the platelets adhered onto the surface. Finally, the present method of assessing platelet adhesion suggested the possibility of its use for kinetic study of platelet adhesion since it allowedus to make numerical evaluation of platelet adhesion as a function of time.

  18. TRPM4 Is a Novel Component of the Adhesome Required for Focal Adhesion Disassembly, Migration and Contractility.

    Directory of Open Access Journals (Sweden)

    Mónica Cáceres

    Full Text Available Cellular migration and contractility are fundamental processes that are regulated by a variety of concerted mechanisms such as cytoskeleton rearrangements, focal adhesion turnover, and Ca2+ oscillations. TRPM4 is a Ca2+-activated non-selective cationic channel (Ca2+-NSCC that conducts monovalent but not divalent cations. Here, we used a mass spectrometry-based proteomics approach to identify putative TRPM4-associated proteins. Interestingly, the largest group of these proteins has actin cytoskeleton-related functions, and among these nine are specifically annotated as focal adhesion-related proteins. Consistent with these results, we found that TRPM4 localizes to focal adhesions in cells from different cellular lineages. We show that suppression of TRPM4 in MEFs impacts turnover of focal adhesions, serum-induced Ca2+ influx, focal adhesion kinase (FAK and Rac activities, and results in reduced cellular spreading, migration and contractile behavior. Finally, we demonstrate that the inhibition of TRPM4 activity alters cellular contractility in vivo, affecting cutaneous wound healing. Together, these findings provide the first evidence, to our knowledge, for a TRP channel specifically localized to focal adhesions, where it performs a central role in modulating cellular migration and contractility.

  19. TRPM4 Is a Novel Component of the Adhesome Required for Focal Adhesion Disassembly, Migration and Contractility

    Science.gov (United States)

    Cáceres, Mónica; Ortiz, Liliana; Recabarren, Tatiana; Romero, Anibal; Colombo, Alicia; Leiva-Salcedo, Elías; Varela, Diego; Rivas, José; Silva, Ian; Morales, Diego; Campusano, Camilo; Almarza, Oscar; Simon, Felipe; Toledo, Hector; Park, Kang-Sik; Trimmer, James S.; Cerda, Oscar

    2015-01-01

    Cellular migration and contractility are fundamental processes that are regulated by a variety of concerted mechanisms such as cytoskeleton rearrangements, focal adhesion turnover, and Ca2+ oscillations. TRPM4 is a Ca2+-activated non-selective cationic channel (Ca2+-NSCC) that conducts monovalent but not divalent cations. Here, we used a mass spectrometry-based proteomics approach to identify putative TRPM4-associated proteins. Interestingly, the largest group of these proteins has actin cytoskeleton-related functions, and among these nine are specifically annotated as focal adhesion-related proteins. Consistent with these results, we found that TRPM4 localizes to focal adhesions in cells from different cellular lineages. We show that suppression of TRPM4 in MEFs impacts turnover of focal adhesions, serum-induced Ca2+ influx, focal adhesion kinase (FAK) and Rac activities, and results in reduced cellular spreading, migration and contractile behavior. Finally, we demonstrate that the inhibition of TRPM4 activity alters cellular contractility in vivo, affecting cutaneous wound healing. Together, these findings provide the first evidence, to our knowledge, for a TRP channel specifically localized to focal adhesions, where it performs a central role in modulating cellular migration and contractility. PMID:26110647

  20. Management of adhesive capsulitis

    Directory of Open Access Journals (Sweden)

    Stupay KL

    2015-08-01

    Full Text Available Kristen L Stupay,1 Andrew S Neviaser2 1Tulane University School of Medicine, New Orleans, LA, USA; 2George Washington University Medical Faculty Associates, Washington, DC, USA Abstract: Adhesive capsulitis of the shoulder is a condition of capsular contracture that reduces both active and passive glenohumeral motion. The cause of adhesive capsulitis is not known but it is strongly associated with endocrine abnormalities such as diabetes. Diverse terminology and the absence of definitive criteria for diagnosis make evaluating treatment modalities difficult. Many treatment methods have been reported, most with some success, but few have been proved to alter the natural course of this disease. Most afflicted patients will achieve acceptable shoulder function without surgery. Those who remain debilitated after 8–12 months are reasonable candidates for invasive treatments. Here, the various treatment methods and the data to support their use are reviewed. Keywords: frozen shoulder, stiff shoulder, periarthritis, painful shoulder 

  1. Both mitogen activated protein kinase and the mammalian target of rapamycin modulate the development of functional renal proximal tubules in matrigel.

    Science.gov (United States)

    Han, Ho Jae; Sigurdson, Wade J; Nickerson, Peter A; Taub, Mary

    2004-04-01

    Tubules may arise during branching morphogenesis through several mechanisms including wrapping, budding, cavitation and cord hollowing. In this report we present evidence that is consistent with renal proximal tubule formation through a process of cord hollowing (a process that requires the concomitant establishment of apicobasal polarity and lumen formation). Pockets of lumen filled with Lucifer Yellow were observed within developing cords of rabbit renal proximal tubule cells in matrigel. The observation of Lucifer Yellow accumulation suggests functional polarization. In the renal proximal tubule Lucifer Yellow is initially transported intracellularly by means of a basolaterally oriented p-aminohippurate transport system, followed by apical secretion into the lumen of the nephron. Consistent with such polarization in developing tubules, Triticum vulgare was observed to bind to the lumenal membranes within pockets of Lucifer Yellow-filled lumens. As this lectin binds apically in the rabbit renal proximal tubule, T. vulgare binding is indicative of the emergence of an apical domain before the formation of a contiguous lumen. Both epidermal growth factor and hepatocyte growth factor stimulated the formation of transporting tubules. The stimulatory effect of both epidermal growth factor and hepatocyte growth factor on tubulogenesis was inhibited by PD98059, a mitogen activated protein kinase kinase inhibitor, rather than by wortmannin, an inhibitor of phosphoinositide 3-kinase. Nevertheless, Lucifer Yellow-filled lumens were observed in tubules that formed in the presence of PD98059 as well as with wortmannin, indicating that these drugs did not prevent the process of cavitation. By contrast, rapamycin, an inhibitor of the mammalian target of rapamycin, prevented the process of cavitation without affecting the frequency of formation of developing cords. Multicellular cysts were observed to form in 8-bromocyclic AMP-treated cultures. As these cysts did not similarly

  2. Arabidopsis Lectin Receptor Kinases LecRK-IX.1 and LecRK-IX.2 Are Functional Analogs in Regulating Phytophthora Resistance and Plant Cell Death.

    Science.gov (United States)

    Wang, Yan; Cordewener, Jan H G; America, Antoine H P; Shan, Weixing; Bouwmeester, Klaas; Govers, Francine

    2015-09-01

    L-type lectin receptor kinases (LecRK) are potential immune receptors. Here, we characterized two closely-related Arabidopsis LecRK, LecRK-IX.1 and LecRK-IX.2, of which T-DNA insertion mutants showed compromised resistance to Phytophthora brassicae and Phytophthora capsici, with double mutants showing additive susceptibility. Overexpression of LecRK-IX.1 or LecRK-IX.2 in Arabidopsis and transient expression in Nicotiana benthamiana increased Phytophthora resistance but also induced cell death. Phytophthora resistance required both the lectin domain and kinase activity, but for cell death, the lectin domain was not needed. Silencing of the two closely related mitogen-activated protein kinase genes NbSIPK and NbNTF4 in N. benthamiana completely abolished LecRK-IX.1-induced cell death but not Phytophthora resistance. Liquid chromatography-mass spectrometry analysis of protein complexes coimmunoprecipitated in planta with LecRK-IX.1 or LecRK-IX.2 as bait, resulted in the identification of the N. benthamiana ABC transporter NbPDR1 as a potential interactor of both LecRK. The closest homolog of NbPDR1 in Arabidopsis is ABCG40, and coimmunoprecipitation experiments showed that ABCG40 associates with LecRK-IX.1 and LecRK-IX.2 in planta. Similar to the LecRK mutants, ABCG40 mutants showed compromised Phytophthora resistance. This study shows that LecRK-IX.1 and LecRK-IX.2 are Phytophthora resistance components that function independent of each other and independent of the cell-death phenotype. They both interact with the same ABC transporter, suggesting that they exploit similar signal transduction pathways.

  3. Kinase activity and specificity assay using synthetic peptides.

    Science.gov (United States)

    Wu, Xu Na; Schulze, Waltraud X

    2015-01-01

    Phosphorylation of substrate proteins by protein kinases can lead to activation or inactivation of signaling pathways or metabolic processes. Precise understanding of activity and specificity of protein kinases are important questions in characterization of kinase functions. Here, we describe a procedure to study kinase activity and specificity using kinase-GFP complexes purified from plant material and synthetic peptides as substrates. Magnetic GFP beads allow purifying receptor-like kinase-GFP complexes from microsomal fractions. Kinase-GFP complexes are then incubated with ATP and the synthetic peptides for kinase reaction. Phosphorylation of substrate peptides is then identified and quantified by mass spectrometry.

  4. Establishing a chemical genetic link between Bruton tyrosine kinase activity in malignant B cells and cell functions involved in the micro-environmental dialogue.

    Science.gov (United States)

    Göckeritz, Elisa; Vondey, Verena; Guastafierro, Anna; Pizevska, Maja; Hassenrück, Floyd; Neumann, Lars; Hallek, Michael; Krause, Günter

    2017-09-01

    To elucidate their mechanism of action, inhibitors of Bruton tyrosine kinase (BTK) and resistant BTK mutants were employed to dissect target-dependent cellular functions. BTK-C481S and -T474I, expressed in Ramos and NALM-6 cells, maintained BTK auto-phosphorylation under treatment with ibrutinib or dasatinib, respectively, which showed only modest cytotoxicity. Retained activity of BTK-T474 partially rescued cell migration from inhibition by dasatinib. Importantly, resistant BTK mutants reconstituted B cell receptor-triggered chemokine secretion in the presence of corresponding inhibitors, demonstrating that BTK activity is connected with cell-intrinsic functions of malignant B cells with importance for their dialogue with the micro-environment. © 2017 John Wiley & Sons Ltd.

  5. Genetic analysis of beta1 integrin function: confirmed, new and revised roles for a crucial family of cell adhesion molecules

    DEFF Research Database (Denmark)

    Brakebusch, C; Hirsch, E; Potocnik, A

    1997-01-01

    . Of more than 20 known integrins, 10 contain the nearly ubiquitously expressed beta1 integrin subunit. Disruption of the beta1 integrin gene by homologous recombination allows us to assess the supposed functions of beta1 containing integrins in vivo in a new way. This review will present and discuss recent...... and new functions found, significantly changing the previous view of beta1 integrin function in vivo....

  6. Protein kinase C δ signaling is required for dietary prebiotic-induced strengthening of intestinal epithelial barrier function

    Science.gov (United States)

    Wu, Richard Y.; Abdullah, Majd; Määttänen, Pekka; Pilar, Ana Victoria C.; Scruten, Erin; Johnson-Henry, Kathene C.; Napper, Scott; O’Brien, Catherine; Jones, Nicola L.; Sherman, Philip M.

    2017-01-01

    Prebiotics are non-digestible oligosaccharides that promote the growth of beneficial gut microbes, but it is unclear whether they also have direct effects on the intestinal mucosal barrier. Here we demonstrate two commercial prebiotics, inulin and short-chain fructo-oligosaccharide (scFOS), when applied onto intestinal epithelia in the absence of microbes, directly promote barrier integrity to prevent pathogen-induced barrier disruptions. We further show that these effects involve the induction of select tight junction (TJ) proteins through a protein kinase C (PKC) δ-dependent mechanism. These results suggest that in the absence of microbiota, prebiotics can directly exert barrier protective effects by activating host cell signaling in the intestinal epithelium, which represents a novel alternative mechanism of action of prebiotics. PMID:28098206

  7. Protein kinase C δ signaling is required for dietary prebiotic-induced strengthening of intestinal epithelial barrier function.

    Science.gov (United States)

    Wu, Richard Y; Abdullah, Majd; Määttänen, Pekka; Pilar, Ana Victoria C; Scruten, Erin; Johnson-Henry, Kathene C; Napper, Scott; O'Brien, Catherine; Jones, Nicola L; Sherman, Philip M

    2017-01-18

    Prebiotics are non-digestible oligosaccharides that promote the growth of beneficial gut microbes, but it is unclear whether they also have direct effects on the intestinal mucosal barrier. Here we demonstrate two commercial prebiotics, inulin and short-chain fructo-oligosaccharide (scFOS), when applied onto intestinal epithelia in the absence of microbes, directly promote barrier integrity to prevent pathogen-induced barrier disruptions. We further show that these effects involve the induction of select tight junction (TJ) proteins through a protein kinase C (PKC) δ-dependent mechanism. These results suggest that in the absence of microbiota, prebiotics can directly exert barrier protective effects by activating host cell signaling in the intestinal epithelium, which represents a novel alternative mechanism of action of prebiotics.

  8. Regorafenib impairs mitochondrial functions, activates AMP-activated protein kinase, induces autophagy, and causes rat hepatocyte necrosis.

    Science.gov (United States)

    Weng, Zuquan; Luo, Yong; Yang, Xi; Greenhaw, James J; Li, Haibo; Xie, Liming; Mattes, William B; Shi, Qiang

    2015-01-02

    The tyrosine kinase inhibitor regorafenib was approved by regulatory agencies for cancer treatment, albeit with strong warnings of severe hepatotoxicity included in the product label. The basis of this toxicity is unknown; one possible mechanism, that of mitochondrial damage, was tested. In isolated rat liver mitochondria, regorafenib directly uncoupled oxidative phosphorylation (OXPHOS) and promoted calcium overload-induced swelling, which were respectively prevented by the recoupler 6-ketocholestanol (KC) and the mitochondrial permeability transition (MPT) pore blocker cyclosporine A (CsA). In primary hepatocytes, regorafenib uncoupled OXPHOS, disrupted mitochondrial inner membrane potential (MMP), and decreased cellular ATP at 1h, and triggered MPT at 3h, which was followed by necrosis but not apoptosis at 7h and 24h, all of which were abrogated by KC. The combination of the glycolysis enhancer fructose plus the mitochondrial ATPase synthase inhibitor oligomycin A abolished regorafenib induced necrosis at 7h. This effect was not seen at 24h nor with the fructose or oligomycin A separately. CsA in combination with trifluoperazine, both MPT blockers, showed similar effects. Two compensatory mechanisms, activation of AMP-activated protein kinase (AMPK) to ameliorate ATP shortage and induction of autophagy to remove dysfunctional mitochondria, were found to be mobilized. Hepatocyte necrosis was enhanced either by the AMPK inhibitor Compound C or the autophagy inhibitor chloroquine, while autophagy inducer rapamycin was strongly cytoprotective. Remarkably, all toxic effects were observed at clinically-relevant concentrations of 2.5-15μM. These data suggest that uncoupling of OXPHOS and the resulting ATP shortage and MPT induction are the key mechanisms for regorafenib induced hepatocyte injury, and AMPK activation and autophagy induction serve as pro-survival pathways against such toxicity. Published by Elsevier Ireland Ltd.

  9. Functional involvement of protein kinase C, Rho-kinase and TRPC3 decreases while PLC increases with advancement of pregnancy in mediating oxytocin-induced myometrial contractions in water buffaloes (Bubalus bubalis).

    Science.gov (United States)

    Sharma, Abhishek; Nakade, Udayraj P; Choudhury, Soumen; Garg, Satish Kumar

    2017-04-01

    Present study unravels the involvement of different calcium signaling pathways in oxytocin-induced contractions in myometrium of non-pregnant and pregnant buffaloes during early and mid-pregnancy stages. Uteri of pregnant animals were more sensitive than of non-pregnant buffaloes. Phasic contractions and frequency of contraction significantly increased with advancement of pregnancy, while tonic contractions non-significantly and amplitude significantly decreased from six months pregnancy onward. Oxytocin produced concentration-dependent-contraction on isolated myometrial strips of pregnant and non-pregnant buffaloes and the dose response curves (DRCs) of oxytocin were significantly (P < 0.05) shifted to right in the presence of nifedipine (1 μM), in Ca 2+ -free Ringer Locke solution (RLS), ruthenium red (30 μM), ruthenium red + nifedipine, cyclopiazonic acid (CPA; Ca 2+ free RLS as well as RLS), CPA (10 μM)+nifedipine, U-73122 (1 μM) + nifedipine and SKF96365 (25 μM) on uteri of non-pregnant and pregnant (early and mid) animals. The DRCs were also significantly shifted towards right in the presence of Y-27632 (10 μM), GF109203X (5 μM) and Pyr3 (10 μM) on uteri of non-pregnant and early pregnancy stage buffaloes while only in the presence of U-73122 (1 μM) on uteri of mid-pregnancy stage buffaloes. Our finding suggest that and L-type Ca 2+ channels, IP3-RyR-gated, and store-operated calcium channels including transient receptor potential channel (TRPC) pathways play significant role in mediating oxytocin-induced contractions in myometrium of pregnant and non-pregnant buffaloes. SERCA plays major role only during early-pregnancy while functional role of protein kinase C (PKC), Rho-kinase and TRPC3 pathways decreased and role of G-protein coupled receptor-phospholipase C (GPCR-PLC) pathway increased with advancement of pregnancy. Copyright © 2016 Elsevier Inc. All rights reserved.

  10. Activation of c-Src and Fyn kinases by protein tyrosine phosphatase RPTPalpha is substrate-specific and compatible with lipid raft localization

    DEFF Research Database (Denmark)

    Vacaresse, Nathalie; Møller, Bente; Danielsen, Erik Michael

    2008-01-01

    Tyrosine kinases of the Src family (SFKs) function in multiple signaling pathways, raising the question of how appropriate regulation and substrate choice are achieved. SFK activity is modulated by several protein tyrosine phosphatases (PTPs), among which RPTPa and SHP2 are the best established. We...... studied how RPTPa affects substrate specificity and regulation of c-Src and Fyn in response to EGF and PDGF. We find that RPTPa, in a growth factor-specific manner, directs the specificity of these kinases towards a specific subset of SFK substrates, particularly the focal adhesion protein Paxillin...

  11. Disruption of zebrafish cyclin G-associated kinase (GAK function impairs the expression of Notch-dependent genes during neurogenesis and causes defects in neuronal development

    Directory of Open Access Journals (Sweden)

    Szeto Daniel P

    2010-01-01

    Full Text Available Abstract Background The J-domain-containing protein auxilin, a critical regulator in clathrin-mediated transport, has been implicated in Drosophila Notch signaling. To ask if this role of auxilin is conserved and whether auxilin has additional roles in development, we have investigated the functions of auxilin orthologs in zebrafish. Results Like mammals, zebrafish has two distinct auxilin-like molecules, auxilin and cyclin G-associated kinase (GAK, differing in their domain structures and expression patterns. Both zebrafish auxilin and GAK can functionally substitute for the Drosophila auxilin, suggesting that they have overlapping molecular functions. Still, they are not completely redundant, as morpholino-mediated knockdown of the ubiquitously expressed GAK alone can increase the specification of neuronal cells, a known Notch-dependent process, and decrease the expression of Her4, a Notch target gene. Furthermore, inhibition of GAK function caused an elevated level of apoptosis in neural tissues, resulting in severe degeneration of neural structures. Conclusion In support of the notion that endocytosis plays important roles in Notch signaling, inhibition of zebrafish GAK function affects embryonic neuronal cell specification and Her4 expression. In addition, our analysis suggests that zebrafish GAK has at least two functions during the development of neural tissues: an early Notch-dependent role in neuronal patterning and a late role in maintaining the survival of neural cells.

  12. Crystal structure of a trimeric archaeal adenylate kinase from the mesophile Methanococcus maripaludis with an unusually broad functional range and thermal stability.

    Science.gov (United States)

    Davlieva, Milya; Shamoo, Yousif

    2010-02-01

    The structure of the trimeric adenylate kinase from the Archaebacteria Methanococcus mariplaludis (AK(MAR)) has been solved to 2.5-A resolution and the temperature dependent stability and kinetics of the enzyme measured. The K(M) and V(max) of AK(MAR) exhibit only modest temperature dependence from 30 degrees -60 degrees C. Although M. mariplaludis is a mesophile with a maximum growth temperature of 43 degrees C, AK(MAR) has a very broad functional range and stability (T(m) = 74.0 degrees C) that are more consistent with a thermophilic enzyme with high thermostability and exceptional activity over a wide range of temperatures, suggesting that this microbe may have only recently invaded a mesophilic niche and has yet to fully adapt. A comparison of the Local Structural Entropy (LSE) for AK(MAR) to the related adenylate kinases from the mesophile Methanococcus voltae and thermophile Methanococcus thermolithotrophicus show that changes in LSE are able to fully account for the intermediate stability of AK(MAR) and highlights a general mechanism for protein adaptation in this class of enzymes. (c) 2009 Wiley-Liss, Inc.

  13. Functional and spatial analysis of C. elegans SYG-1 and SYG-2, orthologs of the Neph/nephrin cell adhesion module directing selective synaptogenesis.

    Directory of Open Access Journals (Sweden)

    Nicola Wanner

    Full Text Available The assembly of specific synaptic connections represents a prime example of cellular recognition. Members of the Ig superfamily are among the most ancient proteins represented in the genomes of both mammalian and invertebrate organisms, where they constitute a trans-synaptic adhesion system. The correct connectivity patterns of the highly conserved immunoglobulin superfamily proteins nephrin and Neph1 are crucial for the assembly of functional neuronal circuits and the formation of the kidney slit diaphragm, a synapse-like structure forming the filtration barrier. Here, we utilize the nematode C. elegans model for studying the requirements of synaptic specificity mediated by nephrin-Neph proteins. In C. elegans, the nephrin/Neph1 orthologs SYG-2 and SYG-1 form intercellular contacts strictly in trans between epithelial guidepost cells and neurons specifying the localization of synapses. We demonstrate a functional conservation between mammalian nephrin and SYG-2. Expression of nephrin effectively compensated loss of syg-2 function in C. elegans and restored defective synaptic connectivity further establishing the C. elegans system as a valuable model for slit diaphragm proteins. Next, we investigated the effect of SYG-1 and SYG-2 trans homodimerization respectively. Strikingly, synapse assembly could be induced by homophilic SYG-1 but not SYG-2 binding indicating a critical role of SYG-1 intracellular signalling for morphogenetic events and pointing toward the dynamic and stochastic nature of extra- and intracellular nephrin-Neph interactions to generate reproducible patterns of synaptic connectivity.

  14. A proteomic approach for comprehensively screening substrates of protein kinases such as Rho-kinase.

    Directory of Open Access Journals (Sweden)

    Mutsuki Amano

    Full Text Available BACKGROUND: Protein kinases are major components of signal transduction pathways in multiple cellular processes. Kinases directly interact with and phosphorylate downstream substrates, thus modulating their functions. Despite the importance of identifying substrates in order to more fully understand the signaling network of respective kinases, efficient methods to search for substrates remain poorly explored. METHODOLOGY/PRINCIPAL FINDINGS: We combined mass spectrometry and affinity column chromatography of the catalytic domain of protein kinases to screen potential substrates. Using the active catalytic fragment of Rho-kinase/ROCK/ROK as the model bait, we obtained about 300 interacting proteins from the rat brain cytosol fraction, which included the proteins previously reported as Rho-kinase substrates. Several novel interacting proteins, including doublecortin, were phosphorylated by Rho-kinase both in vitro and in vivo. CONCLUSIONS/SIGNIFICANCE: This method would enable identification of novel specific substrates for kinases such as Rho-kinase with high sensitivity.

  15. Quantitative proteomic profiling identifies DPYSL3 as pancreatic ductal adenocarcinoma-associated molecule that regulates cell adhesion and migration by stabilization of focal adhesion complex.

    Directory of Open Access Journals (Sweden)

    Takeo Kawahara

    Full Text Available Elucidation of how pancreatic cancer cells give rise to distant metastasis is urgently needed in order to provide not only a better understanding of the underlying molecular mechanisms, but also to identify novel targets for greatly improved molecular diagnosis and therapeutic intervention. We employed combined proteomic technologies including mass spectrometry and isobaric tags for relative and absolute quantification peptide tagging to analyze protein profiles of surgically resected human pancreatic ductal adenocarcinoma tissues. We identified a protein, dihydropyrimidinase-like 3, as highly expressed in human pancreatic ductal adenocarcinoma tissues as well as pancreatic cancer cell lines. Characterization of the roles of dihydropyrimidinase-like 3 in relation to cancer cell adhesion and migration in vitro, and metastasis in vivo was performed using a series of functional analyses, including those employing multiple reaction monitoring proteomic analysis. Furthermore, dihydropyrimidinase-like 3 was found to interact with Ezrin, which has important roles in cell adhesion, motility, and invasion, while that interaction promoted stabilization of an adhesion complex consisting of Ezrin, c-Src, focal adhesion kinase, and Talin1. We also found that exogenous expression of dihydropyrimidinase-like 3 induced activating phosphorylation of Ezrin and c-Src, leading to up-regulation of the signaling pathway. Taken together, the present results indicate successful application of combined proteomic approaches to identify a novel key player, dihydropyrimidinase-like 3, in pancreatic ductal adenocarcinoma tumorigenesis, which may serve as an important biomarker and/or drug target to improve therapeutic strategies.

  16. Quantitative proteomic profiling identifies DPYSL3 as pancreatic ductal adenocarcinoma-associated molecule that regulates cell adhesion and migration by stabilization of focal adhesion complex.

    Science.gov (United States)

    Kawahara, Takeo; Hotta, Naoe; Ozawa, Yukiko; Kato, Seiichi; Kano, Keiko; Yokoyama, Yukihiro; Nagino, Masato; Takahashi, Takashi; Yanagisawa, Kiyoshi

    2013-01-01

    Elucidation of how pancreatic cancer cells give rise to distant metastasis is urgently needed in order to provide not only a better understanding of the underlying molecular mechanisms, but also to identify novel targets for greatly improved molecular diagnosis and therapeutic intervention. We employed combined proteomic technologies including mass spectrometry and isobaric tags for relative and absolute quantification peptide tagging to analyze protein profiles of surgically resected human pancreatic ductal adenocarcinoma tissues. We identified a protein, dihydropyrimidinase-like 3, as highly expressed in human pancreatic ductal adenocarcinoma tissues as well as pancreatic cancer cell lines. Characterization of the roles of dihydropyrimidinase-like 3 in relation to cancer cell adhesion and migration in vitro, and metastasis in vivo was performed using a series of functional analyses, including those employing multiple reaction monitoring proteomic analysis. Furthermore, dihydropyrimidinase-like 3 was found to interact with Ezrin, which has important roles in cell adhesion, motility, and invasion, while that interaction promoted stabilization of an adhesion complex consisting of Ezrin, c-Src, focal adhesion kinase, and Talin1. We also found that exogenous expression of dihydropyrimidinase-like 3 induced activating phosphorylation of Ezrin and c-Src, leading to up-regulation of the signaling pathway. Taken together, the present results indicate successful application of combined proteomic approaches to identify a novel key player, dihydropyrimidinase-like 3, in pancreatic ductal adenocarcinoma tumorigenesis, which may serve as an important biomarker and/or drug target to improve therapeutic strategies.

  17. The effect of dietary red palm oil on the functional recovery of the ischaemic/reperfused isolated rat heart: the involvement of the PI3-Kinase signaling pathway

    Directory of Open Access Journals (Sweden)

    Engelbrecht Anna-Mart

    2009-05-01

    Full Text Available Abstract We have previously shown that dietary red palm oil (RPO supplementation improves functional recovery in hearts subjected to ischaemia/reperfusion-induced injury. Unfortunately, the cellular and molecular mechanisms responsible for this phenomenon are still poorly understood and no knowledge exists regarding the effects of RPO supplementation on the phosphoinositide 3-kinase (PI3-K signaling pathway and apoptosis during ischaemia/reperfusion injury. Therefore, the aims of the present study were three fold: (i to establish the effect of RPO on the functional recovery of the heart after ischaemia/reperfuion injury; (ii to determine the effect of the PI3-K pathway in RPO-induced protection with the aid of an inhibitor (wortmannin; and (iii to evaluate apoptosis in our model. Wistar rats were fed a standard rat chow control diet or a control diet plus 7 g RPO/kg for six weeks. Hearts were excised and mounted on a Langendorff perfusion apparatus. Mechanical function was measured after a 25 min period of total global ischaemia followed by 30 minutes of reperfusion. Hearts subjected to the same conditions were freeze-clamped for biochemical analysis at 10 min during reperfusion to determine the involvement of the PI3-Kinase signaling pathway and apoptosis in our model. Dietary RPO supplementation significantly increased % rate pressure product recovery during reperfusion (71.0 ± 6.3% in control vs 92.36 ± 4.489% in RPO; p vs 75.21 ± 5.26% in RPO + Wm. RPO + Wm also significantly attenuated PI3-K induction compared with the RPO group (59.2 ± 2.8 pixels in RPO vs 37.9 ± 3.4 pixels in RPO + Wm. We have also demonstrated that PI3-K inhibition induced PARP cleavage (marker of apoptosis in the hearts during ischaemia/reperfusion injury and that RPO supplementation counteracted this effect.

  18. Role of protein kinase C in the TBT-induced inhibition of lytic function and MAPK activation in human natural killer cells

    Science.gov (United States)

    Abraha, Abraham B.; Rana, Krupa; Whalen, Margaret M.

    2010-01-01

    Human natural killer (NK) cells are lymphocytes that destroy tumor and virally infected cells. Previous studies have shown that exposures of NK cells to tributyltin (TBT) greatly diminish their ability to destroy tumor cells (lytic function) while activating mitogen-activated protein kinases (MAPK) (p44/42, p38, and JNK) in the NK cells. The signaling pathway that regulates NK lytic function appears to include activation of protein kinase C (PKC) as well as MAPK activity. The TBT-induced activation of MAPKs would trigger a portion of the NK lytic signaling pathway, which would then leave the NK cell unable to trigger this pathway in response to a subsequent encounter with a target cell. In the present study we evaluated the involvement of PKC in the inhibition of NK lysis of tumor cells and activation of MAPKs caused by TBT exposures. TBT caused a 2–3 fold activation of PKC at concentrations ranging from 50–300 nM (16–98 ng/mL), indicating that activation of PKC occurs in response to TBT exposures. This would then leave the NK cell unable to respond to targets. Treatment with the PKC inhibitor, bisindolylmaleimide I, caused an 85% decrease in the ability of NK cells to lyse tumor cells validating the involvement of PKC in the lytic signaling pathway. The role of PKC in the activation of MAPKs by TBT was also investigated using bisindolylmaleimide I. The results indicated that in NK cells where PKC activation was blocked there was no activation of the MAPK, p44/42 in response to TBT. However, TBT-induced activation of the MAPKs, p38 and JNK did not require PKC activation. These results indicate the pivotal role of PKC in the TBT-induced loss of NK lytic function including the activation of p44/42 by TBT in NK cells. PMID:20390410

  19. Role of protein kinase C in TBT-induced inhibition of lytic function and MAPK activation in human natural killer cells.

    Science.gov (United States)

    Abraha, Abraham B; Rana, Krupa; Whalen, Margaret M

    2010-11-01

    Human natural killer (NK) cells are lymphocytes that destroy tumor and virally infected cells. Previous studies have shown that exposure of NK cells to tributyltin (TBT) greatly diminishes their ability to destroy tumor cells (lytic function) while activating mitogen-activated protein kinases (MAPK) (p44/42, p38, and JNK) in NK cells. The signaling pathway that regulates NK lytic function appears to include activation of protein kinase C(PKC) as well as MAPK activity. TBT-induced activation of MAPKs would trigger a portion of the NK lytic signaling pathway, which would then leave the NK cell unable to trigger this pathway in response to a subsequent encounter with a target cell. In the present study we evaluated the involvement of PKC in inhibition of NK lysis of tumor cells and activation of MAPKs caused by TBT exposure. TBT caused a 2–3-fold activation of PKC at concentrations ranging from 50 to 300 nM (16–98 ng/ml),indicating that activation of PKC occurs in response to TBT exposure. This would then leave the NK cell unable to respond to targets. Treatment with the PKC inhibitor, bisindolylmaleimide I, caused an 85% decrease in the ability of NK cells to lyse tumor cells, validating the involvement of PKC in the lytic signaling pathway. The role of PKC in the activation of MAPKs by TBT was also investigated using bisindolylmaleimide I. The results indicated that, in NK cells where PKC activation was blocked, there was no activation of the MAPK, p44/42 in response to TBT.However, TBT-induced activation of the MAPKs, p38 and JNK did not require PKC activation. These results indicate the pivotal role of PKC in the TBT-induced loss of NK lytic function including activation of p44/42 by TBT in NK cells.

  20. Adhesion in microelectronics

    CERN Document Server

    Mittal, K L

    2014-01-01

    This comprehensive book will provide both fundamental and applied aspects of adhesion pertaining to microelectronics in a single and easily accessible source. Among the topics to be covered include; Various theories or mechanisms of adhesionSurface (physical or chemical) characterization of materials as it pertains to adhesionSurface cleaning as it pertains to adhesionWays to improve adhesionUnraveling of interfacial interactions using an array of pertinent techniquesCharacterization of interfaces / interphasesPolymer-polymer adhesionMetal-polymer adhesion  (metallized polymers)Polymer adhesi