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Sample records for adhesion protein n-cadherin

  1. Targeting Cx43 and N-Cadherin, Which Are Abnormally Upregulated in Venous Leg Ulcers, Influences Migration, Adhesion and Activation of Rho GTPases

    PubMed Central

    Mendoza-Naranjo, Ariadna; Cormie, Peter; Serrano, Antonio E.; Hu, Rebecca; O'Neill, Shay; Wang, Chiuhui Mary; Thrasivoulou, Christopher; Power, Kieran T.; White, Alexis; Serena, Thomas; Phillips, Anthony R. J.; Becker, David L.

    2012-01-01

    Background Venous leg ulcers can be very hard to heal and represent a significant medical need with no effective therapeutic treatment currently available. Principal Findings In wound edge biopsies from human venous leg ulcers we found a striking upregulation of dermal N-cadherin, Zonula Occludens-1 and the gap junction protein Connexin43 (Cx43) compared to intact skin, and in stark contrast to the down-regulation of Cx43 expression seen in acute, healing wounds. We targeted the expression of these proteins in 3T3 fibroblasts to evaluate their role in venous leg ulcers healing. Knockdown of Cx43 and N-cadherin, but not Zonula Occludens-1, accelerated cell migration in a scratch wound-healing assay. Reducing Cx43 increased Golgi reorientation, whilst decreasing cell adhesion and proliferation. Furthermore, Connexin43 and N-cadherin knockdown led to profound effects on fibroblast cytoskeletal dynamics after scratch-wounding. The cells exhibited longer lamelipodial protrusions lacking the F-actin belt seen at the leading edge in wounded control cells. This phenotype was accompanied by augmented activation of Rac-1 and RhoA GTPases, as revealed by Förster Resonance Energy Transfer and pull down experiments. Conclusions Cx43 and N-cadherin are potential therapeutic targets in the promotion of healing of venous leg ulcers, by acting at least in part through distinct contributions of cell adhesion, migration, proliferation and cytoskeletal dynamics. PMID:22615994

  2. Epstein–Barr virus LMP1 induces focal adhesions and epithelial cell migration through effects on integrin-α5 and N-cadherin

    PubMed Central

    Wasil, L R; Shair, K H Y

    2015-01-01

    Epstein–Barr virus (EBV) is a γ-herpesvirus associated with human epithelial and B-cell malignancies. The EBV latent membrane protein (LMP) 1 is expressed in nasopharyngeal carcinoma (NPC) and promotes oncogenic intracellular signaling mechanisms. LMP1 also promotes a pro-migratory phenotype through potential effects on cell surface proteins, as expression of LMP1 induces an epithelial–mesenchymal transition (EMT) in epithelial cell lines. In this study, LMP1 was examined for potential effects on cadherin and integrin surface interactions, and assessed for biological effects on adhesion and motility to fibronectin. Expression of LMP1 in the non-tumorigenic epithelial cell line MCF10a induced an EMT-associated cadherin switch. The induced N-cadherin was ligated and localized to the cell surface as determined by triton-solubility and immunofluorescence assays. In addition, LMP1 induced the assembly of focal adhesions (FAs) with increased production of fibronectin in MCF10a and NP460hTERT-immortalized nasopharyngeal cells. Biochemical enrichment of fibronectin-associated proteins indicated that LMP1 selectively promoted the recruitment of integrin-α5 and Src family kinase proteins to FA complexes. Neutralizing antibodies to N-cadherin and integrin-α5, but not integrin-αV, blocked the adhesion and transwell motility of MCF10a cells to fibronectin induced by LMP1. LMP1-induced transwell motility was also decreased by Src inhibition with the PP2 kinase inhibitor and short hairpin RNAs. These studies reveal that LMP1 has multiple mechanisms to promote the adhesive and migratory properties of epithelial cells through induction of fibronectin and modulation of cell surface interactions involving integrin-α5 and N-cadherin, which may contribute to the metastatic potential of NPC. PMID:26479443

  3. N-cadherin prodomain processing regulates synaptogenesis.

    PubMed

    Reinés, Analía; Bernier, Louis-Philippe; McAdam, Robyn; Belkaid, Wiam; Shan, Weisong; Koch, Alexander W; Séguéla, Philippe; Colman, David R; Dhaunchak, Ajit S

    2012-05-01

    Classical cadherins, which are adhesion molecules functioning at the CNS synapse, are synthesized as adhesively inactive precursor proteins in the endoplasmic reticulum (ER). Signal sequence and prodomain cleavage in the ER and Golgi apparatus, respectively, activates their adhesive properties. Here, we provide the first evidence for sorting of nonadhesive precursor N-cadherin (ProN) to the neuronal surface, where it coexists with adhesively competent mature N-cadherin (N-cad), generating a spectrum of adhesive strengths. In cultured hippocampal neurons, a high ProN/N-cad ratio downregulates synapse formation. Neurons expressing genetically engineered uncleavable ProN make markedly fewer synapses. The synapse number can be rescued to normality by depleting surface ProN levels through prodomain cleavage by an exogenous protease. Finally, prodomain processing is developmentally regulated in the rat hippocampus. We conclude that it is the ProN/N-cad ratio and not mature N-cad alone that is critical for regulation of adhesion during synaptogenesis. PMID:22553038

  4. N-cadherin haploinsufficiency affects cardiac gap junctions and arrhythmic susceptibility

    PubMed Central

    Li, Jifen; Levin, Mark D; Xiong, Yanming; Petrenko, Nataliya; Patel, Vickas V.; Radice, Glenn L.

    2008-01-01

    Cardiac-specific deletion of the murine gene (Cdh2) encoding the cell adhesion molecule, N-cadherin, results in disassembly of the intercalated disc (ICD) structure and sudden arrhythmic death. Connexin 43 (Cx43)-containing gap junctions are significantly reduced in the heart after depleting N-cadherin, therefore we hypothesized that animals expressing half the normal levels of N-cadherin would exhibit an intermediate phenotype. We examined the effect of N-cadherin haploinsufficiency on Cx43 expression and susceptibility to induced arrhythmias in mice either wild-type or heterozygous for the Cx43 (Gja1)-null allele. An increase in hypophosphorylated Cx43 accompanied by a modest decrease in total Cx43 protein levels was observed in the N-cadherin heterozygous mice. Consistent with these findings N-cadherin heterozygotes exhibited increased susceptibility to ventricular arrhythmias compared to wild-type mice. Quantitative immunofluorescence microscopy revealed a reduction in size of large Cx43-containing plaques in the N-cadherin heterozygous animals compared to wild-type. Gap junctions were further decreased in number and size in the N-cad/Cx43 compound heterozygous mice with increased arrhythmic susceptibility compared to the single mutants. The scaffold protein, ZO-1, was reduced at the ICD in N-cadherin heterozygous cardiomyocytes providing a possible explanation for the reduction in Cx43 plaque size. These data provide further support for the intimate relationship between N-cadherin and Cx43 in the heart, and suggest that germline mutations in the human N-cadherin (Cdh2) gene may predispose patients to increased risk of cardiac arrhythmias. PMID:18201716

  5. N-Cadherin in Neuroblastoma Disease: Expression and Clinical Significance

    PubMed Central

    Derycke, Lara; De Craemer, Annemie; De Brouwer, Sara; De Preter, Katleen; Van Roy, Nadine; Vandesompele, Jo; Speleman, Frank; Philippé, Jan; Benoit, Yves; Beiske, Klaus; Bracke, Marc; Laureys, Geneviève

    2012-01-01

    One of the first and most important steps in the metastatic cascade is the loss of cell-cell and cell-matrix interactions. N-cadherin, a crucial mediator of homotypic and heterotypic cell-cell interactions, might play a central role in the metastasis of neuroblastoma (NB), a solid tumor of neuroectodermal origin. Using Reverse Transcription Quantitative PCR (RT-qPCR), Western blot, immunocytochemistry and Tissue MicroArrays (TMA) we demonstrate the expression of N-cadherin in neuroblastoma tumors and cell lines. All neuroblastic tumors (n = 356) and cell lines (n = 10) expressed various levels of the adhesion protein. The N-cadherin mRNA expression was significantly lower in tumor samples from patients suffering metastatic disease. Treatment of NB cell lines with the N-cadherin blocking peptide ADH-1 (Exherin, Adherex Technologies Inc.), strongly inhibited tumor cell proliferation in vitro by inducing apoptosis. Our results suggest that N-cadherin signaling may play a role in neuroblastoma disease, marking involvement of metastasis and determining neuroblastoma cell viability. PMID:22355346

  6. P120-Catenin Regulates Early Trafficking Stages of the N-Cadherin Precursor Complex

    PubMed Central

    Wehrendt, Diana P.; Carmona, Fernando; González Wusener, Ana E.; González, Ángela; Martínez, Juan M. Lázaro; Arregui, Carlos O.

    2016-01-01

    It is well established that binding of p120 catenin to the cytoplasmic domain of surface cadherin prevents cadherin endocytosis and degradation, contributing to cell-cell adhesion. In the present work we show that p120 catenin bound to the N-cadherin precursor, contributes to its anterograde movement from the endoplasmic reticulum (ER) to the Golgi complex. In HeLa cells, depletion of p120 expression, or blocking its binding to N-cadherin, increased the accumulation of the precursor in the ER, while it decreased the localization of mature N-cadherin at intercellular junctions. Reconstitution experiments in p120-deficient SW48 cells with all three major isoforms of p120 (1, 3 and 4) had similar capacity to promote the processing of the N-cadherin precursor to the mature form, and its localization at cell-cell junctions. P120 catenin and protein tyrosine phosphatase PTP1B facilitated the recruitment of the N-ethylmaleimide sensitive factor (NSF), an ATPase involved in vesicular trafficking, to the N-cadherin precursor complex. Dominant negative NSF E329Q impaired N-cadherin trafficking, maturation and localization at cell-cell junctions. Our results uncover a new role for p120 catenin bound to the N-cadherin precursor ensuring its trafficking through the biosynthetic pathway towards the cell surface. PMID:27254316

  7. N-cadherin regulates molecular organization of excitatory and inhibitory synaptic circuits in adult hippocampus in vivo

    PubMed Central

    Nikitczuk, Jessica S.; Patil, Shekhar B.; Matikainen-Ankney, Bridget A.; Scarpa, Joseph; Shapiro, Matthew L.

    2016-01-01

    N-cadherin and β-catenin form a transsynaptic adhesion complex required for spine and synapse development. In adulthood, N-cadherin mediates persistent synaptic plasticity, but whether the role of N-cadherin at mature synapses is similar to that at developing synapses is unclear. To address this, we conditionally ablated N-cadherin from excitatory forebrain synapses in mice starting in late postnatal life and examined hippocampal structure and function in adulthood. In the absence of N-cadherin, β-catenin levels were reduced, but numbers of excitatory synapses were unchanged, and there was no impact on number or shape of dendrites or spines. However, the composition of synaptic molecules was altered. Levels of GluA1 and its scaffolding protein PSD95 were diminished and the density of immunolabeled puncta was decreased, without effects on other glutamate receptors and their scaffolding proteins. Additionally, loss of N-cadherin at excitatory synapses triggered increases in the density of markers for inhibitory synapses and decreased severity of hippocampal seizures. Finally, adult mutant mice were profoundly impaired in hippocampal-dependent memory for spatial episodes. These results demonstrate a novel function for the N-cadherin/β-catenin complex in regulating ionotropic receptor composition of excitatory synapses, an appropriate balance of excitatory and inhibitory synaptic proteins and the maintenance of neural circuitry necessary to generate flexible yet persistent cognitive and synaptic function. PMID:24753442

  8. ICAM-2 regulates vascular permeability and N-cadherin localization through ezrin-radixin-moesin (ERM) proteins and Rac-1 signalling

    PubMed Central

    2014-01-01

    Background Endothelial junctions control functions such as permeability, angiogenesis and contact inhibition. VE-Cadherin (VECad) is essential for the maintenance of intercellular contacts. In confluent endothelial monolayers, N-Cadherin (NCad) is mostly expressed on the apical and basal membrane, but in the absence of VECad it localizes at junctions. Both cadherins are required for vascular development. The intercellular adhesion molecule (ICAM)-2, also localized at endothelial junctions, is involved in leukocyte recruitment and angiogenesis. Results In human umbilical vein endothelial cells (HUVEC), both VECad and NCad were found at nascent cell contacts of sub-confluent monolayers, but only VECad localized at the mature junctions of confluent monolayers. Inhibition of ICAM-2 expression by siRNA caused the appearance of small gaps at the junctions and a decrease in NCad junctional staining in sub-confluent monolayers. Endothelioma lines derived from WT or ICAM-2-deficient mice (IC2neg) lacked VECad and failed to form junctions, with loss of contact inhibition. Re-expression of full-length ICAM-2 (IC2 FL) in IC2neg cells restored contact inhibition through recruitment of NCad at the junctions. Mutant ICAM-2 lacking the binding site for ERM proteins (IC2 ΔERM) or the cytoplasmic tail (IC2 ΔTAIL) failed to restore junctions. ICAM-2-dependent Rac-1 activation was also decreased in these mutant cell lines. Barrier function, measured in vitro via transendothelial electrical resistance, was decreased in IC2neg cells, both in resting conditions and after thrombin stimulation. This was dependent on ICAM-2 signalling to the small GTPase Rac-1, since transendothelial electrical resistance of IC2neg cells was restored by constitutively active Rac-1. In vivo, thrombin-induced extravasation of FITC-labeled albumin measured by intravital fluorescence microscopy in the mouse cremaster muscle showed that permeability was increased in ICAM-2-deficient mice compared to controls

  9. N-cadherin expression is regulated by UTP in schwannoma cells.

    PubMed

    Martiáñez, Tania; Lamarca, Aloa; Casals, Nuria; Gella, Alejandro

    2013-06-01

    Schwann cells (SCs) are peripheral myelinating glial cells that express the neuronal Ca(2+)-dependent cell adhesion molecule, neural cadherin (N-cadherin). N-cadherin is involved in glia-glia and axon-glia interactions and participates in many key events, which range from the control of axonal growth and guidance to synapse formation and plasticity. Extracellular UTP activates P2Y purinergic receptors and exerts short- and long-term effects on several tissues to promote wound healing. Nevertheless, the contribution of P2Y receptors in peripheral nervous system functions is not completely understood. The current study demonstrated that UTP induced a dose- and time-dependent increase in N-cadherin expression in SCs. Furthermore, N-cadherin expression was blocked by the P2 purinoceptor antagonist suramin. The increased N-cadherin expression induced by UTP was mediated by phosphorylation of mitogen-activated protein kinases (MAPKs), such as Jun N-terminal kinase, extracellular-regulated kinase and p38 kinase. Moreover, the Rho kinase inhibitor Y27632, the phospholipase C inhibitor U73122 and the protein kinase C inhibitor calphostin C attenuated the UTP-induced activation of MAPKs significantly. Extracellular UTP also modulated increased in the expression of the early transcription factors c-Fos and c-Jun. We also demonstrated that the region of the N-cadherin promoter between nucleotide positions -3698 and -2620, which contained one activator protein-1-binding site, was necessary for UTP-induced gene expression. These results suggest a novel role for P2Y purinergic receptors in the regulation of N-cadherin expression in SCs. PMID:23271561

  10. Cortactin Is Required for N-cadherin Regulation of Kv1.5 Channel Function*

    PubMed Central

    Cheng, Lan; Yung, Aaron; Covarrubias, Manuel; Radice, Glenn L.

    2011-01-01

    The intercalated disc serves as an organizing center for various cell surface components at the termini of the cardiomyocyte, thus ensuring proper mechanoelectrical coupling throughout the myocardium. The cell adhesion molecule, N-cadherin, is an essential component of the intercalated disc. Cardiac-specific deletion of N-cadherin leads to abnormal electrical conduction and sudden arrhythmic death in mice. The mechanisms linking the loss of N-cadherin in the heart and spontaneous malignant ventricular arrhythmias are poorly understood. To investigate whether ion channel remodeling contributes to arrhythmogenesis in N-cadherin conditional knock-out (N-cad CKO) mice, cardiac myocyte excitability and voltage-gated potassium channel (Kv), as well as inwardly rectifying K+ channel remodeling, were investigated in N-cad CKO cardiomyocytes by whole cell patch clamp recordings. Action potential duration was prolonged in N-cad CKO ventricle myocytes compared with wild type. Relative to wild type, IK,slow density was significantly reduced consistent with decreased expression of Kv1.5 and Kv accessory protein, Kcne2, in the N-cad CKO myocytes. The decreased Kv1.5/Kcne2 expression correlated with disruption of the actin cytoskeleton and reduced cortactin at the sarcolemma. Biochemical experiments revealed that cortactin co-immunoprecipitates with Kv1.5. Finally, cortactin was required for N-cadherin-mediated enhancement of Kv1.5 channel activity in a heterologous expression system. Our results demonstrate a novel mechanistic link among the cell adhesion molecule, N-cadherin, the actin-binding scaffold protein, cortactin, and Kv channel remodeling in the heart. These data suggest that in addition to gap junction remodeling, aberrant Kv1.5 channel function contributes to the arrhythmogenic phenotype in N-cad CKO mice. PMID:21507952

  11. Tetraspanin CD82 regulates bone marrow homing of acute myeloid leukemia by modulating the molecular organization of N-cadherin.

    PubMed

    Marjon, K D; Termini, C M; Karlen, K L; Saito-Reis, C; Soria, C E; Lidke, K A; Gillette, J M

    2016-08-01

    Communication between acute myeloid leukemia (AML) and the bone marrow microenvironment is known to control disease progression. Therefore, regulation of AML cell trafficking and adhesion to the bone marrow is of significant interest. In this study, we demonstrate that differential expression of the membrane scaffold CD82 modulates the bone marrow homing of AML cells. By combining mutational analysis and super-resolution imaging, we identify membrane protein clustering by CD82 as a regulator of AML cell adhesion and bone marrow homing. Cluster analysis of super-resolution data indicates that N-linked glycosylation and palmitoylation of CD82 are both critical modifications that control the microdomain organization of CD82 as well as the nanoscale clustering of associated adhesion protein, N-cadherin. We demonstrate that the inhibition of CD82 glycosylation increases the molecular packing of N-cadherin and promotes the bone marrow homing of AML cells. In contrast, we find that the inhibition of CD82 palmitoylation disrupts the formation and organization of N-cadherin clusters and significantly diminishes bone marrow trafficking of AML. Taken together, these data establish a mechanism where the membrane organization of CD82, through specific posttranslational modifications, regulates N-cadherin clustering and membrane density, which impacts the in vivo trafficking of AML cells. As such, these observations provide an alternative model for targeting AML where modulation of protein organization within the membrane may be an effective treatment therapy to disrupt the bone marrow homing potential of AML cells. PMID:26592446

  12. Tetraspanin CD82 regulates bone marrow homing of acute myeloid leukemia by modulating the molecular organization of N-cadherin

    PubMed Central

    Marjon, Kristopher D.; Termini, Christina M.; Karlen, Karin L.; Saito-Reis, Chelsea; Soria, Cesar E.; Lidke, Keith A.; Gillette, Jennifer M.

    2016-01-01

    Communication between acute myeloid leukemia (AML) and the bone marrow microenvironment is known to control disease progression. Therefore, regulation of AML cell trafficking and adhesion to the bone marrow is of significant interest. In this study, we demonstrate that differential expression of the membrane scaffold CD82 modulates the bone marrow homing of AML cells. By combining mutational analysis and super-resolution imaging, we identify membrane protein clustering by CD82 as a regulator of AML cell adhesion and bone marrow homing. Cluster analysis of super-resolution data indicates that N-linked glycosylation and palmitoylation of CD82 are both critical modifications that control the microdomain organization of CD82 as well as the nanoscale clustering of associated adhesion protein, N-cadherin. We demonstrate that inhibition of CD82 glycosylation increases the molecular packing of N-cadherin and promotes the bone marrow homing of AML cells. In contrast, we find that inhibition of CD82 palmitoylation disrupts the formation and organization of N-cadherin clusters and significantly diminishes bone marrow trafficking of AML. Taken together, these data establish a mechanism where the membrane organization of CD82, through specific post-translational modifications, regulates N-cadherin clustering and membrane density, which impacts the in vivo trafficking of AML cells. As such, these observations provide an alternative model for targeting AML where modulation of protein organization within the membrane may be an effective treatment therapy to disrupt the bone marrow homing potential of AML cells. PMID:26592446

  13. N-cadherin relocalization during cardiac trabeculation.

    PubMed

    Cherian, Anoop V; Fukuda, Ryuichi; Augustine, Sruthy Maria; Maischein, Hans-Martin; Stainier, Didier Y R

    2016-07-01

    During cardiac trabeculation, cardiomyocytes delaminate from the outermost (compact) layer to form complex muscular structures known as trabeculae. As these cardiomyocytes delaminate, the remodeling of adhesion junctions must be tightly coordinated so cells can extrude from the compact layer while remaining in tight contact with their neighbors. In this study, we examined the distribution of N-cadherin (Cdh2) during cardiac trabeculation in zebrafish. By analyzing the localization of a Cdh2-EGFP fusion protein expressed under the control of the zebrafish cdh2 promoter, we initially observed Cdh2-EGFP expression along the lateral sides of embryonic cardiomyocytes, in an evenly distributed pattern, and with the occasional appearance of punctae. Within a few hours, Cdh2-EGFP distribution on the lateral sides of cardiomyocytes evolves into a clear punctate pattern as Cdh2-EGFP molecules outside the punctae cluster to increase the size of these aggregates. In addition, Cdh2-EGFP molecules also appear on the basal side of cardiomyocytes that remain in the compact layer. Delaminating cardiomyocytes accumulate Cdh2-EGFP on the surface facing the basal side of compact layer cardiomyocytes, thereby allowing tight adhesion between these layers. Importantly, we find that blood flow/cardiac contractility is required for the transition from an even distribution of Cdh2-EGFP to the formation of punctae. Furthermore, using time-lapse imaging of beating hearts in conjunction with a Cdh2 tandem fluorescent protein timer transgenic line, we observed that Cdh2-EGFP molecules appear to move from the lateral to the basal side of cardiomyocytes along the cell membrane, and that Erb-b2 receptor tyrosine kinase 2 (Erbb2) function is required for this relocalization. PMID:27339140

  14. Linkage of N-cadherin to multiple cytoskeletal elements revealed by a proteomic approach in hippocampal neurons.

    PubMed

    Tanaka, Hidekazu; Takafuji, Kazuaki; Taguchi, Akihiko; Wiriyasermkul, Pattama; Ohgaki, Ryuichi; Nagamori, Shushi; Suh, Pann-Ghill; Kanai, Yoshikatsu

    2012-07-01

    The CNS synapse is an adhesive junction differentiated for chemical neurotransmission and is equipped with presynaptic vesicles and postsynaptic neurotransmitter receptors. Cell adhesion molecule cadherins not only maintain connections between pre- and postsynaptic membranes but also modulate the efficacy of synaptic transmission. Although the components of the cadherin-mediated adhesive apparatus have been studied extensively in various cell systems, the complete picture of these components, particularly at the synaptic junction, remains elusive. Here, we describe the proteomic assortment of the N-cadherin-mediated synaptic adhesion apparatus in cultured hippocampal neurons. N-cadherin immunoprecipitated from Triton X-100-solubilized neuronal extract contained equal amounts of β- and α-catenins, as well as F-actin-related membrane anchor proteins such as integrins bridged with α-actinin-4, and Na(+)/K(+)-ATPase bridged with spectrins. A close relative of β-catenin, plakoglobin, and its binding partner, desmoplakin, were also found, suggesting that a subset of the N-cadherin-mediated adhesive apparatus also anchors intermediate filaments. Moreover, dynein heavy chain and LEK1/CENPF/mitosin were found. This suggests that internalized pools of N-cadherin in trafficking vesicles are conveyed by dynein motors on microtubules. In addition, ARVCF and NPRAP/neurojungin/δ2-catenin, but not p120ctn/δ1-catenin or plakophilins-1, -2, -3, -4 (p0071), were found, suggesting other possible bridges to microtubules. Finally, synaptic stimulation by membrane depolarization resulted in an increased 93-kDa band, which corresponded to proteolytically truncated β-catenin. The integration of three different classes of cytoskeletal systems found in the synaptic N-cadherin complex may imply a dynamic switching of adhesive scaffolds in response to synaptic activity. PMID:22609377

  15. N-cadherin prodomain cleavage regulates synapse formation in vivo.

    PubMed

    Latefi, Nazlie S; Pedraza, Liliana; Schohl, Anne; Li, Ziwei; Ruthazer, Edward S

    2009-07-01

    Cadherins are initially synthesized bearing a prodomain that is thought to limit adhesion during early stages of biosynthesis. Functional cadherins lack this prodomain, raising the intriguing possibility that cells may utilize prodomain cleavage as a means to temporally or spatially regulate adhesion after delivery of cadherin to the cell surface. In support of this idea, immunostaining for the prodomain of zebrafish N-cadherin revealed enriched labeling at neuronal surfaces at the soma and along axonal processes. To determine whether post-translational cleavage of the prodomain affects synapse formation, we imaged Rohon-Beard cells in zebrafish embryos expressing GFP-tagged wild-type N-cadherin (NCAD-GFP) or a GFP-tagged N-cadherin mutant expressing an uncleavable prodomain (PRON-GFP) rendering it nonadhesive. NCAD-GFP accumulated at synaptic microdomains in a developmentally regulated manner, and its overexpression transiently accelerated synapse formation. PRON-GFP was much more diffusely distributed along the axon and its overexpression delayed synapse formation. Our results support the notion that N-cadherin serves to stabilize pre- to postsynaptic contacts early in synapse development and suggests that regulated cleavage of the N-cadherin prodomain may be a mechanism by which the kinetics of synaptogenesis are regulated. PMID:19365814

  16. N-cadherin is essential for retinal lamination in the zebrafish.

    PubMed

    Erdmann, Bettina; Kirsch, Frank-P; Rathjen, Fritz G; Moré, Margret I

    2003-03-01

    N-cadherin is one of the major Ca(2+)-dependent cell adhesion proteins in the developing nervous system. Here, we analyze eye development in the zebrafish N-cadherin loss-of-function mutant parachute(paR2.10) (pac(paR2.10)). The zebrafish visual system is fully developed by the time pac(paR2.10) mutants show lethality at day 5. Already at 24 hr postfertilization (hpf), mutant retinal cells are more disorganized and more rounded than in wild-type. At later stages, mutant retinae display a severe lamination defect with rosette formation (mostly islands of plexiform layer tissue surrounded by inner nuclear layer or photoreceptor cells), even though all major classes of cell types appear to be present as determined by histology. Of interest, electron microscopy reveals that the islands of plexiform layer tissue contain a normal amount of synapses with normal morphology. Although mutant photoreceptor cells are sometimes deformed, all typical structural components are present, including the membranous discs for rhodopsin storage. The lens fibers of the pac(paR2.10) mutants develop completely normally, but in some cases, lens epithelial cells round up and become multilayered. We conclude that cell adhesion mediated by N-cadherin is of major importance for retinal lamination and involved in maintenance of the lens epithelial sheet, but is not essential for the formation of photoreceptor ultrastructure or for synaptogenesis. PMID:12619142

  17. Effects of N-Cadherin Disruption on Spine Morphological Dynamics

    PubMed Central

    Mysore, Shreesh P.; Tai, Chin-Yin; Schuman, Erin M.

    2007-01-01

    Structural changes at synapses are thought to be a key mechanism for the encoding of memories in the brain. Recent studies have shown that changes in the dynamic behavior of dendritic spines accompany bidirectional changes in synaptic plasticity, and that the disruption of structural constraints at synapses may play a mechanistic role in spine plasticity. While the prolonged disruption of N-cadherin, a key synaptic adhesion molecule, has been shown to alter spine morphology, little is known about the short-term regulation of spine morphological dynamics by N-cadherin. With time-lapse, confocal imaging in cultured hippocampal neurons, we examined the progression of structural changes in spines following an acute treatment with AHAVD, a peptide known to interfere with the function of N-cadherin. We characterized fast and slow timescale spine dynamics (minutes and hours, respectively) in the same population of spines. We show that N-cadherin disruption leads to enhanced spine motility and reduced length, followed by spine loss. The structural effects are accompanied by a loss of functional connectivity. Further, we demonstrate that early structural changes induced by AHAVD treatment, namely enhanced motility and reduced length, are indicators for later spine fate, i.e., spines with the former changes are more likely to be subsequently lost. Our results thus reveal the short-term regulation of synaptic structure by N-cadherin and suggest that some forms of morphological dynamics may be potential readouts for subsequent, stimulus-induced rewiring in neuronal networks. PMID:18946519

  18. Role of N-cadherin in proliferation, migration, and invasion of germ cell tumours

    PubMed Central

    Jarry, Hubertus; Küffer, Stefan; Kaulfuss, Silke; Burfeind, Peter; Strauβ, Arne; Thelen, Paul; Radzun, Heinz Joachim; Ströbel, Philipp; Honecker, Friedemann; Behnes, Carl Ludwig

    2015-01-01

    Germ cell tumors (GCTs) are the most common malignancies in young men. Most patients with GCT can be cured with cisplatin-based combination chemotherapy, even in metastatic disease. In case of therapy resistance, prognosis is usually poor. We investigated the potential of N-cadherin inhibition as a therapeutic strategy. We analyzed the GCT cell lines NCCIT, NTERA-2, TCam-2, and the cisplatin-resistant sublines NCCIT-R and NTERA-2R. Effects of a blocking antibody or siRNA against N-cadherin on proliferation, migration, and invasion were investigated. Mouse xenografts of GCT cell lines were analyzed by immunohistochemistry for N-cadherin expression. All investigated GCT cell lines were found to express N-cadherin protein in vitro and in vivo. Downregulation of N-cadherin in vitro leads to a significant inhibition of proliferation, migration, and invasion. N-cadherin-downregulation leads to a significantly higher level of pERK. N-cadherin-inhibition resulted in significantly higher rates of apoptotic cells in caspase-3 staining. Expression of N-cadherin is preserved in cisplatin-resistant GCT cells, pointing to an important physiological role in cell survival. N-cadherin-downregulation results in a significant decrease of proliferation, migration, and invasion and stimulates apoptosis in cisplatin-naive and resistant GCT cell lines. Therefore, targeting N-cadherin may be a promising therapeutic approach, particularly in cisplatin-resistant, therapy refractory and metastatic GCT. PMID:26451610

  19. A novel o-naphtoquinone inhibits N-cadherin expression and blocks melanoma cell invasion via AKT signaling.

    PubMed

    Montenegro, Raquel Carvalho; de Vasconcellos, Marne Carvalho; Barbosa, Gleyce Dos Santos; Burbano, Rommel M R; Souza, Luciana G S; Lemos, Telma L G; Costa-Lotufo, Letícia V; de Moraes, Manoel Odorico

    2013-10-01

    The down-regulation or loss of epithelial markers is often accompanied by the up-regulation of mesenchymal markers. E-cadherin generally suppresses invasiveness, whereas N-cadherin promotes invasion and metastasis in vitro. The aim of this work is to investigate the role of biflorin, a naphthoquinone with proven anticancer properties, on the expression of N-cadherin and AKT proteins in MDA-MB-435 invasive melanoma cancer cells after 12h of exposure to 1, 2.5 and 5 μM biflorin. Biflorin inhibited MDA-MB-435 invasion in a dose-dependent manner (p<0.01). Likewise, biflorin down-regulated N-cadherin and AKT-1 expression in a dose-dependent manner. Biflorin did not inhibit the adhesion of MDA-MB-435 cells to any tested substrates. Additionally, biflorin blocked the invasiveness of cells by down-regulating N-cadherin, most likely via AKT-1 signaling. As such, biflorin may be a novel anticancer agent and a new prototype for drug design. PMID:23912027

  20. N-cadherin-catenin complexes form prior to cleavage of the proregion and transport to the plasma membrane.

    PubMed

    Wahl, James K; Kim, Young J; Cullen, Janet M; Johnson, Keith R; Wheelock, Margaret J

    2003-05-01

    Cadherins are calcium-dependent glycoproteins that function as cell-cell adhesion molecules and are linked to the actin cytoskeleton via catenins. Newly synthesized cadherins contain a prosequence that must be proteolytically removed to generate a functional adhesion molecule. The goal of this study was to examine the proteolytic processing of N-cadherin and the assembly of the cadherin-catenin complex in cells that express endogenous N-cadherin. A monoclonal antibody specific for the proregion of human N-cadherin was generated and used to examine N-cadherin processing. Our data show that newly synthesized proN-cadherin is phosphorylated and proteolytically processed prior to transport to the plasma membrane. In addition, we show that beta-catenin and plakoglobin associate only with phosphorylated proN-cadherin, whereas p120(ctn) can associate with both phosphorylated and non-phosphorylated proN-cadherin. Immunoprecipitations using anti-proN-cadherin showed that cadherin-catenin complexes are assembled prior to localization at the plasma membrane. These data suggest that a core N-cadherin-catenin complex assembles in the endoplasmic reticulum or Golgi compartment and is transported to the plasma membrane where linkage to the actin cytoskeleton can be established. PMID:12604612

  1. Essential cooperation of N-cadherin and neuroligin-1 in the transsynaptic control of vesicle accumulation.

    PubMed

    Stan, A; Pielarski, K N; Brigadski, T; Wittenmayer, N; Fedorchenko, O; Gohla, A; Lessmann, V; Dresbach, T; Gottmann, K

    2010-06-15

    Cell adhesion molecules are key players in transsynaptic communication, precisely coordinating presynaptic differentiation with postsynaptic specialization. At glutamatergic synapses, their retrograde signaling has been proposed to control presynaptic vesicle clustering at active zones. However, how the different types of cell adhesion molecules act together during this decisive step of synapse maturation is largely unexplored. Using a knockout approach, we show that two synaptic adhesion systems, N-cadherin and neuroligin-1, cooperate to control vesicle clustering at nascent synapses. Live cell imaging and fluorescence recovery after photobleaching experiments at individual synaptic boutons revealed a strong impairment of vesicle accumulation in the absence of N-cadherin, whereas the formation of active zones was largely unaffected. Strikingly, also the clustering of synaptic vesicles triggered by neuroligin-1 overexpression required the presence of N-cadherin in cultured neurons. Mechanistically, we found that N-cadherin acts by postsynaptically accumulating neuroligin-1 and activating its function via the scaffolding molecule S-SCAM, leading, in turn, to presynaptic vesicle clustering. A similar cooperation of N-cadherin and neuroligin-1 was observed in immature CA3 pyramidal neurons in an organotypic hippocampal network. Moreover, at mature synapses, N-cadherin was required for the increase in release probability and miniature EPSC frequency induced by expressed neuroligin-1. This cooperation of two cell adhesion systems provides a mechanism for coupling bidirectional synapse maturation mediated by neuroligin-1 to cell type recognition processes mediated by classical cadherins. PMID:20534458

  2. Identification of plakoglobin domains required for association with N-cadherin and alpha-catenin.

    PubMed

    Sacco, P A; McGranahan, T M; Wheelock, M J; Johnson, K R

    1995-08-25

    Cadherins are calcium-dependent, cell surface glycoproteins involved in cell-cell adhesion. To function in cell-cell adhesion, the transmembrane cadherin molecule must be associated with the cytoskeleton via cytoplasmic proteins known as catenins. Three catenins, alpha-catenin, beta-catenin, and gamma-catenin (also known as plakoglobin), have been identified. The domain of the cadherin molecule important for its interaction with the catenins has been mapped to the COOH-terminal 70 amino acids, but less is known about regions of the catenins that allow them to associate with one another or with the cadherin molecule. In this study we have transfected carboxyl-terminal deletions of plakoglobin into the human fibrosarcoma HT-1080 and used immunofluorescence localization and co-immunoprecipitation to map the regions of plakoglobin that allow it to associate with N-cadherin and with alpha-catenin. Plakoglobin is an armadillo family member containing 13 weakly similar internal repeats. These data show that the alpha-catenin-binding region maps within the first repeat and the N-cadherin-binding region maps within repeats 7 and 8. PMID:7650039

  3. Sphingosine 1-phosphate receptor regulation of N-cadherin mediates vascular stabilization

    PubMed Central

    Paik, Ji-Hye; Skoura, Athanasia; Chae, Sung-Suk; Cowan, Ann E.; Han, David K.; Proia, Richard L.; Hla, Timothy

    2004-01-01

    Vascular stabilization, a process by which nascent vessels are invested with mural cells, is important in angiogenesis. Here we describe the molecular basis of vascular stabilization regulated by sphingosine 1-phosphate (S1P), a platelet-derived lipid mediator. S1P1 receptor-dependent cell-surface trafficking and activation of the cell-cell adhesion molecule N-cadherin is essential for interactions between endothelial and mural cells. Endothelial cell S1P1/Gi/Rac pathway induces microtubule polymerization, resulting in trafficking of N-cadherin to polarized plasma membrane domains. S1P treatment modulated the phosphorylation of N-cadherin as well as p120-catenin and induced the formation of cadherin/catenin/actin complexes containing novel regulatory and trafficking factors. The net result of endothelial cell S1P1 receptor activation is the proper trafficking and strengthening of N-cadherin-dependent cell-cell adhesion with mural cells. Perturbation of N-cadherin expression with small interfering RNA profoundly attenuated vascular stabilization in vitro and in vivo. S1P-induced trafficking and activation of N-cadherin provides a novel mechanism for the stabilization of nascent blood vessels by mural cells and may be exploited to control angiogenesis and vascular diseases. PMID:15371328

  4. MT5-MMP, ADAM-10, and N-Cadherin Act in Concert To Facilitate Synapse Reorganization after Traumatic Brain Injury

    PubMed Central

    Warren, Kelly M.; Reeves, Thomas M.

    2012-01-01

    Abstract Matrix metalloproteinases (MMPs) influence synaptic recovery following traumatic brain injury (TBI). Membrane type 5-matrix metalloproteinase (MT5-MMP) and a distintegrin and metalloproteinase-10 (ADAM-10) are membrane-bound MMPs that cleave N-cadherin, a protein critical to synapse stabilization. This study examined protein and mRNA expression of MT5-MMP, ADAM-10, and N-cadherin after TBI, contrasting adaptive and maladaptive synaptogenesis. The effect of MMP inhibition on MT5-MMP, ADAM-10, and N-cadherin was assessed during maladaptive plasticity and correlated with synaptic function. Rats were subjected to adaptive unilateral entorhinal cortical lesion (UEC) or maladaptive fluid percussion TBI+bilateral entorhinal cortical lesion (TBI+BEC). Hippocampal MT5-MMP and ADAM-10 protein was significantly elevated 2 and 7 days post-injury. At 15 days after UEC, each MMP returned to control level, while TBI+BEC ADAM-10 remained elevated. At 2 and 7 days, N-cadherin protein was below control. By the 15-day synapse stabilization phase, UEC N-cadherin rose above control, a shift not seen for TBI+BEC. At 7 days, increased TBI+BEC ADAM-10 transcript correlated with protein elevation. UEC ADAM-10 mRNA did not change, and no differences in MT5-MMP or N-cadherin mRNA were detected. Confocal imaging showed MT5-MMP, ADAM-10, and N-cadherin localization within reactive astrocytes. MMP inhibition attenuated ADAM-10 protein 15 days after TBI+BEC and increased N-cadherin. This inhibition partially restored long-term potentiation induction, but did not affect paired-pulse facilitation. Our results confirm time- and injury-dependent expression of MT5-MMP, ADAM-10, and N-cadherin during reactive synaptogenesis. Persistent ADAM-10 expression was correlated with attenuated N-cadherin level and reduced functional recovery. MMP inhibition shifted ADAM-10 and N-cadherin toward adaptive expression and improved synaptic function. PMID:22489706

  5. N-cadherin mediated distribution of beta-catenin alters MAP kinase and BMP-2 signaling on chondrogenesis-related gene expression.

    PubMed

    Modarresi, Rozbeh; Lafond, Toulouse; Roman-Blas, Jorge A; Danielson, Keith G; Tuan, Rocky S; Seghatoleslami, M Reza

    2005-05-01

    We have examined the effect of calcium-dependent adhesion, mediated by N-cadherin, on cell signaling during chondrogenesis of multipotential embryonic mouse C3H10T1/2 cells. The activity of chondrogenic genes, type II collagen, aggrecan, and Sox9 were examined in monolayer (non-chondrogenic), and micromass (chondrogenic) cultures of parental C3H10T1/2 cells and altered C3H10T1/2 cell lines that express a dominant negative form of N-cadherin (delta390-T1/2) or overexpress normal N-cadherin (MNCD2-T1/2). Our findings show that missexpression or inhibition of N-cadherin in C3H10T1/2 cells results in temporal and spatial changes in expression of the chondrogenic genes Sox9, aggrecan, and collagen type II. We have also analyzed activity of the serum response factor (SRF), a nuclear target of MAP kinase signaling implicated in chondrogenesis. In semi-confluent monolayer cultures (minimum cell-cell contact) of C3H10T1/2, MNCD2-T1/2, or delta390-T1/2 cells, there was no significant change in the pattern of MAP kinase or bone morphogenetic protein-2 (BMP-2) regulation of SRF. However, in micromass cultures, the effect of MAP kinase and BMP-2 on SRF activity was proportional to the nuclear localization of beta-catenin, a Wnt stabilized cytoplasmic factor that can associate with lymphoid enhancer-binding factor (LEF) to serve as a transcription factor. Our findings suggest that the extent of adherens junction formation mediated by N-cadherin can modulate the potential Wnt-induced nuclear activity of beta-catenin. PMID:15723280

  6. Myosin X regulates neuronal radial migration through interacting with N-cadherin

    PubMed Central

    Lai, Mingming; Guo, Ye; Ma, Jun; Yu, Huali; Zhao, Dongdong; Fan, Wenqiang; Ju, Xingda; Sheikh, Muhammad A.; Malik, Yousra S.; Xiong, Wencheng; Guo, Weixiang; Zhu, Xiaojuan

    2015-01-01

    Proper brain function depends on correct neuronal migration during development, which is known to be regulated by cytoskeletal dynamics and cell-cell adhesion. Myosin X (Myo10), an uncharacteristic member of the myosin family, is an important regulator of cytoskeleton that modulates cell motilities in many different cellular contexts. We previously reported that Myo10 was required for neuronal migration in the developing cerebral cortex, but the underlying mechanism was still largely unknown. Here, we found that knockdown of Myo10 expression disturbed the adherence of migrating neurons to radial glial fibers through abolishing surface Neuronal cadherin (N-cadherin) expression, thereby impaired neuronal migration in the developmental cortex. Next, we found Myo10 interacted with N-cadherin cellular domain through its FERM domain. Furthermore, we found knockdown of Myo10 disrupted N-cadherin subcellular distribution and led to localization of N-cadherin into Golgi apparatus and endosomal sorting vesicle. Taking together, these results reveal a novel mechanism of Myo10 interacting with N-cadherin and regulating its cell-surface expression, which is required for neuronal adhesion and migration. PMID:26347613

  7. Protein mediated membrane adhesion

    NASA Astrophysics Data System (ADS)

    Carlson, Andreas; Mahadevan, L.

    2015-05-01

    Adhesion in the context of mechanical attachment, signaling, and movement in cellular dynamics is mediated by the kinetic interactions between membrane-embedded proteins in an aqueous environment. Here, we present a minimal theoretical framework for the dynamics of membrane adhesion that accounts for the kinetics of protein binding, the elastic deformation of the membrane, and the hydrodynamics of squeeze flow in the membrane gap. We analyze the resulting equations using scaling estimates to characterize the spatiotemporal features of the adhesive patterning and corroborate them using numerical simulations. In addition to characterizing aspects of cellular dynamics, our results might also be applicable to a range of phenomena in physical chemistry and materials science where flow, deformation, and kinetics are coupled to each other in slender geometries.

  8. Conserved alternative splicing and expression patterns of arthropod N-cadherin.

    PubMed

    Hsu, Shu-Ning; Yonekura, Shinichi; Ting, Chun-Yuan; Robertson, Hugh M; Iwai, Youichi; Uemura, Tadashi; Lee, Chi-Hon; Chiba, Akira

    2009-04-01

    Metazoan development requires complex mechanisms to generate cells with diverse function. Alternative splicing of pre-mRNA not only expands proteomic diversity but also provides a means to regulate tissue-specific molecular expression. The N-Cadherin gene in Drosophila contains three pairs of mutually-exclusive alternatively-spliced exons (MEs). However, no significant differences among the resulting protein isoforms have been successfully demonstrated in vivo. Furthermore, while the N-Cadherin gene products exhibit a complex spatiotemporal expression pattern within embryos, its underlying mechanisms and significance remain unknown. Here, we present results that suggest a critical role for alternative splicing in producing a crucial and reproducible complexity in the expression pattern of arthropod N-Cadherin. We demonstrate that the arthropod N-Cadherin gene has maintained the three sets of MEs for over 400 million years using in silico and in vivo approaches. Expression of isoforms derived from these MEs receives precise spatiotemporal control critical during development. Both Drosophila and Tribolium use ME-13a and ME-13b in "neural" and "mesodermal" splice variants, respectively. As proteins, either ME-13a- or ME-13b-containing isoform can cell-autonomously rescue the embryonic lethality caused by genetic loss of N-Cadherin. Ectopic muscle expression of either isoform beyond the time it normally ceases leads to paralysis and lethality. Together, our results offer an example of well-conserved alternative splicing increasing cellular diversity in metazoans. PMID:19343204

  9. Recruitment of β-catenin to N-cadherin is necessary for smooth muscle contraction.

    PubMed

    Wang, Tao; Wang, Ruping; Cleary, Rachel A; Gannon, Olivia J; Tang, Dale D

    2015-04-01

    β-Catenin is a key component that connects transmembrane cadherin with the actin cytoskeleton at the cell-cell interface. However, the role of the β-catenin/cadherin interaction in smooth muscle has not been well characterized. Here stimulation with acetylcholine promoted the recruitment of β-catenin to N-cadherin in smooth muscle cells/tissues. Knockdown of β-catenin by lentivirus-mediated shRNA attenuated smooth muscle contraction. Nevertheless, myosin light chain phosphorylation at Ser-19 and actin polymerization in response to contractile activation were not reduced by β-catenin knockdown. In addition, the expression of the β-catenin armadillo domain disrupted the recruitment of β-catenin to N-cadherin. Force development, but not myosin light chain phosphorylation and actin polymerization, was reduced by the expression of the β-catenin armadillo domain. Furthermore, actin polymerization and microtubules have been implicated in intracellular trafficking. In this study, the treatment with the inhibitor latrunculin A diminished the interaction of β-catenin with N-cadherin in smooth muscle. In contrast, the exposure of smooth muscle to the microtubule depolymerizer nocodazole did not affect the protein-protein interaction. Together, these findings suggest that smooth muscle contraction is mediated by the recruitment of β-catenin to N-cadherin, which may facilitate intercellular mechanotransduction. The association of β-catenin with N-cadherin is regulated by actin polymerization during contractile activation. PMID:25713069

  10. N-Cadherin-Mediated Signaling Regulates Cell Phenotype for Nucleus Pulposus Cells of the Intervertebral Disc

    PubMed Central

    Hwang, Priscilla Y.; Jing, Liufang; Michael, Keith W.; Richardson, William J.; Chen, Jun; Setton, Lori A.

    2015-01-01

    Juvenile nucleus pulposus (NP) cells of the intervertebral disc (IVD) are large, vacuolated cells that form cell clusters with strong cell–cell interactions. With maturation and aging, NP cells lose their ability to form these cell clusters, with aging-associated changes in NP cell phenotype, morphology, and proteoglycan synthesis that may contribute to IVD degeneration. Therefore, it is important to understand the mechanisms governing juvenile NP cell cluster behavior towards the goal of revealing factors that can promote juvenile, healthy NP cell phenotypes. N-cadherin has been identified as a cell–cell adhesion marker that is present in juvenile NP cells, but disappears with age. The goal of this study was to reveal the importance of N-cadherin in regulating cell–cell interactions in juvenile NP cell cluster formation and test for a regulatory role in maintaining a juvenile NP phenotype in vitro. Juvenile porcine IVD cells, of notochordal origin, were promoted to form cell clusters in vitro, and analyzed for preservation of the juvenile NP phenotype. Additionally, cadherin-blocking experiments were performed to prevent cluster formation in order to study the importance of cluster formation in NP cell signaling. Findings reveal N-cadherin-mediated cell–cell contacts promote cell clustering behavior and regulate NP cell matrix production and preservation of NP-specific markers. Inhibition of N-cadherin-mediated contacts resulted in loss of all features of the juvenile NP cell. These results establish a regulatory role for N-cadherin in juvenile NP cells, and suggest that preservation of the N-cadherin mediated cell–cell contact is important for preserving juvenile NP cell phenotype and morphology. PMID:25848407

  11. N-cadherin Determines Individual Variations in the Therapeutic Efficacy of Human Umbilical Cord Blood-derived Mesenchymal Stem Cells in a Rat Model of Myocardial Infarction

    PubMed Central

    Lee, Eun Ju; Choi, Eue-Keun; Kang, Soo Kyoung; Kim, Gi-Hwan; Park, Ju Young; Kang, Hyun-Jae; Lee, Sae-Won; Kim, Keum-Hyun; Kwon, Jin Sook; Lee, Ki Hong; Ahn, Youngkeun; Lee, Ho-Jae; Cho, Hyun-Jai; Choi, Soo Jin; Oh, Won Il; Park, Young-Bae; Kim, Hyo-Soo

    2012-01-01

    In this study, we established and characterized human umbilical cord blood-derived mesenchymal stem cells (hUCB-MSCs) from four different donors. However, the hUCB-MSCs showed remarkable variations in their therapeutic efficacy for repairing rat infarcted myocardium (including the process of angiogenesis) 8 weeks after transplantation. In addition, we observed that the level of vascular endothelial growth factor (VEGF) is correlated with the therapeutic efficacy of the four hUCB-MSCs. Next, to investigate the practical application of hUCB-MSCs, we searched for surface signature molecules that could serve as indicators of therapeutic efficacy. The gene for N-cadherin was the only cell surface gene that was highly expressed in the most effective hUCB-MSCs, both at the transcriptional and translational levels. We observed downregulation and upregulation of VEGF in response to N-cadherin blocking and N-cadherin overexpression, respectively. Activation of extracellular signal-regulated kinase (ERK), but not protein kinase B, was increased when N-cadherin expression was increased, whereas disruption of N-cadherin-mediated cell–cell contact induced suppression of ERK activation and led to VEGF downregulation. Moreover, by investigating hUCB-MSCs overexpressing N-cadherin or N-cadherin knockdown hUCB-MSCs, we confirmed the in vivo function of N-cadherin. In addition, we observed that DiI-labeled hUCB-MSCs express N-cadherin in the peri-infarct area and interact with cardiomyocytes. PMID:22068423

  12. Lateral assembly of N-cadherin drives tissue integrity by stabilizing adherens junctions

    PubMed Central

    Garg, S.; Fischer, S. C.; Schuman, E. M.; Stelzer, E. H. K.

    2015-01-01

    Cadherin interactions ensure the correct registry and anchorage of cells during tissue formation. Along the plasma membrane, cadherins form inter-junctional lattices via cis- and trans-dimerization. While structural studies have provided models for cadherin interactions, the molecular nature of cadherin binding in vivo remains unexplored. We undertook a multi-disciplinary approach combining live cell imaging of three-dimensional cell assemblies (spheroids) with a computational model to study the dynamics of N-cadherin interactions. Using a loss-of-function strategy, we demonstrate that each N-cadherin interface plays a distinct role in spheroid formation. We found that cis-dimerization is not a prerequisite for trans-interactions, but rather modulates trans-interfaces to ensure tissue stability. Using a model of N-cadherin junction dynamics, we show that the absence of cis-interactions results in low junction stability and loss of tissue integrity. By quantifying the binding and unbinding dynamics of the N-cadherin binding interfaces, we determined that mutating either interface results in a 10-fold increase in the dissociation constant. These findings provide new quantitative information on the steps driving cadherin intercellular adhesion and demonstrate the role of cis-interactions in junction stability. PMID:25589573

  13. Adhesives from modified soy protein

    DOEpatents

    Sun, Susan; Wang, Donghai; Zhong, Zhikai; Yang, Guang

    2008-08-26

    The, present invention provides useful adhesive compositions having similar adhesive properties to conventional UF and PPF resins. The compositions generally include a protein portion and modifying ingredient portion selected from the group consisting of carboxyl-containing compounds, aldehyde-containing compounds, epoxy group-containing compounds, and mixtures thereof. The composition is preferably prepared at a pH level at or near the isoelectric point of the protein. In other preferred forms, the adhesive composition includes a protein portion and a carboxyl-containing group portion.

  14. E-cadherin, N-cadherin Expression and Histologic Characterization of Canine Choroid Plexus Tumors.

    PubMed

    Reginato, A; Girolami, D; Menchetti, L; Foiani, G; Mandara, M T

    2016-07-01

    Choroid plexus tumors (CPTs) are reported with an increasing incidence in dogs, and they call for a reexamination of histologic features and criteria of classification corresponding to their biological behavior. In this study, the human World Health Organization classification was applied to 16 canine CPTs, and the expression of molecules involved in neoplastic cell adhesion (E-cadherin, N-cadherin), invasion (doublecortin), and proliferation (Ki-67) was investigated. Mitotic index was found to be the main criterion for grading CPTs. Cell density and multilayering of papillae were also statistically associated with histologic grade. Intraventricular spread and parenchymal invasion was observed for tumors showing histologic benign features. E-cadherin was expressed in all CPT grades, independent of tumor invasion. N-cadherin immunolabeling was more expressed in grade I than high-grade CPTs, whereas doublecortin expression was not detected in CPTs. An increasing proliferative activity was observed in relation with histologic grade. PMID:26792846

  15. Glioma stem cell invasion through regulation of the interconnected ERK, integrin α6 and N-cadherin signaling pathway.

    PubMed

    Velpula, Kiran Kumar; Rehman, Azeem A; Chelluboina, Bharath; Dasari, Venkata Ramesh; Gondi, Christopher S; Rao, Jasti S; Veeravalli, Krishna Kumar

    2012-11-01

    The recent characterization of glioma stem cells (GSCs) prompts a necessary examination of the signaling pathways that facilitate invasiveness. Molecular crosstalk between expression mechanisms has been identified in a range of cancers, including glioblastoma multiforme. However, hardly any literature exists that addresses whether cancer stem cells utilize these same interconnected pathways. Protein factors commonly implicated in malignant tumors include extracellular signal-regulated kinase (ERK), N-cadherin, and integrin α6. Although studies have reported the molecular crosstalk involved among these proteins, the present study illustrates the importance of the ERK transduction pathway in N-cadherin and integrin α6 regulated invasion in GSCs. Conversely, the data also suggests that GSCs rely on N-cadherin and integrin α6 interaction to regulate ERK signaling. Moreover, confocal visualization revealed the co-localization of N-cadherin and integrin α6 in GSCs and clinical surgical biopsies extracted from glioma patients. Interestingly, ERK knockdown reduced this co-localization. Upon co-culturing GSCs with human umbilical cord blood stem cells (hUCBSCs), we observed a subsequent decrease in pERK, N-cadherin and integrin α6 expression. In addition, co-culturing hUCBSCs with GSCs decreased co-localization of N-cadherin and integrin α6 in GSCs. Our results demonstrate the dynamic interplay among ERK, N-cadherin and integrin α6 in GSC invasion and also reveal the therapeutic potential of hUCBSCs in treating the molecular crosstalk observed in GSC-regulated invasion. PMID:22789454

  16. Opposite Roles of Furin and PC5A in N-Cadherin Processing12

    PubMed Central

    Maret, Deborah; Sadr, Mohamad Seyed; Sadr, Emad Seyed; Colman, David R; Del Maestro, Rolando F; Seidah, Nabil G

    2012-01-01

    We recently demonstrated that lack of Furin-processing of the N-cadherin precursor (proNCAD) in highly invasive melanoma and brain tumor cells results in the cell-surface expression of a nonadhesive protein favoring cell migration and invasion in vitro. Quantitative polymerase chain reaction analysis of malignant human brain tumor cells revealed that of all proprotein convertases (PCs) only the levels of Furin and PC5A are modulated, being inversely (Furin) or directly (PC5A) correlated with brain tumor invasive capacity. Intriguingly, the N-terminal sequence following the Furin-activated NCAD site (RQKR↓DW161, mouse nomenclature) reveals a second putative PC-processing site (RIRSDR↓DK189) located in the first extracellular domain. Cleavage at this site would abolish the adhesive functions of NCAD because of the loss of the critical Trp161. This was confirmed upon analysis of the fate of the endogenous prosegment of proNCAD in human malignant glioma cells expressing high levels of Furin and low levels of PC5A (U343) or high levels of PC5A and negligible Furin levels (U251). Cellular analyses revealed that Furin is the best activating convertase releasing an ∼17-kDa prosegment, whereas PC5A is the major inactivating enzyme resulting in the secretion of an ∼20-kDa product. Like expression of proNCAD at the cell surface, cleavage of the NCAD molecule at RIRSDR↓DK189 renders the U251 cancer cells less adhesive to one another and more migratory. Our work modifies the present view on posttranslational processing and surface expression of classic cadherins and clarifies how NCAD possesses a range of adhesive potentials and plays a critical role in tumor progression. PMID:23097623

  17. Hydrogels functionalized with N-cadherin mimetic peptide enhance osteogenesis of hMSCs by emulating the osteogenic niche.

    PubMed

    Zhu, Meiling; Lin, Sien; Sun, Yuxin; Feng, Qian; Li, Gang; Bian, Liming

    2016-01-01

    N-cadherin is considered to be the key factor in directing cell-cell interactions during mesenchymal condensation, which is essential to osteogenesis. In this study, hyaluronic acid (HA) hydrogels are biofunctionalized with an N-cadherin mimetic peptide to mimic the pro-osteogenic niche in the endosteal space to promote the osteogenesis of human mesenchymal stem cells (hMSCs). Results show that the conjugation of the N-cadherin peptide in the HA hydrogels enhances the expression of the osteogenic marker genes in the seeded hMSCs. Furthermore, the biofunctionalized HA hydrogels promote the alkaline phosphatase activity, type I collagen deposition, and matrix mineralization by the seeded hMSCs under both in vitro and in vivo condition. We postulate that the biofunctionalized hydrogels emulates the N-cadherin-mediated homotypic cell-cell adhesion among MSCs and the "orthotypic" interaction between the osteoblasts and MSCs. These findings demonstrate that the biofunctionalized HA hydrogels provide a supportive niche microenvironment for the osteogenesis of hMSCs. PMID:26580785

  18. Myocardial connexin-43 and N-Cadherin decrease during vanadium inhalation.

    PubMed

    Fortoul, Teresa I; Soto-Mota, Adrian; Rojas-Lemus, Marcela; Rodriguez-Lara, Vianey; Gonzalez-Villalva, Adriana; Montaño, Luis F; Paez, Araceli; Colin-Barenque, Laura; López-Valdez, Nelly; Cano-Gutiérrez, Gumaro; Bizarro-Nevares, Patricia; Ustarroz-Cano, Martha

    2016-04-01

    Particulate matter air pollution has considerably increased during the last decades; vanadium is a transition element adhered to this particulate matter, and the combustion of fossil fuels is the main source in the atmosphere. It has been reported that air pollution and specifically vanadium exposure increases the probability of suffering arrhythmias; however the biological mechanism of such a relationship remains unknown. It has been established that a diminished presence of N-Cadherin alters the Connexin-43 arrangement, and the consequent altered presence of these proteins predisposes to ventricular heart rate problems. We analyzed myocardial histology and the expression of N-Cadherin and Connexin-43 by immunohistochemistry in mouse that inhaled vanadium. Our results showed a significant and progressive reduction in both N-Cadherin and Connexin-43, as well as the presence of meganucleus; myofibrils disruption, and clumping in the exposed groups were also observed. Our findings add more information about a possible explanation for the arrythmogenic effect observed in dwellers of cities with high particulate matter atmospheric pollution. PMID:26568576

  19. Identification of two structural types of calcium-dependent adhesion molecules in the chicken embryo.

    PubMed Central

    Crittenden, S L; Rutishauser, U; Lilien, J

    1988-01-01

    By using an immunological and peptide mapping approach two calcium-dependent cell-cell adhesion molecules (calCAMs) in the embryonic chicken are compared. A third closely related molecule is identified and compared to the two calCAMs. One of the calCAMs appears to be identical to the previously identified adhesion molecule N-cadherin, originally identified in chicken retina and localized to neural tissues. The second is the same as L-CAM, originally identified in chicken liver but localized to a variety of epithelial tissues. The third, also found in liver, is similar to L-CAM but is much closer in structure to N-cadherin. It is, however, immunologically distinct from N-cadherin. We therefore refer to this newly identified molecule as CRM-L for cadherin-related molecule in liver. CRM-L, N-cadherin, and L-CAM are all cell-surface proteins with a similar stability to tryptic digestion in the presence of calcium. CRM-L has the same molecular mass and isoelectric point as N-cadherin but is distinct from L-CAM in these properties. Two-dimensional peptide maps of complete tryptic digests reveal that CRM-L shares 69% of its peptides with N-cadherin and 20% with L-CAM. On the basis of these data, we suggest that there are at least two distinguishable types of calCAMs in the chicken embryo: one represented by the closely related molecules N-cadherin and CRM-L, and another represented by L-CAM. Images PMID:3368455

  20. Cortical interneurons migrating on a pure substrate of N-cadherin exhibit fast synchronous centrosomal and nuclear movements and reduced ciliogenesis

    PubMed Central

    Luccardini, Camilla; Leclech, Claire; Viou, Lucie; Rio, Jean-Paul; Métin, Christine

    2015-01-01

    The embryonic development of the cortex involves a phase of long distance migration of interneurons born in the basal telencephalon. Interneurons first migrate tangentially and then reorient their trajectories radially to enter the developing cortex. We have shown that migrating interneurons can assemble a primary cilium, which maintains the centrosome to the plasma membrane and processes signals to control interneuron trajectory (Baudoin et al., 2012). In the developing cortex, N-cadherin is expressed by migrating interneurons and by cells in their migratory pathway. N-cadherin promotes the motility and maintains the polarity of tangentially migrating interneurons (Luccardini et al., 2013). Because N-cadherin is an important factor that regulates the migration of medial ganglionic eminence (MGE) cells in vivo, we further characterized the motility and polarity of MGE cells on a substrate that only comprises this protein. MGE cells migrating on a N-cadherin substrate were seven times faster than on a laminin substrate and two times faster than on a substrate of cortical cells. A primary cilium was much less frequently observed on MGE cells migrating on N-cadherin than on laminin. Nevertheless, the mature centriole (MC) frequently docked to the plasma membrane in MGE cells migrating on N-cadherin, suggesting that plasma membrane docking is a basic feature of the centrosome in migrating MGE cells. On the N-cadherin substrate, centrosomal and nuclear movements were remarkably synchronous and the centrosome remained near the nucleus. Interestingly, MGE cells with cadherin invalidation presented centrosomal movements no longer coordinated with nuclear movements. In summary, MGE cells migrating on a pure substrate of N-cadherin show fast, coordinated nuclear and centrosomal movements, and rarely present a primary cilium. PMID:26283922

  1. Surface Expression of Precursor N-cadherin Promotes Tumor Cell Invasion12

    PubMed Central

    Maret, Deborah; Gruzglin, Eugenia; Sadr, Mohamad Seyed; Siu, Vincent; Shan, Weisong; Koch, Alexander W; Seidah, Nabil G; Del Maestro, Rolando F; Colman, David R

    2010-01-01

    The expression of N-cadherin (NCAD) has been shown to correlate with increased tumor cell motility and metastasis. However, NCAD-mediated adhesion is a robust phenomenon and therefore seems to be inconsistent with the “release” from intercellular adhesion required for invasion. We show that in the most invasive melanoma and brain tumor cells, altered posttranslational processing results in abundant nonadhesive precursor N-cadherin (proNCAD) at the cell surface, although total NCAD levels remain constant. We demonstrate that aberrantly processed proNCAD promotes cell migration and invasion in vitro. Furthermore, in human tumor specimens, we find high levels of proNCAD as well, supporting an overall conclusion that proNCAD and mature NCAD coexist on these tumor cell surfaces and that it is the ratio between these functionally antagonistic moieties that directly correlates with invasion potential. Our work provides insight into what may be a widespread mechanism for invasion and metastasis and challenges the current dogma of the functional roles played by classic cadherins in tumor progression. PMID:21170270

  2. Differential Function of N-Cadherin and Cadherin-7 in the Control of Embryonic Cell Motility

    PubMed Central

    Dufour, Sylvie; Beauvais-Jouneau, Alice; Delouvée, Annie; Thiery, Jean Paul

    1999-01-01

    Similar amounts of N-cadherin and cadherin-7, the prototypes of type I and type II cadherin, induced cell-cell adhesion in murine sarcoma 180 transfectants, Ncad-1 and cad7-29, respectively. However, in the initial phase of aggregation, Ncad-1 cells aggregated more rapidly than cad7-29 cells. Isolated Ncad-1 and cad7-29 cells adhered and spread in a similar manner on fibronectin (FN), whereas aggregated cad7-29 cells were more motile and dispersed than aggregated Ncad-1 cells. cad7-29 cells established transient contacts with their neighbors which were stabilized if FN-cell interactions were perturbed. In contrast, Ncad-1 cells remained in close contact when they migrated on FN. Both β-catenin and cadherin were more rapidly downregulated in cad7-29 than in Ncad-1 cells treated with cycloheximide, suggesting a higher turnover rate for cadherin-7–mediated cell-cell contacts than for those mediated by N-cadherin. The extent of FN-dependent focal adhesion kinase phosphorylation was much lower if the cells had initiated N-cadherin–mediated rather than cadherin-7–mediated cell adhesion before plating. On grafting into the embryo, Ncad-1 cells did not migrate and remained at or close to the graft site, even after 48 h, whereas grafted cad7-29 cells dispersed efficiently into embryonic structures. Thus, the adhesive phenotype of cadherin-7–expressing cells is regulated by the nature of the extracellular matrix environment which also controls the migratory behavior of the cells. In addition, adhesions mediated by different cadherins differentially regulate FN-dependent signaling. The transient contacts specifically observed in cadherin- 7–expressing cells may also be important in the control of cell motility. PMID:10427101

  3. N-Cadherin Aided in Maintaining the Characteristics of Leukemic Stem Cells.

    PubMed

    Zhi, Lei; Gao, Ying; Yu, Chunyan; Zhang, Yi; Zhang, Bo; Yang, Jie; Yao, Zhi

    2016-07-01

    In our previous study, it has been revealed that N-cadherin(+) and leukemic stem cells (LSCs, CD34(+) /CD38(-) /CD123(+) ) could be enriched by chemotherapy because of their resistance to chemotherapy. In this study, we found that N-cadherin mRNA was highly expressed in the bone marrow mononuclear cells (BMMNCs) of patients with t(8;21) translocation. To determine the role of N-cadherin in maintaining LSCs self-renewal and stationary properties, colony-forming assay, cell cycle analysis, and engraftment in nonobese diabetic/severe combined immunodeficiency (NOD/SCID) mice were used to compare N-cadherin(+) and N-cadherin(-) cells. Both leukemic cell lines KG1a and CD34(+) /CD38(-) BMMNCs derived from acute myeloid leukemia patients were used, and cells were divided into N-cadherin(+) and N-cadherin(-) fraction after sorting by FACS. The results showed that N-cadherin(+) cells had remarkable increased numbers of colonies with cytokines stimulation when compared with the negative control, suggesting a higher proliferative capacity of N-cadherin(+) cells with cytokines stimulation. The results also showed that most cells in N-cadherin(+) fraction stayed in the G0 -G1 stage, indicating the involvement of N-cadherin in maintaining the quiescent state of LSCs in niche. The results of engraftment showed that there was a higher proportion of hCD45(+) cells in mice transplanted with N-cadherin(+) cells than N-cadherin(-) cells. In addition, it was obvious that NOD/SCID mice transplanted with N-cadherin(+) cells had a shorter lifetime than the negative control, suggesting that LSCs self-renewal capacity resides predominantly in N-cadherin(+) fraction. In summary, N-cadherin might play an important role in maintaining the self-renewal and stationary properties of LSCs. Anat Rec, 299:990-998, 2016. © 2016 Wiley Periodicals, Inc. PMID:27064800

  4. Alterations in cell adhesion proteins and cardiomyopathy

    PubMed Central

    Li, Jifen

    2014-01-01

    Cell adhesive junction is specialized intercellular structure composed of cell adhesion proteins. They are essential to connect adjacent heart muscle cell and make heart contraction effectively and properly. Clinical and genetic studies have revealed close relationship between cell adhesive proteins and the occurrence of various cardiomyopathies. Here we will review recent development on the disease phenotype, potential cellular and molecular mechanism related to cell adhesion molecules, with particular disease pathogenesis learned from genetic manipulated murine models. PMID:24944760

  5. Loss of N-Cadherin Expression in Tumor Transplants Produced From As+3- and Cd+2-Transformed Human Urothelial (UROtsa) Cell Lines

    PubMed Central

    Sandquist, Elizabeth J.; Somji, Seema; Dunlevy, Jane R.; Garrett, Scott H.; Zhou, Xu Dong; Slusser-Nore, Andrea

    2016-01-01

    Background Epithelial to mesenchymal transition is a process in which a cell experiences a loss of epithelial cell characteristics and acquires a more mesenchymal cell phenotype. In cancer, epithelial to mesenchymal transition has been proposed to play an important role during specific stages of tumor progression. The role epithelial to mesenchymal transition and mesenchymal to epithelial transition might play in toxicant-induced urothelial cancer is unknown. Methods Real-time PCR, Western blotting, immuno-histochemistry and immuno-fluorescence were used to determine the expression of E- and N-cadherin in the UROtsa parent, the As+3- and Cd+2-transformed cell lines, the spheroids isolated from these cell lines as well as the tumor heterotransplants that were produced by the injection of the transformed cells into immune compromised mice. Results This study showed that N-cadherin expression was increased in 6 As+3- and 7 Cd+2- transformed cell lines generated from human urothelial cells (UROtsa). The expression varied within each cell line, with 10% to 95% of the cells expressing N-cadherin. Tumors produced from these cell lines showed no expression of the N-cadherin protein. Spheroids which are made up of putative cancer initiating cells produced from these cell lines showed only background expression of N-cadherin mRNA, increased expression of aldehyde dehydrogenase 1 mRNA and produced tumors which did not express N-cadherin. There was no change in the expression of E-cadherin in the tumors, and the tumors formed by all the As+3 and Cd+2-transformed cell lines and cancer initiating cells stained intensely and uniformly for E-cadherin. Conclusions The finding that the cells expressing N-cadherin gave rise to tumors with no expression of N-cadherin is in agreement with the classical view of epithelial to mesenchymal transition. Epithelial to mesenchymal transition and N-cadherin are associated with dissemination and not with the ability to establish new tumor growth

  6. Hyaluronan suppresses prostate tumor cell proliferation through diminished expression of N-cadherin and aberrant growth factor receptor signaling

    SciTech Connect

    Bharadwaj, Alamelu G.; Goodrich, Nathaniel P.; McAtee, Caitlin O.; Haferbier, Katie; Oakley, Gregory G.; Wahl, James K.; Simpson, Melanie A.

    2011-05-01

    Hyaluronan (HA) production has been functionally implicated in prostate tumorigenesis and metastasis. We previously used prostate tumor cells overexpressing the HA synthesizing enzyme HAS3 or the clinically relevant hyaluronidase Hyal1 to show that excess HA production suppresses tumor growth, while HA turnover accelerates spontaneous metastasis from the prostate. Here, we examined pathways responsible for effects of HAS3 and Hyal1 on tumor cell phenotype. Detailed characterization of cell cycle progression revealed that expression of Hyal1 accelerated cell cycle re-entry following synchronization, whereas HAS3 alone delayed entry. Hyal1 expressing cells exhibited a significant reduction in their ability to sustain ERK phosphorylation upon stimulation by growth factors, and in their expression of the cyclin-dependent kinase inhibitor p21. In contrast, HAS3 expressing cells showed prolonged ERK phosphorylation and increased expression of both p21 and p27, in asynchronous and synchronized cultures. Changes in cell cycle regulatory proteins were accompanied by HA-induced suppression of N-cadherin, while E-cadherin expression and {beta}-catenin expression and distribution remained unchanged. Our results are consistent with a model in which excess HA synthesis suppresses cell proliferation by promoting homotypic E-cadherin mediated cell-cell adhesion, consequently signaling to elevate cell cycle inhibitor expression and suppress G1- to S-phase transition.

  7. Plakoglobin Reduces the in vitro Growth, Migration and Invasion of Ovarian Cancer Cells Expressing N-Cadherin and Mutant p53

    PubMed Central

    Alaee, Mahsa; Danesh, Ghazal; Pasdar, Manijeh

    2016-01-01

    Aberrant expression of cadherins and catenins plays pivotal roles in ovarian cancer development and progression. Plakoglobin (PG, γ-catenin) is a paralog of β-catenin with dual adhesive and signaling functions. While β-catenin has known oncogenic function, PG generally acts as a tumor/metastasis suppressor. We recently showed that PG interacted with p53 and that its growth/metastasis inhibitory function may be mediated by this interaction. Very little is known about the role of PG in ovarian cancer. Here, we investigated the in vitro tumor/metastasis suppressor effects of PG in ovarian cancer cell lines with mutant p53 expression and different cadherin profiles. We showed that the N-cadherin expressing and E-cadherin and PG deficient ES-2 cells were highly migratory and invasive, whereas OV-90 cells that express E-cadherin, PG and very little/no N-cadherin were not. Exogenous expression of PG or E-cadherin or N-cadherin knockdown in ES-2 cells (ES-2-E-cad, ES-2-PG and ES-2-shN-cad) significantly reduced their migration and invasion. Also, PG expression or N-cadherin knockdown significantly decreased ES-2 cells growth. Furthermore, PG interacted with both cadherins and with wild type and mutant p53 in normal ovarian and ES-2-PG cell lines, respectively. PMID:27144941

  8. An occludin-focal adhesion kinase protein complex at the blood-testis barrier: a study using the cadmium model.

    PubMed

    Siu, Erica R; Wong, Elissa W P; Mruk, Dolores D; Sze, K L; Porto, Catarina S; Cheng, C Yan

    2009-07-01

    Several integral membrane proteins that constitute the blood-testis barrier (BTB) in mammalian testes, in particular rodents, are known to date. These include tight junction (TJ) proteins (e.g. occludin, junctional adhesion molecule-A, claudins), basal ectoplasmic specialization proteins (e.g. N-cadherin), and gap junction proteins (e.g. connexin43). However, the regulators (e.g. protein kinases and phosphatases) that affect these proteins, such as their interaction with the cytoskeletal actin, which in turn confer cell adhesion at the TJ, remain largely unknown. We report herein that focal adhesion kinase (FAK) is a putative interacting partner of occludin, but not claudin-11 or junctional adhesion molecule-A. Immunohistochemistry and fluorescence microscopy studies illustrated that the expression of FAK in the seminiferous epithelium of adult rat testes was stage specific. FAK colocalized with occludin at the BTB in virtually all stages of the seminiferous epithelial cycle but considerably diminished in stages VIII-IX, at the time of BTB restructuring to facilitate the transit of primary leptotene spermatocytes. Using Sertoli cells cultured in vitro with established TJ-permeability barrier and ultrastructures of TJ, basal ectoplasmic specialization and desmosome-like junction that mimicked the BTB in vivo, FAK was shown to colocalize with occludin and zonula occludens-1 (ZO-1) at the Sertoli-Sertoli cell interface. When these Sertoli cell cultures were treated with CdCl(2) to perturb the TJ-barrier function, occludin underwent endocytic-mediated internalization in parallel with FAK and ZO-1. Thus, these findings demonstrate that FAK is an integrated regulatory component of the occludin-ZO-1 protein complex, suggesting that functional studies can be performed to study the role of FAK in BTB dynamics. PMID:19213829

  9. An Occludin-Focal Adhesion Kinase Protein Complex at the Blood-Testis Barrier: A Study Using the Cadmium Model

    PubMed Central

    Siu, Erica R.; Wong, Elissa W. P.; Mruk, Dolores D.; Sze, K. L.; Porto, Catarina S.; Cheng, C. Yan

    2009-01-01

    Several integral membrane proteins that constitute the blood-testis barrier (BTB) in mammalian testes, in particular rodents, are known to date. These include tight junction (TJ) proteins (e.g. occludin, junctional adhesion molecule-A, claudins), basal ectoplasmic specialization proteins (e.g. N-cadherin), and gap junction proteins (e.g. connexin43). However, the regulators (e.g. protein kinases and phosphatases) that affect these proteins, such as their interaction with the cytoskeletal actin, which in turn confer cell adhesion at the TJ, remain largely unknown. We report herein that focal adhesion kinase (FAK) is a putative interacting partner of occludin, but not claudin-11 or junctional adhesion molecule-A. Immunohistochemistry and fluorescence microscopy studies illustrated that the expression of FAK in the seminiferous epithelium of adult rat testes was stage specific. FAK colocalized with occludin at the BTB in virtually all stages of the seminiferous epithelial cycle but considerably diminished in stages VIII–IX, at the time of BTB restructuring to facilitate the transit of primary leptotene spermatocytes. Using Sertoli cells cultured in vitro with established TJ-permeability barrier and ultrastructures of TJ, basal ectoplasmic specialization and desmosome-like junction that mimicked the BTB in vivo, FAK was shown to colocalize with occludin and zonula occludens-1 (ZO-1) at the Sertoli-Sertoli cell interface. When these Sertoli cell cultures were treated with CdCl2 to perturb the TJ-barrier function, occludin underwent endocytic-mediated internalization in parallel with FAK and ZO-1. Thus, these findings demonstrate that FAK is an integrated regulatory component of the occludin-ZO-1 protein complex, suggesting that functional studies can be performed to study the role of FAK in BTB dynamics. PMID:19213829

  10. Control of the collective migration of enteric neural crest cells by the Complement anaphylatoxin C3a and N-cadherin.

    PubMed

    Broders-Bondon, Florence; Paul-Gilloteaux, Perrine; Gazquez, Elodie; Heysch, Julie; Piel, Matthieu; Mayor, Roberto; Lambris, John D; Dufour, Sylvie

    2016-06-01

    We analyzed the cellular and molecular mechanisms governing the adhesive and migratory behavior of enteric neural crest cells (ENCCs) during their collective migration within the developing mouse gut. We aimed to decipher the role of the complement anaphylatoxin C3a during this process, because this well-known immune system attractant has been implicated in cephalic NCC co-attraction, a process controlling directional migration. We used the conditional Ht-PA-cre transgenic mouse model allowing a specific ablation of the N-cadherin gene and the expression of a fluorescent reporter in migratory ENCCs without affecting the central nervous system. We performed time-lapse videomicroscopy of ENCCs from control and N-cadherin mutant gut explants cultured on fibronectin (FN) and micropatterned FN-stripes with C3a or C3aR antagonist, and studied cell migration behavior with the use of triangulation analysis to quantify cell dispersion. We performed ex vivo gut cultures with or without C3aR antagonist to determine the effect on ENCC behavior. Confocal microscopy was used to analyze the cell-matrix adhesion properties. We provide the first demonstration of the localization of the complement anaphylatoxin C3a and its receptor on ENCCs during their migration in the embryonic gut. C3aR receptor inhibition alters ENCC adhesion and migration, perturbing directionality and increasing cell dispersion both in vitro and ex vivo. N-cadherin-null ENCCs do not respond to C3a co-attraction. These findings indicate that C3a regulates cell migration in a N-cadherin-dependent process. Our results shed light on the role of C3a in regulating collective and directional cell migration, and in ganglia network organization during enteric nervous system ontogenesis. The detection of an immune system chemokine in ENCCs during ENS development may also shed light on new mechanisms for gastrointestinal disorders. PMID:27041467

  11. Molecular mechanics of mussel adhesion proteins

    NASA Astrophysics Data System (ADS)

    Qin, Zhao; Buehler, Markus J.

    2014-01-01

    Mussel foot protein (mfp), a natural glue produced by marine mussel, is an intriguing material because of its superior ability for adhesion in various environments. For example, a very small amount of this material is sufficient to affix a mussel to a substrate in water, providing structural support under extreme forces caused by the dynamic effects of waves. Towards a more complete understanding of its strength and underwater workability, it is necessary to understand the microscropic mechanisms by which the protein structure interacts with various substrates. However, none of the mussel proteins' structure is known, preventing us from directly using atomistic modeling to probe their structural and mechanical properties. Here we use an advanced molecular sampling technique to identify the molecular structures of two mussel foot proteins (mfp-3 and mfp-5) and use those structures to study their mechanics of adhesion, which is then incorporated into a continuum model. We calculate the adhesion energy of the mussel foot protein on a silica substrate, compute the adhesion strength based on results obtained from molecular modeling, and compare with experimental data. Our results show good agreement with experimental measurements, which validates the multiscale model. We find that the molecular structure of the folded mussel foot protein (ultimately defined by its genetic sequence) favors strong adhesion to substrates, where L-3,4-dihydroxyphenylalanine (or DOPA) protein subunits work in a cooperative manner to enhance adhesion. Our experimental data suggests a peak attachment force of 0.4±0.1 N, which compares favorably with the prediction from the multiscale model of Fc=0.21-0.33 N. The principles learnt from those results could guide the fabrication of new interfacial materials (e.g. composites) to integrate organic with inorganic surfaces in an effective manner.

  12. The planar cell polarity protein Vangl2 is involved in postsynaptic compartmentalization.

    PubMed

    Nagaoka, Tadahiro; Kishi, Masashi

    2016-01-26

    The excitatory postsynaptic region of the vertebrate hippocampus is usually compartmentalized into the postsynaptic density (PSD) and N-cadherin-rich domain, which is important for synaptic adhesion. However, the molecular mechanisms underlying the compartment formation are unknown. In the present report, we show that the planar cell polarity (PCP) protein Van Gogh-like 2 (Vangl2) plays a role in this regionalization. In cultured rat hippocampal neurons that were subjected to Vangl2 expression silencing, the formed clusters of PSD-95, one of the major scaffolding proteins in PSD, tended to overlap with those of N-cadherin. Further, in the dendrites of these neurons, the immunofluorescence of PSD-95 was to some extent diffused, without a significant change in the total signal. Because Vangl2 physically interacts with both PSD-95 and N-cadherin in vivo, these results suggest that a PCP-related direct molecular mechanism underlies the horizontal polarization of the postsynaptic regions. PMID:26683906

  13. A self-renewing division of zebrafish Müller glial cells generates neuronal progenitors that require N-cadherin to regenerate retinal neurons

    PubMed Central

    Nagashima, Mikiko; Barthel, Linda K.; Raymond, Pamela A.

    2013-01-01

    Müller glia function as retinal stem cells in adult zebrafish. In response to loss of retinal neurons, Müller glia partially dedifferentiate, re-express neuroepithelial markers and re-enter the cell cycle. We show that the immunoglobulin superfamily adhesion molecule Alcama is a novel marker of multipotent retinal stem cells, including injury-induced Müller glia, and that each Müller glial cell divides asymmetrically only once to produce an Alcama-negative, proliferating retinal progenitor. The initial mitotic division of Müller glia involves interkinetic nuclear migration, but mitosis of retinal progenitors occurs in situ. Rapidly dividing retinal progenitors form neurogenic clusters tightly associated with Alcama/N-cadherin-labeled Müller glial radial processes. Genetic suppression of N-cadherin function interferes with basal migration of retinal progenitors and subsequent regeneration of HuC/D+ inner retinal neurons. PMID:24154521

  14. Soy and cottonseed protein blends as wood adhesives

    Technology Transfer Automated Retrieval System (TEKTRAN)

    As an environmentally friendlier alternative to adhesives from petroleum feedstock, soy proteins are currently being formulated as wood adhesives. Cottonseed proteins have also been found to provide good adhesive properties. In at least some cases, cottonseed proteins appear to form greater shear ...

  15. Angiomotin like-1 is a novel component of the N-cadherin complex affecting endothelial/pericyte interaction in normal and tumor angiogenesis

    PubMed Central

    Zheng, Yujuan; Zhang, Yuanyuan; Barutello, Giuseppina; Chiu, Kungchun; Arigoni, Maddalena; Giampietro, Costanza; Cavallo, Federica; Holmgren, Lars

    2016-01-01

    Transmission of mechanical force via cell junctions is an important component that molds cells into shapes consistent with proper organ function. Of particular interest are the cadherin transmembrane proteins, which play an essential role in connecting cell junctions to the intra-cellular cytoskeleton. Understanding how these biomechanical complexes orchestrate intrinsic and extrinsic forces is important for our understanding of the underlying mechanisms driving morphogenesis. We have previously identified the Amot protein family, which are scaffold proteins that integrate polarity, junctional, and cytoskeletal cues to modulate cellular shape in endothelial as well as epithelial cells. In this report, we show that AmotL1 is a novel partner of the N-cadherin protein complex. We studied the role of AmotL1 in normal retinal as well as tumor angiogenesis using inducible endothelial-specific knock-out mice. We show that AmotL1 is essential for normal establishment of vascular networks in the post-natal mouse retina as well as in a transgenic breast cancer model. The observed phenotypes were consistent with a non-autonomous pericyte defect. We show that AmotL1 forms a complex with N-cadherin present on both endothelial cells and pericytes. We propose that AmotL1 is an essential effector of the N-cadherin mediated endothelial/pericyte junctional complex. PMID:27464479

  16. N-cadherin regulates primary motor axon growth and branching during zebrafish embryonic development.

    PubMed

    Brusés, Juan L

    2011-06-15

    N-cadherin is a classical type I cadherin that contributes to the formation of neural circuits by regulating growth cone migration and the formation of synaptic contacts. This study analyzed the role of N-cadherin in primary motor axons growth during development of the zebrafish (Danio rerio) embryo. After exiting the spinal cord, primary motor axons migrate ventrally through a common pathway and form the first neuromuscular junction with the muscle pioneer cells located at the horizontal myoseptum, which serves as a choice point for cell-type-specific pathway selection. Analysis of N-cadherin mutants (cdh2(hi3644Tg) ) and embryos injected with N-cadherin antisense morpholinos showed primary motor axons extending aberrant axonal branches at the choice point in ∼40% of the somitic hemisegments and an ∼150% increase in the number of branches per axon length within the ventral myotome. Analysis of individual axons trajectories showed that the caudal (CaP) and rostral (RoP) motor neurons axons formed aberrant branches at the choice point that abnormally extended in the rostrocaudal axis and ventrally to the horizontal myoseptum. Expression of a dominant-interfering N-cadherin cytoplasmic domain in primary motor neurons caused some axons to stall abnormally at the horizontal myoseptum and to impair their migration into the ventral myotome. However, in N-cadherin-depleted embryos, the majority of primary motor axons innervated their appropriate myotomal territories, indicating that N-cadherin regulates motor axon growth and branching without severely affecting the mechanisms that control axonal target selection. PMID:21452216

  17. N-cadherin regulates primary motor axons growth and branching during zebrafish embryonic development

    PubMed Central

    Brusés, Juan L

    2013-01-01

    N-cadherin is a classical type I cadherin that contributes to the formation of neural circuits by regulating growth cone migration and the formation of synaptic contacts. This study analyzed the role of N-cadherin in primary motor axons growth during development of the zebrafish (Danio rerio) embryo. After exiting the spinal cord, primary motor axons migrate ventrally through a common pathway and form the first neuromuscular junction with the muscle pioneer cells located at the horizontal myoseptum, which serves as a choice point for cell-type specific pathway selection. Analysis of N-cadherin mutants (cdh2hi3644Tg) and embryos injected with N-cadherin antisense morpholinos showed primary motor axons extending aberrant axonal branches at the choice point in ~40% of the somitic hemisegments, and an ~150% increase in the number of branches per axon length within the ventral myotome. Analysis of individual axons trajectories showed that the caudal (CaP) and rostral (RoP) motor neurons axons formed aberrant branches at the choice point which abnormally extended in the rostrocaudal axis and ventrally to the horizontal myoseptum. Expression of a dominant-interfering N-cadherin cytoplasmic domain in primary motor neurons caused some axons to abnormally stall at the horizontal myoseptum and to impair their migration into the ventral myotome. However, in N-cadherin depleted embryos the majority of primary motor axons innervated their appropriate myotomal territories indicating that N-cadherin regulates motor axon growth and branching without severely affecting the mechanisms that control axonal target selection. PMID:21452216

  18. How to develop globular proteins into adhesives.

    PubMed

    van der Leeden, M C; Rutten, A A; Frens, G

    2000-05-26

    To make globular proteins suitable for application in adhesives, the specific bonds and interactions which shape their structure have to broken. Only then, a layer of relatively large, flexible and interwoven polymer chains, which are firmly attached to the solid surface by adsorption, can be created. Such a network layer is essential to save the adhesive bond under an applied force, because it can distribute the concentration of stresses generated at the interface into the bulk. Unfolding and swelling of a protein can be achieved by changing the solvent quality. For the globular whey protein beta-lactoglobulin, the optimal conditions for unfolding and swelling is found with 98% formic acid as a solvent. In formic acid, beta-lactoglobulin looses its amphoteric character (it is protonated, probably for approximately 20%). In addition, formic acid is less polar than water and thus a better solvent for the apolar parts of the protein. The swelling and unfolding behaviour of beta-lactoglobulin is studied by viscosity and CD-spectroscopy measurements. For the interpretation of the results we apply the Kuhn formalism that the conformation of a protein can be described in terms of a statistical chain which consists of segments of an average persistence length P. The statistical segment length P, which varies with the experimental conditions, is directly related to the adsorption energy required for a strong adhesion between coil and surface. It determines the depletion energy kT P(-2) m(-2) which must be overcome by specific attraction between side groups of the protein chain and the surface. For beta-lactoglobulin in 98% formic acid, we find a P value of approximately 2.2 nm, pointing at a relatively flexible chain. The minimum net adsorption energy kT P(-2) is then approximately 1 mJ m(-2), a relatively small value to be exceeded. Preliminary results of destructive adhesion tests on beech wood lap-shear joints reveal promising tensile strengths of approximately 2

  19. N-cadherin deficiency impairs pericyte recruitment, and not endothelial differentiation or sprouting, in embryonic stem cell-derived angiogenesis

    SciTech Connect

    Tillet, Emmanuelle . E-mail: emmanuelle.tillet@cea.fr; Vittet, Daniel; Feraud, Olivier; Moore, Robert; Kemler, Rolf; Huber, Philippe

    2005-11-01

    Endothelial cells express two classical cadherins, VE-cadherin and N-cadherin. VE-cadherin is absolutely required for vascular morphogenesis, but N-cadherin is thought to participate in vessel stabilization by interacting with periendothelial cells during vessel formation. However, recent data suggest a more critical role for N-cadherin in endothelium that would regulate angiogenesis, in part by controlling VE-cadherin expression. In this study, we have assessed N-cadherin function in vascular development using an in vitro model derived from embryonic stem (ES) cell differentiation. We show that pluripotent ES cells genetically null for N-cadherin can differentiate normally into endothelial cells. In addition, sprouting angiogenesis was unaltered, suggesting that N-cadherin is not essential for the early events of angiogenesis. However, the lack of N-cadherin led to an impairment in pericyte covering of endothelial outgrowths. We conclude that N-cadherin is necessary neither for vasculogenesis nor proliferation and migration of endothelial cells but is required for the subsequent maturation of endothelial sprouts by interacting with pericytes.

  20. Mussel adhesion is dictated by time-regulated secretion and molecular conformation of mussel adhesive proteins

    PubMed Central

    Petrone, Luigi; Kumar, Akshita; Sutanto, Clarinda N.; Patil, Navinkumar J.; Kannan, Srinivasaraghavan; Palaniappan, Alagappan; Amini, Shahrouz; Zappone, Bruno; Verma, Chandra; Miserez, Ali

    2015-01-01

    Interfacial water constitutes a formidable barrier to strong surface bonding, hampering the development of water-resistant synthetic adhesives. Notwithstanding this obstacle, the Asian green mussel Perna viridis attaches firmly to underwater surfaces via a proteinaceous secretion (byssus). Extending beyond the currently known design principles of mussel adhesion, here we elucidate the precise time-regulated secretion of P. viridis mussel adhesive proteins. The vanguard 3,4-dihydroxy-L-phenylalanine (Dopa)-rich protein Pvfp-5 acts as an adhesive primer, overcoming repulsive hydration forces by displacing surface-bound water and generating strong surface adhesion. Using homology modelling and molecular dynamics simulations, we find that all mussel adhesive proteins are largely unordered, with Pvfp-5 adopting a disordered structure and elongated conformation whereby all Dopa residues reside on the protein surface. Time-regulated secretion and structural disorder of mussel adhesive proteins appear essential for optimizing extended nonspecific surface interactions and byssus' assembly. Our findings reveal molecular-scale principles to help the development of wet-resistant adhesives. PMID:26508080

  1. Mussel adhesion is dictated by time-regulated secretion and molecular conformation of mussel adhesive proteins.

    PubMed

    Petrone, Luigi; Kumar, Akshita; Sutanto, Clarinda N; Patil, Navinkumar J; Kannan, Srinivasaraghavan; Palaniappan, Alagappan; Amini, Shahrouz; Zappone, Bruno; Verma, Chandra; Miserez, Ali

    2015-01-01

    Interfacial water constitutes a formidable barrier to strong surface bonding, hampering the development of water-resistant synthetic adhesives. Notwithstanding this obstacle, the Asian green mussel Perna viridis attaches firmly to underwater surfaces via a proteinaceous secretion (byssus). Extending beyond the currently known design principles of mussel adhesion, here we elucidate the precise time-regulated secretion of P. viridis mussel adhesive proteins. The vanguard 3,4-dihydroxy-L-phenylalanine (Dopa)-rich protein Pvfp-5 acts as an adhesive primer, overcoming repulsive hydration forces by displacing surface-bound water and generating strong surface adhesion. Using homology modelling and molecular dynamics simulations, we find that all mussel adhesive proteins are largely unordered, with Pvfp-5 adopting a disordered structure and elongated conformation whereby all Dopa residues reside on the protein surface. Time-regulated secretion and structural disorder of mussel adhesive proteins appear essential for optimizing extended nonspecific surface interactions and byssus' assembly. Our findings reveal molecular-scale principles to help the development of wet-resistant adhesives. PMID:26508080

  2. Mussel adhesion is dictated by time-regulated secretion and molecular conformation of mussel adhesive proteins

    NASA Astrophysics Data System (ADS)

    Petrone, Luigi; Kumar, Akshita; Sutanto, Clarinda N.; Patil, Navinkumar J.; Kannan, Srinivasaraghavan; Palaniappan, Alagappan; Amini, Shahrouz; Zappone, Bruno; Verma, Chandra; Miserez, Ali

    2015-10-01

    Interfacial water constitutes a formidable barrier to strong surface bonding, hampering the development of water-resistant synthetic adhesives. Notwithstanding this obstacle, the Asian green mussel Perna viridis attaches firmly to underwater surfaces via a proteinaceous secretion (byssus). Extending beyond the currently known design principles of mussel adhesion, here we elucidate the precise time-regulated secretion of P. viridis mussel adhesive proteins. The vanguard 3,4-dihydroxy-L-phenylalanine (Dopa)-rich protein Pvfp-5 acts as an adhesive primer, overcoming repulsive hydration forces by displacing surface-bound water and generating strong surface adhesion. Using homology modelling and molecular dynamics simulations, we find that all mussel adhesive proteins are largely unordered, with Pvfp-5 adopting a disordered structure and elongated conformation whereby all Dopa residues reside on the protein surface. Time-regulated secretion and structural disorder of mussel adhesive proteins appear essential for optimizing extended nonspecific surface interactions and byssus' assembly. Our findings reveal molecular-scale principles to help the development of wet-resistant adhesives.

  3. Investigation of modified cottonseed protein adhesives for wood composites

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Several modified cottonseed protein isolates were studied and compared to corresponding soy protein isolates for their adhesive properties when bonded to wood composites. Modifications included treatments with alkali, guanidine hydrochloride, sodium dodecyl sulfate (SDS), and urea. Wood composites...

  4. Adhesion of mussel foot proteins to different substrate surfaces

    PubMed Central

    Lu, Qingye; Danner, Eric; Waite, J. Herbert; Israelachvili, Jacob N.; Zeng, Hongbo; Hwang, Dong Soo

    2013-01-01

    Mussel foot proteins (mfps) have been investigated as a source of inspiration for the design of underwater coatings and adhesives. Recent analysis of various mfps by a surface forces apparatus (SFA) revealed that mfp-1 functions as a coating, whereas mfp-3 and mfp-5 resemble adhesive primers on mica surfaces. To further refine and elaborate the surface properties of mfps, the force–distance profiles of the interactions between thin mfp (i.e. mfp-1, mfp-3 or mfp-5) films and four different surface chemistries, namely mica, silicon dioxide, polymethylmethacrylate and polystyrene, were measured by an SFA. The results indicate that the adhesion was exquisitely dependent on the mfp tested, the substrate surface chemistry and the contact time. Such studies are essential for understanding the adhesive versatility of mfps and related/similar adhesion proteins, and for translating this versatility into a new generation of coatings and (including in vivo) adhesive materials. PMID:23173195

  5. Soy protein isolate molecular level contributions to bulk adhesive properties

    NASA Astrophysics Data System (ADS)

    Shera, Jeanne Norton

    Increasing environmental awareness and the recognized health hazards of formaldehyde-based resins has prompted a strong demand for environmentally-responsible adhesives for wood composites. Soy protein-based adhesives have been shown to be commercially viable with 90-day shelf stability and composite physical properties comparable to those of commercial formaldehyde-based particleboards. The main research focus is to isolate and characterize the molecular level features in soy protein isolate responsible for providing mechanical properties, storage stability, and water resistance during adhesive formulation, processing, and wood composite fabrication. Commercial composite board will be reviewed to enhance our understanding of the individual components and processes required for particleboard production. The levels of protein structure will be defined and an overview of current bio-based technology will be presented. In the process, the logic for utilizing soy protein as a sole binder in the adhesive will be reinforced. Variables such as adhesive components, pH, divalent ions, blend aging, protein molecular weight, formulation solids content, and soy protein functionalization will relate the bulk properties of soy protein adhesives to the molecular configuration of the soybean protein. This work has demonstrated that when intermolecular beta-sheet interactions and protein long-range order is disrupted, viscosity and mechanical properties decrease. Storage stability can be maintained through the stabilization of intermolecular beta-sheet interactions. When molecular weight is reduced through enzymatic digestion, long-range order is disrupted and viscosity and mechanical properties decrease accordingly. Processibility and physical properties must be balanced to increase solids while maintaining low viscosity, desirable mechanical properties, and adequate storage stability. The structure of the soybean protein must be related to the particleboard bulk mechanical

  6. N-cadherin{sup +} HSCs in fetal liver exhibit higher long-term bone marrow reconstitution activity than N-cadherin{sup -} HSCs

    SciTech Connect

    Toyama, Hirofumi; Arai, Fumio; Hosokawa, Kentaro; Ikushima, Yoshiko Matsumoto; Suda, Toshio

    2012-11-23

    Highlights: Black-Right-Pointing-Pointer High N-cad expression was detected in E12.5 mouse FL LT-HSCs (EPCR{sup +} LSK cells). Black-Right-Pointing-Pointer Immunohistochemically, N-cad{sup +} HSCs co-localized with sinusoidal ECs (Lyve-1{sup +} cells) in E12.5 FL, but these gradually detached in E15.5 and E18.5 FL. Black-Right-Pointing-Pointer N-cad{sup +} LSK cells in E12.5 FL exhibited higher LTR activity versus N-cad{sup -} LSK cells, which decreased in E15.5 and E18.5. Black-Right-Pointing-Pointer N-cad expression may confer high LTR activity to HSCs by facilitating interactions with the perisinusoidal niche in FL. -- Abstract: Adult hematopoietic stem cells (HSCs) are maintained in a microenvironment known as the stem cell niche. The regulation of HSCs in fetal liver (FL) and their niche, however, remains to be elucidated. In this study, we investigated the role of N-cadherin (N-cad) in the maintenance of HSCs during FL hematopoiesis. By using anti-N-cad antibodies (Abs) produced by our laboratory, we detected high N-cad expression in embryonic day 12.5 (E12.5) mouse FL HSCs, but not in E15.5 and E18.5 FL. Immunofluorescence staining revealed that N-cad{sup +}c-Kit{sup +} and N-cad{sup +} endothelial protein C receptor (EPCR){sup +} HSCs co-localized with Lyve-1{sup +} sinusoidal endothelial cells (ECs) in E12.5 FL and that some of these cells also expressed N-cad. However, N-cad{sup +} HSCs were also observed to detach from the perisinusoidal niche at E15.5 and E18.5, concomitant with a down-regulation of N-cad and an up-regulation of E-cadherin (E-cad) in hepatic cells. Moreover, EPCR{sup +} long-term (LT)-HSCs were enriched in the N-cad{sup +}Lin{sup -}Sca-1{sup +}c-Kit{sup +} (LSK) fraction in E12.5 FL, but not in E15.5 or E18.5 FL. In a long-term reconstitution (LTR) activity assay, higher engraftment associated with N-cad{sup +} LSK cells versus N-cad{sup -} LSK cells in E12.5 FL when transplanted into lethally irradiated recipient mice. However, the

  7. c-Yes regulates cell adhesion at the apical ectoplasmic specialization-blood-testis barrier axis via its effects on protein recruitment and distribution

    PubMed Central

    Xiao, Xiang; Mruk, Dolores D.

    2013-01-01

    During spermatogenesis, extensive restructuring takes place at the cell-cell interface since developing germ cells migrate progressively from the basal to the adluminal compartment of the seminiferous epithelium. Since germ cells per se are not motile cells, their movement relies almost exclusively on the Sertoli cell. Nonetheless, extensive exchanges in signaling take place between these cells in the seminiferous epithelium. c-Yes, a nonreceptor protein tyrosine kinase belonging to the Src family kinases (SFKs) and a crucial signaling protein, was recently shown to be upregulated at the Sertoli cell-cell interface at the blood-testis barrier (BTB) at stages VIII–IX of the seminiferous epithelial cycle of spermatogenesis. It was also highly expressed at the Sertoli cell-spermatid interface known as apical ectoplasmic specialization (apical ES) at stage V to early stage VIII of the epithelial cycle during spermiogenesis. Herein, it was shown that the knockdown of c-Yes by RNAi in vitro and in vivo affected both Sertoli cell adhesion at the BTB and spermatid adhesion at the apical ES, causing a disruption of the Sertoli cell tight junction-permeability barrier function, germ cell loss from the seminiferous epithelium, and also a loss of spermatid polarity. These effects were shown to be mediated by changes in distribution and/or localization of adhesion proteins at the BTB (e.g., occludin, N-cadherin) and at the apical ES (e.g., nectin-3) and possibly the result of changes in the underlying actin filaments at the BTB and the apical ES. These findings implicate that c-Yes is a likely target of male contraceptive research. PMID:23169788

  8. Spatial distribution of proteins in the quagga mussel adhesive apparatus.

    PubMed

    Rees, David J; Hanifi, Arash; Manion, Joseph; Gantayet, Arpita; Sone, Eli D

    2016-01-01

    The invasive freshwater mollusc Dreissena bugensis (quagga mussel) sticks to underwater surfaces via a proteinacious 'anchor' (byssus), consisting of a series of threads linked to adhesive plaques. This adhesion results in the biofouling of crucial underwater industry infrastructure, yet little is known about the proteins responsible for the adhesion. Here the identification of byssal proteins extracted from freshly secreted byssal material is described. Several new byssal proteins were observed by gel electrophoresis. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry was used to characterize proteins in different regions of the byssus, particularly those localized to the adhesive interface. Byssal plaques and threads contain in common a range of low molecular weight proteins, while several proteins with higher mass were observed only in the plaque. At the adhesive interface, a plaque-specific ~8.1 kDa protein had a relative increase in signal intensity compared to the bulk of the plaque, suggesting it may play a direct role in adhesion. PMID:26825294

  9. Intrinsic Surface-Drying Properties of Bio-adhesive Proteins

    PubMed Central

    Akdogan, Yasar; Wei, Wei; Huang, Kuo-Ying; Kageyama, Yoshiyuki; Danner, Eric W.; Miller, Dusty R.; Martinez Rodriguez, Nadine R.; Herbert Waite, J.

    2014-01-01

    Sessile marine mussels must “dry” underwater surfaces before adhering to them. Synthetic adhesives have yet to overcome this fundamental challenge. Previous studies of bio-inspired adhesion have largely been performed under applied compressive forces but these are poor predictors of an adhesive’s ability to spontaneously penetrate surface hydration layers. In a force-free approach to measuring molecular-level interaction via the surface water diffusivity, different mussel foot proteins were found to have differential abilities to evict hydration layers from the surfaces—a necessary step for adsorption and adhesion. It was anticipated that Dopa would mediate dehydration given its efficacy forbio-inspired wet adhesion. Instead, hydrophobic side-chains are found to be a critical component in bringing about protein-surface intimacy. This is the first direct measurement of interfacial water dynamics during force-free adsorptive interactions at solid surfaces, and offers guidance for engineering wet adhesives and coatings. PMID:25168789

  10. Control of the collective migration of enteric neural crest cells by the Complement anaphylatoxin C3a and N-cadherin

    PubMed Central

    Broders-Bondon, Florence; Paul-Gilloteaux, Perrine; Gazquez, Elodie; Heysch, Julie; Piel, Matthieu; Mayor, Roberto; Lambris, John D.; Dufour, Sylvie

    2016-01-01

    We analyzed the cellular and molecular mechanisms governing the adhesive and migratory behavior of enteric neural crest cells (ENCCs) during their collective migration within the developing mouse gut. We aimed to decipher the role of the complement anaphylatoxin C3a during this process, because this well-known immune system attractant has been implicated in cephalic NCC co-attraction, a process controlling directional migration. We used the conditional Ht-PA-cre transgenic mouse model allowing a specific ablation of the N-cadherin gene and the expression of a fluorescent reporter in migratory ENCCs without affecting the central nervous system. We performed time-lapse videomicroscopy of ENCCs from control and N-cad-herin mutant gut explants cultured on fibronectin (FN) and micropatterned FN-stripes with C3a or C3aR antagonist, and studied cell migration behavior with the use of triangulation analysis to quantify cell dispersion. We performed ex vivo gut cultures with or without C3aR antagonist to determine the effect on ENCC behavior. Confocal microscopy was used to analyze the cell-matrix adhesion properties. We provide the first demonstration of the localization of the complement anaphylatoxin C3a and its receptor on ENCCs during their migration in the embryonic gut. C3aR receptor inhibition alters ENCC adhesion and migration, perturbing directionality and increasing cell dispersion both in vitro and ex vivo. N-cad-herin-null ENCCs do not respond to C3a co-attraction. These findings indicate that C3a regulates cell migration in a N-cadherin-dependent process. Our results shed light on the role of C3a in regulating collective and directional cell migration, and in ganglia network organization during enteric nervous system ontogenesis. The detection of an immune system chemokine in ENCCs during ENS development may also shed light on new mechanisms for gastrointestinal disorders. PMID:27041467

  11. Characteristics of bladder transitional cell carcinoma with E-cadherin and N-cadherin double-negative expression

    PubMed Central

    LUO, YANG; ZHU, YONG-TONG; MA, LI-LI; PANG, SHI-YU; WEI, LI-JIE; LEI, CHENG-YONG; HE, CHENG-WU; TAN, WAN-LONG

    2016-01-01

    The aim of the present study was to examine the characteristics of bladder transitional cell carcinoma with E-cadherin and N-cadherin double-negative expression. An immunofluorescence assay was used to detect E-cadherin and N-cadherin expression in infiltrative bladder cancer tissues, and immunofluorescence and western blot analysis were used to detect E-cadherin and N-cadherin expression in human urinary bladder grade II carcinoma 5637, transitional cell carcinoma UMUC-3 and invasive bladder carcinoma EJ cells. Cell proliferation, migration, invasion and plate colony formation assays were used to detect the proliferative, migratory and invasive abilities and the efficiency of plate colony formation of 5637, UMUC3 and EJ cells. A tumor xenograft formation assay was used to evaluate the tumorigenic abilities of 5637, UMUC-3 and EJ cells in vivo. E-cadherin and N-cadherin double-negative expression was identified in various pathological grades of infiltrative bladder cancers. E-cadherin positive and N-cadherin negative expression was exhibited by 5637 cells. By contrast, E-cadherin negative and N-cadherin positive expression was exhibited by EJ cells, and E-cadherin and N-cadherin double-negative expression was exhibited by UMUC-3 cells. The ability of cells to proliferate, migrate, invade, and the efficiency of plate colony formation and tumorigenic abilities of the cells were significantly different among 5637, UMUC-3 and EJ cells. These cell characteristics were significantly increased in UMUC-3 cells compared with 5637 cells; however, the characteristics were significantly decreased compared with EJ cells. The biological characteristics of bladder cancer cells with E-cadherin and N-cadherin double-negative expression was between bladder cancer cells that exhibited a E-cadherin positive and N-cadherin negative expression, and bladder cancer cells that exhibited E-cadherin negative and N-cadherin positive expression. The present study deduces that the status of E

  12. Mussel-mimetic protein-based adhesive hydrogel.

    PubMed

    Kim, Bum Jin; Oh, Dongyeop X; Kim, Sangsik; Seo, Jeong Hyun; Hwang, Dong Soo; Masic, Admir; Han, Dong Keun; Cha, Hyung Joon

    2014-05-12

    Hydrogel systems based on cross-linked polymeric materials which could provide both adhesion and cohesion in wet environment have been considered as a promising formulation of tissue adhesives. Inspired by marine mussel adhesion, many researchers have tried to exploit the 3,4-dihydroxyphenylalanine (DOPA) molecule as a cross-linking mediator of synthetic polymer-based hydrogels which is known to be able to achieve cohesive hardening as well as adhesive bonding with diverse surfaces. Beside DOPA residue, composition of other amino acid residues and structure of mussel adhesive proteins (MAPs) have also been considered important elements for mussel adhesion. Herein, we represent a novel protein-based hydrogel system using DOPA-containing recombinant MAP. Gelation can be achieved using both oxdiation-induced DOPA quinone-mediated covalent and Fe(3+)-mediated coordinative noncovalent cross-linking. Fe(3+)-mediated hydrogels show deformable and self-healing viscoelastic behavior in rheological analysis, which is also well-reflected in bulk adhesion strength measurement. Quinone-mediated hydrogel has higher cohesive strength and can provide sufficient gelation time for easier handling. Collectively, our newly developed MAP hydrogel can potentially be used as tissue adhesive and sealant for future applications. PMID:24650082

  13. Polymer adhesion at surfaces: biological adhesive proteins and their synthetic mimics

    NASA Astrophysics Data System (ADS)

    Messersmith, Phillip

    2008-03-01

    Mussels are famous for their ability to permanently adhere to a wide variety of wet surfaces, such as rocks, metal and polymer ship hulls, and wood structures. They accomplish this through specialized proteins collectively referred to as mussel adhesive proteins (MAPs). The biophysical aspects of MAP adhesion is being revealed through the use of single molecule force measurements. The results provide insight into the adhesive roles of key amino acids found in these proteins, including the magnitude of adhesive forces, cooperative effects, and their self-healing properties. This molecular-level information is being incorporated into designs of biomimetic polymer coatings for a variety of applications. Our biomimetic approach to polymer design will be illustrated by a few examples where adhesive constituents found in MAPs are exploited to make wet-adhesive polymer coatings. In addition, small molecule analogs of MAPs can be used to apply thin functional films onto virtually any material surface using a facile approach. These coatings have a variety of potential uses in microelectronics, water treatment, prevention of environmental biofouling, and for control of biointerfacial phenomena at the surfaces of medical/diagnostic devices.

  14. Novel protein-repellent dental adhesive containing 2-methacryloyloxyethyl phosphorylcholine

    PubMed Central

    Zhang, Ning; Melo, Mary Anne S.; Bai, Yuxing; Xu, Hockin H. K.

    2015-01-01

    Objectives Biofilms at tooth-restoration margins can produce acids and cause secondary caries. A protein-repellent adhesive resin can potentially inhibition bacteria attachment and biofilm growth. However, there has been no report on protein-repellent dental resins. The objectives of this study were to develop a protein-repellent bonding agent incorporating 2-methacryloyloxyethyl phosphorylcholine (MPC), and to investigate its resistance to protein adsorption and biofilm growth for the first time. Methods MPC was incorporated into Scotchbond Multi-Purpose (SBMP) at 0%, 3.75%, 7.5%, 11.25%, and 15% by mass. Extracted human teeth were used to measure dentin shear bond strengths. Protein adsorption onto resins was determined by a micro bicinchoninic acid (BCA) method. A dental plaque microcosm biofilm model with human saliva as inoculum was used to measure biofilm metabolic activity and colony-forming unit (CFU) counts. Results Adding 7.5% MPC into primer and adhesive did not decrease the dentin bond strength, compared to control (p > 0.1). Incorporation of 7.5% of MPC achieved the lowest protein adsorption, which was 20-fold less than that of control. Incorporation of 7.5% of MPC greatly reduced bacterial adhesion, yielding biofilm total microorganism, total streptococci, and mutans streptococci CFU that were an order of magnitude less than control. Conclusions A protein-repellent dental adhesive resin was developed for the first time. Incorporation of MPC into primer and adhesive at 7.5% by mass greatly reduced the protein adsorption and bacterial adhesion, without compromising the dentin bond strength. The novel protein-repellent primer and adhesive are promising to inhibit biofilm formation and acid production, to protect the tooth-restoration margins and prevent secondary caries. PMID:25234652

  15. The focal adhesion protein PINCH-1 associates with EPLIN at integrin adhesion sites

    PubMed Central

    Karaköse, Esra; Geiger, Tamar; Flynn, Kevin; Lorenz-Baath, Katrin; Zent, Roy; Mann, Matthias; Fässler, Reinhard

    2015-01-01

    ABSTRACT PINCH-1 is a LIM-only domain protein that forms a ternary complex with integrin-linked kinase (ILK) and parvin (to form the IPP complex) downstream of integrins. Here, we demonstrate that PINCH-1 (also known as Lims1) gene ablation in the epidermis of mice caused epidermal detachment from the basement membrane, epidermal hyperthickening and progressive hair loss. PINCH-1-deficient keratinocytes also displayed profound adhesion, spreading and migration defects in vitro that were substantially more severe than those of ILK-deficient keratinocytes indicating that PINCH-1 also exerts functions in an ILK-independent manner. By isolating the PINCH-1 interactome, the LIM-domain-containing and actin-binding protein epithelial protein lost in neoplasm (EPLIN, also known as LIMA1) was identified as a new PINCH-1-associated protein. EPLIN localized, in a PINCH-1-dependent manner, to integrin adhesion sites of keratinocytes in vivo and in vitro and its depletion severely attenuated keratinocyte spreading and migration on collagen and fibronectin without affecting PINCH-1 levels in focal adhesions. Given that the low PINCH-1 levels in ILK-deficient keratinocytes were sufficient to recruit EPLIN to integrin adhesions, our findings suggest that PINCH-1 regulates integrin-mediated adhesion of keratinocytes through the interactions with ILK as well as EPLIN. PMID:25609703

  16. ADP Ribosylation Factor 6 Regulates Neuronal Migration in the Developing Cerebral Cortex through FIP3/Arfophilin-1-dependent Endosomal Trafficking of N-cadherin.

    PubMed

    Hara, Yoshinobu; Fukaya, Masahiro; Hayashi, Kanehiro; Kawauchi, Takeshi; Nakajima, Kazunori; Sakagami, Hiroyuki

    2016-01-01

    During neural development, endosomal trafficking controls cell shape and motility through the polarized transport of membrane proteins related to cell-cell and cell-extracellular matrix interactions. ADP ribosylation factor 6 (Arf6) is a critical small GTPase that regulates membrane trafficking between the plasma membrane and endosomes. We herein demonstrated that the knockdown of endogenous Arf6 in mouse cerebral cortices led to impaired neuronal migration in the intermediate zone and cytoplasmic retention of N-cadherin and syntaxin12 in migrating neurons. Rescue experiments with separation-of-function Arf6 mutants identified Rab11 family-interacting protein 3 (FIP3)/Arfophilin-1, a dual effector for Arf6 and Rab11, as a downstream effector of Arf6 in migrating neurons. The knockdown of FIP3 led to impaired neuronal migration in the intermediate zone and cytoplasmic retention of N-cadherin in migrating neurons, similar to that of Arf6, which could be rescued by the coexpression of wild-type FIP3 but not FIP3 mutants lacking the binding site for Arf6 or Rab11. These results suggest that Arf6 regulates cortical neuronal migration in the intermediate zone through the FIP3-dependent endosomal trafficking. PMID:27622210

  17. ADP Ribosylation Factor 6 Regulates Neuronal Migration in the Developing Cerebral Cortex through FIP3/Arfophilin-1-dependent Endosomal Trafficking of N-cadherin

    PubMed Central

    Hara, Yoshinobu; Fukaya, Masahiro

    2016-01-01

    Abstract During neural development, endosomal trafficking controls cell shape and motility through the polarized transport of membrane proteins related to cell–cell and cell–extracellular matrix interactions. ADP ribosylation factor 6 (Arf6) is a critical small GTPase that regulates membrane trafficking between the plasma membrane and endosomes. We herein demonstrated that the knockdown of endogenous Arf6 in mouse cerebral cortices led to impaired neuronal migration in the intermediate zone and cytoplasmic retention of N-cadherin and syntaxin12 in migrating neurons. Rescue experiments with separation-of-function Arf6 mutants identified Rab11 family-interacting protein 3 (FIP3)/Arfophilin-1, a dual effector for Arf6 and Rab11, as a downstream effector of Arf6 in migrating neurons. The knockdown of FIP3 led to impaired neuronal migration in the intermediate zone and cytoplasmic retention of N-cadherin in migrating neurons, similar to that of Arf6, which could be rescued by the coexpression of wild-type FIP3 but not FIP3 mutants lacking the binding site for Arf6 or Rab11. These results suggest that Arf6 regulates cortical neuronal migration in the intermediate zone through the FIP3-dependent endosomal trafficking. PMID:27622210

  18. Thrombomodulin reduces tumorigenic and metastatic potential of lung cancer cells by up-regulation of E-cadherin and down-regulation of N-cadherin expression.

    PubMed

    Zheng, Nana; Huo, Zihe; Zhang, Bin; Meng, Mei; Cao, Zhifei; Wang, Zhiwei; Zhou, Quansheng

    2016-08-01

    Thrombomodulin (TM) is an endothelial cell membrane protein and plays critical roles in anti-thrombosis, anti-inflammation, vascular endothelial protection, and is traditionally regarded as a "vascular protection god". In recent years, although TM has been reported to be down-regulated in a variety of malignant tumors including lung cancer, the role and mechanism of TM in lung cancer are enigmatic. In this study, we found that induction of TM overexpression by cholesterol-reducing drug atorvastatin significantly diminished the tumorigenic capability of the lung cancer cells. Moreover, we demonstrated that TM overexpression caused G0/G1 phase arrest and markedly reduced the colony forming capability of the cells. Furthermore, overexpression of TM inhibited cell migration and invasion. Consistently, depletion of TM promoted cell growth, reduced the cell population at the G0/G1 phase, and enhanced cell migratory ability. Mechanistic study revealed that TM up-regulated E-cadherin but down-regulated N-cadherin expression, resulting in reversal of epithelial-mesenchymal transition (EMT) in the lung cancer cells. Moreover, silencing TM expression led to decreased E-cadherin and increased N-cadherin. Taken together, our study suggests that TM functions as a tumor suppressive protein, providing a conceptual framework for inducing TM overexpression as a sensible strategy and approach for novel anti-lung cancer drug discovery. PMID:27223053

  19. MODULATION OF N-CADHERIN JUNCTIONS AND THEIR ROLE AS EPICENTERS OF DIFFERENTIATION-SPECIFIC ACTIN REGULATION IN THE DEVELOPING LENS

    PubMed Central

    Leonard, Michelle; Zhang, Liping; Zhai, Ni; Cader, Ahmad; Chan, Yim; Nowak, Roberta B.; Fowler, Velia M.; Menko, A. Sue

    2010-01-01

    Extensive elongation of lens fiber cells is a central feature of lens morphogenesis. Our study investigates the role of N-cadherin junctions in this process in vivo. We investigate both the molecular players involved in N-cadherin junctional maturation and the subsequent function of these junctions as epicenters for the assembly of an actin cytoskeleton that drives morphogenesis. We present the first evidence of nascent cadherin junctions in vivo, and show they are a prominent feature along lateral interfaces of undifferentiated lens epithelial cells. Maturation of these N-cadherin junctions, required for lens cell differentiation, preceded organization of a cortical actin cytoskeleton along the cells’ lateral borders, but was linked to recruitment of α-catenin and dephosphorylation of N-cadherin-linked β-catenin. Biochemical analysis revealed differentiation-specific recruitment of actin regulators cortactin and Arp3 to maturing N-cadherin junctions of differentiating cells, linking N-cadherin junctional maturation with actin cytoskeletal assembly during fiber cell elongation. Blocking formation of mature N-cadherin junctions led to reduced association of α-catenin with N-cadherin, prevented organization of actin along lateral borders of differentiating lens fiber cells and blocked their elongation. These studies provide a molecular link between N-cadherin junctions and the organization of an actin cytoskeleton that governs lens fiber cell morphogenesis in vivo. PMID:20969840

  20. Cranial sensory ganglia neurons require intrinsic N-cadherin function for guidance of afferent fibers to their final targets

    PubMed Central

    LaMora, Angela; Voigt, Mark M.

    2009-01-01

    Cell adhesion molecules, such as N-cadherin (cdh2), are essential for normal neuronal development, and as such have been implicated in an array of processes including neuronal differentiation and migration, and axon growth and fasciculation. Cdh2 is expressed in neurons of the peripheral nervous system during development, but its role in these cells during this time is poorly understood. Using the transgenic zebrafish line, tg(p2xr3.2:eGFPsl1), we have examined the involvement of cdh2 in the formation of sensory circuits by the peripheral nervous system. The tg(p2xr3.2:eGFPsl1) fish allows visualization of neurons comprising gV, gVII, gIX and gX and their axons throughout development. Reduction of cdh2 in this line was achieved by either crosses to the cdh2-mutant strain, glass onion (glo) or injection of a cdh2 morpholino (MO) into single-cell embryos. Here we show that cdh2 function is required to alter the directional vectors of growing axons upon reaching intermediate targets. The central axons enter the hindbrain appropriately but fail to turn caudally towards their final targets. Similarly, the peripheral axons extend ventrally, but fail to turn and project along a rostral/caudal axis. Furthermore, by expressing dominant negative cdh2 constructs selectively within cranial sensory ganglia (CSG) neurons, we found that cdh2 function is necessary within the axons to elicit these stereotypic turns, thus demonstrating that cdh2 acts cell autonomously. Together, our in vivo data reveal a novel role for cdh2 in the establishment of circuits by peripheral sensory neurons. PMID:19356698

  1. Slit1b-Robo3 signaling and N-cadherin regulate apical process retraction in developing retinal ganglion cells.

    PubMed

    Wong, Grace K W; Baudet, Marie-Laure; Norden, Caren; Leung, Louis; Harris, William A

    2012-01-01

    When neurons exit the cell cycle after their terminal mitosis, they detach from the apical surface of the neuroepithelium. Despite the fact that this detachment is crucial for further neurogenesis and neuronal migration, the underlying mechanisms are still not understood. Here, taking advantage of the genetics and imaging possibilities of the zebrafish retina as a model system, we show by knockdown experiments that the guidance molecule Slit1b and its receptor Robo3 are required for apical retraction of retinal ganglion cells (RGCs). In contrast, N-cadherin seems to be responsible for maintenance of apical attachment, as expression of dominant-negative N-cadherin causes RGCs to lose apical attachments prematurely and rescues retraction in slit1b morphants. These results suggest that Slit-Robo signaling downregulates N-cadherin activity to allow apical retraction in newly generated RGCs. PMID:22219284

  2. Slit1b-Robo3 signaling and N-cadherin regulate apical process retraction in developing retinal ganglion cells

    PubMed Central

    Wong, Grace K W; Baudet, Marie-Laure; Norden, Caren; Leung, Louis; Harris, William A.

    2012-01-01

    When neurons exit the cell cycle after their terminal mitosis, they detach from the apical surface of the neuroepithelium. Despite the fact that this detachment is crucial for further neurogenesis and neuronal migration, the underlying mechanisms are still not understood. Here, taking advantage of the genetics and imaging possibilities of the zebrafish retina as a model system, we show by knock down experiments that the guidance molecule Slit1b as well as its receptor Robo3 are required for apical retraction of retinal ganglion cells (RGCs). In contrast, N-cadherin seems to be responsible for maintenance of apical attachment as expression of dominant-negative N-cadherin causes RGCs to lose apical attachments prematurely and rescues retraction in slit1b morphants. These results suggest that Slit-Robo signaling downregulates N-cadherin activity to allow apical retraction in newly generated RGCs. PMID:22219284

  3. Dancing to Another Tune—Adhesive Moonlighting Proteins in Bacteria

    PubMed Central

    Kainulainen, Veera; Korhonen, Timo K.

    2014-01-01

    Biological moonlighting refers to proteins which express more than one function. Moonlighting proteins occur in pathogenic and commensal as well as in Gram-positive and Gram-negative bacteria. The canonical functions of moonlighting proteins are in essential cellular processes, i.e., glycolysis, protein synthesis, chaperone activity, and nucleic acid stability, and their moonlighting functions include binding to host epithelial and phagocytic cells, subepithelia, cytoskeleton as well as to mucins and circulating proteins of the immune and hemostatic systems. Sequences of the moonlighting proteins do not contain known motifs for surface export or anchoring, and it has remained open whether bacterial moonlighting proteins are actively secreted to the cell wall or whether they are released from traumatized cells and then rebind onto the bacteria. In lactobacilli, ionic interactions with lipoteichoic acids and with cell division sites are important for surface localization of the proteins. Moonlighting proteins represent an abundant class of bacterial adhesins that are part of bacterial interactions with the environment and in responses to environmental changes. Multifunctionality in bacterial surface proteins appears common: the canonical adhesion proteins fimbriae express also nonadhesive functions, whereas the mobility organelles flagella as well as surface proteases express adhesive functions. PMID:24833341

  4. Fibroblast adhesion to recombinant tropoelastin expressed as a protein A-fusion protein.

    PubMed Central

    Grosso, L E; Parks, W C; Wu, L J; Mecham, R P

    1991-01-01

    A bovine tropoelastin cDNA encoding exons 15-36 that includes the elastin-receptor binding site was expressed in Escherichia coli as a fusion protein with Protein A from Staphylococcus aureus. After isolation of the fusion protein by affinity chromatography on Ig-Sepharose, the tropoelastin domain was separated from plasmid-pR1T2T-encoded Protein A (Protein A') by CNBr cleavage. Cell-adhesion assays demonstrated specific adhesion to the recombinant tropoelastin. Furthermore, the data indicate that interactions involving the bovine elastin receptor mediate nuchalligament fibroblast adhesion to the recombinant protein. In agreement with earlier studies of fibroblast chemotaxis to bovine tropoelastin, nuchal-ligament fibroblast adhesion demonstrated developmental regulation of the elastin receptor. Images Fig. 2. Fig. 3. PMID:1996952

  5. Glycosylated Hydroxytryptophan in a Mussel Adhesive Protein from Perna viridis*

    PubMed Central

    Zhao, Hua; Sagert, Jason; Hwang, Dong Soo; Waite, J. Herbert

    2009-01-01

    The 3,4-dihydroxyphenyl-l-alanine (Dopa)-containing proteins of mussel byssus play a critical role in wet adhesion and have inspired versatile new synthetic strategies for adhesives and coatings. Apparently, however, not all mussel adhesive proteins are beholden to Dopa chemistry. The cDNA-deduced sequence of Pvfp-1, a highly aromatic and redox active byssal coating protein in the green mussel Perna viridis, suggests that Dopa may be replaced by a post-translational modification of tryptophan. The N-terminal tryptophan-rich domain of Pvfp-1 contains 42 decapeptide repeats with the consensus sequences ATPKPW1TAW2K and APPPAW1TAW2K. A small collagen domain (18 Gly-X-Y repeats) is also present. Tandem mass spectrometry of isolated tryptic decapeptides has detected both C2-hexosylated tryptophan (W1) and C2-hexosylated hydroxytryptophan (W2), the latter of which is redox active. The UV absorbance spectrum of W2 is consistent with 7-hydroxytryptophan, which represents an intriguing new theme for bioinspired opportunistic wet adhesion. PMID:19584055

  6. Soluble N-cadherin: A novel inhibitor of VSMC proliferation and intimal thickening

    PubMed Central

    Lyon, Cressida A.; Wadey, Kerry S.; George, Sarah J.

    2016-01-01

    Reoccurrence of symptoms occurs in 30–50% of coronary artery disease patients receiving vein grafts or bare-metal stents due to intimal thickening (restenosis). Restenosis is caused by vascular smooth muscle cell (VSMC) migration and proliferation. New therapeutic approaches that reduce VSMC migration and proliferation while promoting endothelial cell (EC) coverage are required. We assessed the effect of a soluble form of N-cadherin (SNC-Fc, a fusion of the extracellular portion of N-Cadherin to a mutated Fc fragment of IgG), a cell–cell junction molecule, on human saphenous VSMC proliferation and migration in vitro. We also assessed its effect on intimal thickening in a validated human ex vivo organ culture model. We observed that SNC-Fc significantly inhibited VSMC proliferation and to a lesser extent migration. The anti-proliferative effect of SNC-Fc was mediated by the interaction of SNC-Fc with the FGFR, rather than through inhibition of β-catenin signalling. SNC-Fc also significantly reduced intimal thickening by ~ 85% in the ex vivo organ culture model. SNC-Fc treatment inhibited proliferation of the intimal cells but did not affect migration. SNC-Fc reduced EC apoptosis, without detrimental effects on EC proliferation and migration in vitro. Importantly SNC-Fc increased EC coverage in the ex vivo model of intimal thickening. In conclusion, we suggest that SNC-Fc may have potential as an anti-proliferative therapeutic agent for reducing restenosis which has no detrimental effects on endothelial cells. PMID:26586312

  7. Adhesion

    MedlinePlus

    ... adhesions Ovarian cyst References Munireddy S, Kavalukas SL, Barbul A. Intra-abdominal healing: gastrointestinal tract and adhesions. Surg Clin N Am Kulaylat MN, Dayton, MT. Surgical complications. In: Townsend CM Jr, Beauchamp RD, Evers BM, Mattox KL, ...

  8. Silk Fibroin Aqueous-Based Adhesives Inspired by Mussel Adhesive Proteins.

    PubMed

    Burke, Kelly A; Roberts, Dane C; Kaplan, David L

    2016-01-11

    Silk fibroin from the domesticated silkworm Bombyx mori is a naturally occurring biopolymer with charged hydrophilic terminal regions that end-cap a hydrophobic core consisting of repeating sequences of glycine, alanine, and serine residues. Taking inspiration from mussels that produce proteins rich in L-3,4-dihydroxyphenylalanine (DOPA) to adhere to a variety of organic and inorganic surfaces, the silk fibroin was functionalized with catechol groups. Silk fibroin was selected for its high molecular weight, tunable mechanical and degradation properties, aqueous processability, and wide availability. The synthesis of catechol-functionalized silk fibroin polymers containing varying amounts of hydrophilic polyethylene glycol (PEG, 5000 g/mol) side chains was carried out to balance silk hydrophobicity with PEG hydrophilicity. The efficiency of the catechol functionalization reaction did not vary with PEG conjugation over the range studied, although tuning the amount of PEG conjugated was essential for aqueous solubility. Adhesive bonding and cell compatibility of the resulting materials were investigated, where it was found that incorporating as little as 6 wt % PEG prior to catechol functionalization resulted in complete aqueous solubility of the catechol conjugates and increased adhesive strength compared with silk lacking catechol functionalization. Furthermore, PEG-silk fibroin conjugates maintained their ability to form β-sheet secondary structures, which can be exploited to reduce swelling. Human mesenchymal stem cells (hMSCs) proliferated on the silks, regardless of PEG and catechol conjugation. These materials represent a protein-based approach to catechol-based adhesives, which we envision may find applicability as biodegradable adhesives and sealants. PMID:26674175

  9. M protein mediates streptococcal adhesion to HEp-2 cells.

    PubMed

    Wang, J R; Stinson, M W

    1994-02-01

    Streptococcus pyogenes adheres to human epithelial cells in vitro and in vivo. To identify adhesins, cell wall components were extracted from S. pyogenes M6 with alkali or by treatment with mutanolysin and lysozyme. HEp-2 cells were incubated with extracts of S. pyogenes M6 and then analyzed by Western blot (immunoblot) assays, using antibodies to S. pyogenes. Only one streptococcal component (62 kDa) was bound to HEp-2 cells and was identified serologically as M6 protein. Experiments with pepsin-cleaved fragments of M protein indicated that the binding site was located at the N-terminal half of the molecule. M protein was bound selectively to two trypsin-sensitive surface components, 97 and 205 kDa, of HEp-2 cells on nitrocellulose blots of sodium dodecyl sulfate-polyacrylamide gels. Tritium-labeled lipoteichoic acid bound to different HEp-2 cell components, 34 and 35 kDa, in a parallel experiment, indicating that lipoteichoic acid was not complexed with M protein and does not mediate M-protein binding. The four HEp-2 components were unrelated to fibronectin since they did not react with specific antibodies. An M-protein-deficient (M-) strain of streptococcus (JRS75), grown in chemically defined medium, showed 73% less adhesion activity to HEp-2 monolayers than an M+ strain (JRS4). Streptococcal adhesion was insensitive to competitive inhibition by selected monosaccharides. These results indicate that M protein binds directly to certain HEp-2 cell membrane components and mediates streptococcal adhesion. PMID:8300205

  10. LINKIN, a new transmembrane protein necessary for cell adhesion

    PubMed Central

    Kato, Mihoko; Chou, Tsui-Fen; Yu, Collin Z; DeModena, John; Sternberg, Paul W

    2014-01-01

    In epithelial collective migration, leader and follower cells migrate while maintaining cell–cell adhesion and tissue polarity. We have identified a conserved protein and interactors required for maintaining cell adhesion during a simple collective migration in the developing C. elegans male gonad. LINKIN is a previously uncharacterized, transmembrane protein conserved throughout Metazoa. We identified seven atypical FG–GAP domains in the extracellular domain, which potentially folds into a β-propeller structure resembling the α-integrin ligand-binding domain. C. elegans LNKN-1 localizes to the plasma membrane of all gonadal cells, with apical and lateral bias. We identified the LINKIN interactors RUVBL1, RUVBL2, and α-tubulin by using SILAC mass spectrometry on human HEK 293T cells and testing candidates for lnkn-1-like function in C. elegans male gonad. We propose that LINKIN promotes adhesion between neighboring cells through its extracellular domain and regulates microtubule dynamics through RUVBL proteins at its intracellular domain. DOI: http://dx.doi.org/10.7554/eLife.04449.001 PMID:25437307

  11. Evaluation of photodynamic therapy in adhesion protein expression

    PubMed Central

    PACHECO-SOARES, CRISTINA; MAFTOU-COSTA, MAIRA; DA CUNHA MENEZES COSTA, CAROLINA GENÚNCIO; DE SIQUEIRA SILVA, ANDREZA CRISTINA; MORAES, KAREN C.M.

    2014-01-01

    Photodynamic therapy (PDT) is a treatment modality that has clinical applications in both non-neoplastic and neoplastic diseases. PDT involves a light-sensitive compound (photosensitizer), light and molecular oxygen. This procedure may lead to several different cellular responses, including cell death. Alterations in the attachment of cancer cells to the substratum and to each other are important consequences of photodynamic treatment. PDT may lead to changes in the expression of cellular adhesion structure and cytoskeleton integrity, which are key factors in decreasing tumor metastatic potential. HEp-2 cells were photosensitized with aluminum phthalocyanine tetrasulfonate and zinc phthalocyanine, and the proteins β1-integrin and focal adhesion kinase (FAK) were assayed using fluorescence microscopy. The verification of expression changes in the genes for FAK and β1 integrin were performed by reverse transcription-polymerase chain reaction (RT-PCR). The results revealed that HEp-2 cells do not express β-integrin or FAK 12 h following PDT. It was concluded that the PDT reduces the adhesive ability of HEp-2 cells, inhibiting their metastatic potential. The present study aimed to analyze the changes in the expression and organization of cellular adhesion elements and the subsequent metastatic potential of HEp-2 cells following PDT treatment. PMID:25013490

  12. Hexavalent chromium at low concentration alters Sertoli cell barrier and connexin 43 gap junction but not claudin-11 and N-cadherin in the rat seminiferous tubule culture model

    SciTech Connect

    Carette, Diane; Perrard, Marie-Hélène; Prisant, Nadia; Gilleron, Jérome; Pointis, Georges; Segretain, Dominique; Durand, Philippe

    2013-04-01

    Exposure to toxic metals, specifically those belonging to the nonessential group leads to human health defects and among them reprotoxic effects. The mechanisms by which these metals produce their negative effects on spermatogenesis have not been fully elucidated. By using the Durand's validated seminiferous tubule culture model, which mimics the in vivo situation, we recently reported that concentrations of hexavalent chromium, reported in the literature to be closed to that found in the blood circulation of men, increase the number of germ cell cytogenetic abnormalities. Since this metal is also known to affect cellular junctions, we investigated, in the present study, its potential influence on the Sertoli cell barrier and on junctional proteins present at this level such as connexin 43, claudin-11 and N-cadherin. Cultured seminiferous tubules in bicameral chambers expressed the three junctional proteins and ZO-1 for at least 12 days. Exposure to low concentrations of chromium (10 μg/l) increased the trans-epithelial resistance without major changes of claudin-11 and N-cadherin expressions but strongly delocalized the gap junction protein connexin 43 from the membrane to the cytoplasm of Sertoli cells. The possibility that the hexavalent chromium-induced alteration of connexin 43 indirectly mediates the effect of the toxic metal on the blood–testis barrier dynamic is postulated. - Highlights: ► Influence of Cr(VI) on the Sertoli cell barrier and on junctional proteins ► Use of cultured seminiferous tubules in bicameral chambers ► Low concentrations of Cr(VI) (10 μg/l) altered the trans-epithelial resistance. ► Cr(VI) did not alter claudin-11 and N-cadherin. ► Cr(VI) delocalized connexin 43 from the membrane to the cytoplasm of Sertoli cells.

  13. N-Cadherin Regulates Cell Migration Through a Rab5-Dependent Temporal Control of Macropinocytosis.

    PubMed

    Wen, Meng-Hsuan; Wang, Jen-Yeu; Chiu, Yu-Ting; Wang, Mei-Pin; Lee, Sue-Ping; Tai, Chin-Yin

    2016-07-01

    Macropinocytosis is a clathrin-independent endocytic pathway implicated in fluid uptake, pathogen invasion and cell migration. During collective cell migration, macropinocytosis occurs primarily at membrane ruffles arising from the leading edges of migrating cells. We report here that N-cadherin (Ncad) regulates the tempo of macropinocytosis and thereby influences wound-induced collective cell migration. Using live-cell and super-resolution imaging techniques, we observed that Ncad formed clusters at the membrane ruffles and macropinosomes. De-clustering of Ncad by an interfering antibody impaired the recruitment of Rab5-an early endosomal marker-to the macropinosomes. Moreover, we demonstrated that Ncad interacts with Rab5, and laser ablation of Ncad caused Rab5 to dissociate from the macropinosomes. Although Rab5 detached from macropinosomes upon the de-clustering of Ncad, the recruitment of late endosomal marker Rab7 occurred earlier. Consequently, both centripetal trafficking of macropinosomes and collective migration were accelerated due to de-clustering of Ncad. Thus, our results suggest that Ncad is involved in the maturation of macropinocytosis through Rab5 recruitment, linking macropinocytosis and cell migration through a novel function of Ncad. PMID:27062132

  14. Expression of E-, P- and N-Cadherin and Its Clinical Significance in Cervical Squamous Cell Carcinoma and Precancerous Lesions

    PubMed Central

    Li, Baohua; Shi, Haiyan; Wang, Fenfen; Hong, Die; Lv, Weiguo; Xie, Xing; Cheng, Xiaodong

    2016-01-01

    Aberrant expression of classical cadherins has been observed in tumor invasion and metastasis, but its involvement in cervical carcinogenesis and cancer progression is not clear. We investigated E-, P- and N-cadherin expression and its significance in cervical squamous cell carcinoma (SCC) and cervical intraepithelial neoplasia (CIN). This retrospective study enrolled 508 patients admitted to Women's Hospital, School of Medicine, Zhejiang University with cervical lesions between January 2006 and December 2010. Immunochemical staining was performed in 98 samples of normal cervical epithelium (NC), 283 of CIN, and 127 of early-stage SCC. The association of cadherin staining with clinical characteristics and survival of the patients was evaluated by univariate and multivariate analysis. We found gradients of decreasing E-cadherin expression and increasing P-cadherin expression from NC through CIN to SCC. Aberrant E-cadherin and P-cadherin expression were significantly associated with clinical parameters indicating poor prognosis and shorter patient survival. Interestingly, we found very low levels of positive N-cadherin expression in CIN and SCC tissues that were not related to CIN or cancer. Pearson chi-square tests showed that E-cadherin expression in SCC was inversely correlated with P-cadherin expression (E-P switch), and was not correlated with N-cadherin expression. More important, patients with tissues exhibiting an E-P switch in expression had highly aggressive phenotypes and poorer prognosis than those without E-P switch expression. Our findings suggest that E-cadherin and P-cadherin, but not N-cadherin staining, might be useful in diagnosing CIN and for predicting prognosis in patients with early-stage SCC. PMID:27223886

  15. Developmental changes in expression, subcellular distribution, and function of Drosophila N-cadherin, guided by a cell-intrinsic program during neuronal differentiation.

    PubMed

    Kurusu, Mitsuhiko; Katsuki, Takeo; Zinn, Kai; Suzuki, Emiko

    2012-06-15

    Cell adhesion molecules (CAMs) perform numerous functions during neural development. An individual CAM can play different roles during each stage of neuronal differentiation; however, little is known about how such functional switching is accomplished. Here we show that Drosophila N-cadherin (CadN) is required at multiple developmental stages within the same neuronal population and that its sub-cellular expression pattern changes between the different stages. During development of mushroom body neurons and motoneurons, CadN is expressed at high levels on growing axons, whereas expression becomes downregulated and restricted to synaptic sites in mature neurons. Phenotypic analysis of CadN mutants reveals that developing axons require CadN for axon guidance and fasciculation, whereas mature neurons for terminal growth and receptor clustering. Furthermore, we demonstrate that CadN downregulation can be achieved in cultured neurons without synaptic contact with other cells. Neuronal silencing experiments using Kir(2.1) indicate that neuronal excitability is also dispensable for CadN downregulation in vivo. Interestingly, downregulation of CadN can be prematurely induced by ectopic expression of a nonselective cation channel, dTRPA1, in developing neurons. Together, we suggest that switching of CadN expression during neuronal differentiation involves regulated cation influx within neurons. PMID:22542600

  16. Adhesion and structure properties of protein nanomaterials containing hydrophobic and charged amino acids.

    PubMed

    Shen, Xinchun; Mo, Xiaoqun; Moore, Robyn; Frazier, Shawnalea J; Iwamoto, Takeo; Tomich, John M; Sun, Xiuzhi Susan

    2006-03-01

    Protein polymers are being used or considered for biobased adhesives and coating materials. Most adhesives derived from macro protein molecules work through receptors or cross-links to bring about adhesion. The adhesion mechanism of protein polymers would lead to better understanding of adhesives and the discovery of new practical properties of protein polymers at both nano- and macro-scales. The objective of this research work was to study adhesion properties of protein polymers at nanoscale (a peptide adhesive with nanometer-scale units that range in size of several nanometers, defined as protein nanomaterial). Seven protein nanomaterial samples with different degrees of adhesive strength were designed and synthesized using solid phase chemistries. All protein nanomaterials contain a common hydrophobic core flanked by charged amino acid sequences. The adhesion properties of the protein nanomaterials were investigated at different pH values and curing temperatures. The protein nanomaterials self aggregate and interact with the wood surface. The protein nanomaterial KKK-FLIVIGSII-KKK identified in this study had high adhesive strength toward wood. It had the highest shear strength at pH 12, with an amino acid sequence that was very hydrophobic and uncharged. This protein nanomaterial underwent structural analyses using circular dichroism, laser-Fourier transform infrared, and laser desorption mass spectrometry. At pH 12 this peptide adopted a pH-induced beta-like conformation. Adhesive strength reflects contributions of both hydrogen bonding and van der Waals interactions. Ionic and covalent bonds do not appear to be significant factors for adhesion in this study. PMID:16573147

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

    PubMed Central

    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

  18. Use of additives to enhance the properties of cottonseed protein as wood adhesives

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Soy protein is currently being used commercially as a “green” wood adhesive. Previous work in this laboratory has shown that cottonseed protein isolate, tested on maple wood veneer, produced higher adhesive strength and hot water resistance relative to soy protein. In the present study, cottonseed...

  19. Focal adhesion protein abnormalities in myelodysplastic mesenchymal stromal cells

    SciTech Connect

    Aanei, Carmen Mariana; Eloae, Florin Zugun; Flandrin-Gresta, Pascale; Tavernier, Emmanuelle; Carasevici, Eugen; Guyotat, Denis; Campos, Lydia

    2011-11-01

    Direct cell-cell contact between haematopoietic progenitor cells (HPCs) and their cellular microenvironment is essential to maintain 'stemness'. In cancer biology, focal adhesion (FA) proteins are involved in survival signal transduction in a wide variety of human tumours. To define the role of FA proteins in the haematopoietic microenvironment of myelodysplastic syndromes (MDS), CD73-positive mesenchymal stromal cells (MSCs) were immunostained for paxillin, pFAK [Y{sup 397}], and HSP90{alpha}/{beta} and p130CAS, and analysed for reactivity, intensity and cellular localisation. Immunofluorescence microscopy allowed us to identify qualitative and quantitative differences, and subcellular localisation analysis revealed that in pathological MSCs, paxillin, pFAK [Y{sup 397}], and HSP90{alpha}/{beta} formed nuclear molecular complexes. Increased expression of paxillin, pFAK [Y{sup 397}], and HSP90{alpha}/{beta} and enhanced nuclear co-localisation of these proteins correlated with a consistent proliferative advantage in MSCs from patients with refractory anaemia with excess blasts (RAEB) and negatively impacted clonogenicity of HPCs. These results suggest that signalling via FA proteins could be implicated in HPC-MSC interactions. Further, because FAK is an HSP90{alpha}/{beta} client protein, these results suggest the utility of HSP90{alpha}/{beta} inhibition as a target for adjuvant therapy for myelodysplasia.

  20. Expression of epithelial adhesion proteins and integrins in chronic inflammation.

    PubMed Central

    Haapasalmi, K.; Mäkelä, M.; Oksala, O.; Heino, J.; Yamada, K. M.; Uitto, V. J.; Larjava, H.

    1995-01-01

    Epithelial cell behavior in chronic inflammation is poorly characterized. During inflammation of tooth-supporting structures (periodontal disease), increased proliferation of epithelial cells into the inflamed connective tissue stroma is commonly seen. In some areas ulceration and degeneration take place. We studied alterations in the expression of adhesion molecules and integrins during chronic periodontal inflammation. In inflamed tissue, laminin-1 and type IV collagen were still present in the basement membrane and surrounding blood vessels, but they were also found extravascularly in inflamed connective tissue stroma. Type VII collagen and laminin-5 (also known as kalinin, epiligrin, or nicein) were poorly preserved in the basement membrane zone, but both were found in unusual streak-like distributions in the subepithelial connective tissue stroma in inflamed tissue. Both fibronectin and tenascin were substantially decreased in chronically inflamed connective tissue, showing only punctate staining at the basement membrane zone. Integrins of the beta 1 family showed two distinct staining patterns in epithelial cells during chronic inflammation; focal losses of beta 1 integrins (alpha 2 beta 1 and alpha 3 beta 1) were found in most areas, while in other areas the entire pocket epithelium was found to be strongly positive for beta 1 integrins. No members of the alpha v integrin family were found in any epithelia studied. Expression of the alpha 6 beta 4 integrin was high in basal cells of healthy tissue, but weak in epithelium associated with chronic inflammation. Chronic inflammation therefore involves alterations in both adhesion proteins and integrins expressed by epithelial cells. Basement membrane components found at abnormal sites in stroma in chronic inflammation might serve as new adhesive ligands for various cell types in inflamed stroma. Images Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 PMID:7541610

  1. Boronate Complex Formation with Dopa Containing Mussel Adhesive Protein Retards pH-Induced Oxidation and Enables Adhesion to Mica

    PubMed Central

    Israelachvili, Jacob N.; Chen, Yunfei; Waite, J. Herbert

    2014-01-01

    The biochemistry of mussel adhesion has inspired the design of surface primers, adhesives, coatings and gels for technological applications. These mussel-inspired systems often focus on incorporating the amino acid 3,4-dihydroxyphenyl-L-alanine (Dopa) or a catecholic analog into a polymer. Unfortunately, effective use of Dopa is compromised by its susceptibility to auto-oxidation at neutral pH. Oxidation can lead to loss of adhesive function and undesired covalent cross-linking. Mussel foot protein 5 (Mfp-5), which contains ∼30 mole % Dopa, is a superb adhesive under reducing conditions but becomes nonadhesive after pH-induced oxidation. Here we report that the bidentate complexation of borate by Dopa to form a catecholato-boronate can be exploited to retard oxidation. Although exposure of Mfp-5 to neutral pH typically oxidizes Dopa, resulting in a>95% decrease in adhesion, inclusion of borate retards oxidation at the same pH. Remarkably, this Dopa-boronate complex dissociates upon contact with mica to allow for a reversible Dopa-mediated adhesion. The borate protection strategy allows for Dopa redox stability and maintained adhesive function in an otherwise oxidizing environment. PMID:25303409

  2. Interaction of the enteropathogenic Escherichia coli protein, translocated intimin receptor (Tir), with focal adhesion proteins.

    PubMed

    Freeman, N L; Zurawski, D V; Chowrashi, P; Ayoob, J C; Huang, L; Mittal, B; Sanger, J M; Sanger, J W

    2000-12-01

    When enteropathogenic Escherichia coli (EPEC) attach and infect host cells, they induce a cytoskeletal rearrangement and the formation of cytoplasmic columns of actin filaments called pedestals. The attached EPEC and pedestals move over the surface of the host cell in an actin-dependent reaction [Sanger et al., 1996: Cell Motil Cytoskeleton 34:279-287]. The discovery that EPEC inserts the protein, translocated intimin receptor (Tir), into the membrane of host cells, where it binds the EPEC outer membrane protein, intimin [Kenny et al., 1997: Cell 91:511-520], suggests Tir serves two functions: tethering the bacteria to the host cell and providing a direct connection to the host's cytoskeleton. The sequence of Tir predicts a protein of 56.8 kD with three domains separated by two predicted trans-membrane spanning regions. A GST-fusion protein of the N-terminal 233 amino acids of Tir (Tir1) binds to alpha-actinin, talin, and vinculin from cell extracts. GST-Tir1 also coprecipitates purified forms of alpha-actinin, talin, and vinculin while GST alone does not bind these three focal adhesion proteins. Biotinylated probes of these three proteins also bound Tir1 cleaved from GST. Similar associations of alpha-actinin, talin, and vinculin were also detected with the C-terminus of Tir, i.e., Tir3, the last 217 amino acids. Antibody staining of EPEC-infected cultured cells reveals the presence of focal adhesion proteins beneath the attached bacteria. Our experiments support a model in which the cytoplasmic domains of Tir recruit a number of focal adhesion proteins that can bind actin filaments to form pedestals. Since pedestals also contain villin, tropomyosin and myosin II [Sanger et al., 1996: Cell Motil. Cytoskeleton 34:279-287], the pedestals appear to be a novel structure sharing properties of both focal adhesions and microvilli. PMID:11093251

  3. Allosteric Coupling in the Bacterial Adhesive Protein FimH*

    PubMed Central

    Rodriguez, Victoria B.; Kidd, Brian A.; Interlandi, Gianluca; Tchesnokova, Veronika; Sokurenko, Evgeni V.; Thomas, Wendy E.

    2013-01-01

    The protein FimH is expressed by the majority of commensal and uropathogenic strains of Escherichia coli on the tips of type 1 fimbriae and mediates adhesion via a catch bond to its ligand mannose. Crystal structures of FimH show an allosteric conformational change, but it remains unclear whether all of the observed structural differences are part of the allosteric mechanism. Here we use the protein structural analysis tool RosettaDesign combined with human insight to identify and synthesize 10 mutations in four regions that we predicted would stabilize one of the conformations of that region. The function of each variant was characterized by measuring binding to the ligand mannose, whereas the allosteric state was determined using a conformation-specific monoclonal antibody. These studies demonstrated that each region investigated was indeed part of the FimH allosteric mechanism. However, the studies strongly suggested that some regions were more tightly coupled to mannose binding and others to antibody binding. In addition, we identified many FimH variants that appear locked in the low affinity state. Knowledge of regulatory sites outside the active and effector sites as well as the ability to make FimH variants locked in the low affinity state may be crucial to the future development of novel antiadhesive and antimicrobial therapies using allosteric regulation to inhibit FimH. PMID:23821547

  4. N-Cadherin Expression Is Associated with Acquisition of EMT Phenotype and with Enhanced Invasion in Erlotinib-Resistant Lung Cancer Cell Lines

    PubMed Central

    Zhang, Xiaoju; Liu, Guangzhi; Kang, Yi; Dong, Zhaogang; Qian, Qiyu; Ma, Xitao

    2013-01-01

    Background The epidermal growth-factor receptor tyrosine kinase inhibitors have been effective in non-small cell lung cancer patients. However, acquired resistance eventually develops in most patients despite an initial positive response. Emerging evidence suggests that there is a molecular connection between acquired resistance and the epithelial–mesenchymal transition (EMT). N-cadherin is involved in the EMT and in the metastasis of cancer cells. Here, we analyzed N-cadherin expression and function in erlotinib-resistant lung cancer cell lines. Methods H1650 cell lines were used to establish the subline resistant to erlotinib(H1650ER). Then, induction of the EMT was analyzed using immunostaining and western blots in H1650ER cells. N-cadherin expression in the resistant cells was examined using FACS and western blot. In addition, an invasion assay was performed to characterize the resistant cells. The effects of N-cadherin on cell proliferation and invasion were analyzed. The association of N-cadherin expression with the EMT phenotype was investigated using immunohistochemical analysis of 13 archived, lung adenocarcinoma tissues, before and after treatment with erlotinib. Results In H1650ER cells, N-cadherin expression was upregulated, paralleled by the reduced expression of E-cadherin. The marked histological change and the development of a spindle-like morphology suggest that H1650ER cells underwent an EMT, accompanied by a decrease in E-cadherin and an increase in vimentin. A change in the EMT status between pre-and post-treatment was observed in 11 out of 13 cases (79%). In biopsies of resistant cancers, N-cadherin expression was increased in 10 out of 13 cases. Induction of the EMT was consistent with aggressive characteristics. Inhibition of N-cadherin expression by siRNA was tested to reduce proliferation and invasion of H1650ER cells in vitro. Conclusions Our data provide evidence that induction of the EMT contributes to the acquired resistance to EGFR

  5. Biomimetic soy protein nanocomposites with calcium carbonate crystalline arrays for use as wood adhesive.

    PubMed

    Liu, Dagang; Chen, Huihuang; Chang, Peter R; Wu, Qinglin; Li, Kaifu; Guan, Litao

    2010-08-01

    Despite the biodegradability, non-toxicity, and renewability, commercially available soy protein-based adhesives still have not been widely adopted by industry, partially due to their disappointing performances, i.e., low glue strength in the dry state and no glue strength in the wet state. In this study, biomimetic soy protein/CaCO(3) hybrid wood glue was devised and an attempt made to improve the adhesion strength. The structure and morphology of the adhesive and its fracture bonding interface and adhesion strength were investigated. Results showed that the compact rivets or interlocking links, and ion crosslinking of calcium, carbonate, hydroxyl ions in the adhesive greatly improving the water-resistance and bonding strength of soy protein adhesives. Glue strength of soy protein hybrid adhesive was higher than 6 MPa even after three water-immersion cycles. This green and sustainable proteinous hybrid adhesive, with high glue strength and good water-resistance, is a good substitute for formaldehyde wood glues. PMID:20307978

  6. p38 mitogen-activated protein kinase interacts with vinculin at focal adhesions during fatty acid-stimulated cell adhesion

    PubMed Central

    George, Margaret D.; Wine, Robert N.; Lackford, Brad; Kissling, Grace E.; Akiyama, Steven K.; Olden, Kenneth; Roberts, John D.

    2014-01-01

    Arachidonic acid stimulates cell adhesion by activating α2β1 integrins in a process that depends on protein kinases, including p38 mitogen activated protein kinase. Here, we describe the interaction of cytoskeletal components with key signaling molecules that contribute to spreading of, and morphological changes in, arachidonic acid-treated MDA-MB-435 human breast carcinoma cells. Arachidonic acid-treated cells showed increased attachment and spreading on collagen type IV as measured by electric cell-substrate impedance sensing. Fatty acid-treated cells displayed short cortical actin filaments associated with an increased number of β1 integrin-containing pseudopodia whereas untreated cells displayed elongated stress fibers and fewer clusters of β1 integrins. Confocal microscopy of arachidonic acid-treated cells showed that vinculin and phospho-p38 both appeared enriched in pseudopodia and at the tips of actin filaments, and fluorescence ratio imaging indicated the increase was specific for the phospho-(active) form of p38. Immunoprecipitates of phospho-p38 from extracts of arachidonic acid-treated cells contained vinculin, and GST-vinculin fusion proteins carrying the central region of vinculin bound phospho-p38, whereas fusion proteins expressing the terminal portions of vinculin did not. These data suggest that phospho-p38 associates with particular domains on critical focal adhesion proteins that are involved in tumor cell adhesion and spreading and that this association can be regulated by factors in the tumor microenvironment. PMID:24219282

  7. Nucleation and growth of cadherin adhesions

    SciTech Connect

    Lambert, Mireille; Thoumine, Olivier; Brevier, Julien; Choquet, Daniel; Riveline, Daniel; Mege, Rene-Marc

    2007-11-15

    Cell-cell contact formation relies on the recruitment of cadherin molecules and their anchoring to actin. However, the precise chronology of events from initial cadherin trans-interactions to adhesion strengthening is unclear, in part due to the lack of access to the distribution of cadherins within adhesion zones. Using N-cadherin expressing cells interacting with N-cadherin coated surfaces, we characterized the formation of cadherin adhesions at the ventral cell surface. TIRF and RIC microscopies revealed streak-like accumulations of cadherin along actin fibers. FRAP analysis indicated that engaged cadherins display a slow turnover at equilibrium, compatible with a continuous addition and removal of cadherin molecules within the adhesive contact. Association of cadherin cytoplasmic tail to actin as well as actin cables and myosin II activity are required for the formation and maintenance of cadherin adhesions. Using time lapse microscopy we deciphered how cadherin adhesions form and grow. As lamellipodia protrude, cadherin foci stochastically formed a few microns away from the cell margin. Neo-formed foci coalesced aligned and coalesced with preformed foci either by rearward sliding or gap filling to form cadherin adhesions. Foci experienced collapse at the rear of cadherin adhesions. Based on these results, we present a model for the nucleation, directional growth and shrinkage of cadherin adhesions.

  8. Focal adhesion linker proteins expression of fibroblast related to adhesion in response to different transmucosal abutment surfaces

    PubMed Central

    Moon, Yeon-Hee; Yoon, Mi-Kyeong; Moon, Jung-Sun; Kang, Jee-Hae; Kim, Sun-Hun; Yang, Hong-Seo

    2013-01-01

    PURPOSE To evaluate adherence of human gingival fibroblasts (HGFs) to transmucosal abutment of dental implant with different surface conditions with time and to investigate the roles of focal adhesion linker proteins (FALPs) involved in HGFs adhesion to abutment surfaces. MATERIALS AND METHODS Morphologies of cultured HGFs on titanium and ceramic discs with different surface were observed by scanning electron microscopy. Biocompatibility and focal adhesion were evaluated by ultrasonic wave application and cell viability assay. FALPs expression levels were assessed by RT-PCR and western blot. RESULTS There seemed to be little difference in biocompatibility and adhesion strength of HGFs depending on the surface conditions and materials. In all experimental groups, the number of cells remaining on the disc surface after ultrasonic wave application increased more than 2 times at 3 days after seeding compared to 1-day cultured cells and this continued until 7 days of culture. FALPs expression levels, especially of vinculin and paxillin, also increased in 5-day cultured cells compared to 1-day cultured fibroblasts on the disc surface. CONCLUSION These results might suggest that the strength of adhesion of fibroblasts to transmucosal abutment surfaces increases with time and it seemed to be related to expressions of FALPs. PMID:24049577

  9. Protein Recovery from Secondary Paper Sludge and Its Potential Use as Wood Adhesive

    NASA Astrophysics Data System (ADS)

    Pervaiz, Muhammad

    Secondary sludge is an essential part of biosolids produced through the waste treatment plant of paper mills. Globally paper mills generate around 3.0 million ton of biosolids and in the absence of beneficial applications, the handling and disposal of this residual biomass poses a serious environmental and economic proposition. Secondary paper sludges were investigated in this work for recovery of proteins and their use as wood adhesive. After identifying extracellular polymeric substances as adhesion pre-cursors through analytical techniques, studies were carried out to optimize protein recovery from SS and its comprehensive characterization. A modified physicochemical protocol was developed to recover protein from secondary sludge in substantial quantities. The combined effect of French press and sonication techniques followed by alkali treatment resulted in significant improvement of 44% in the yield of solubilized protein compared to chemical methods. The characterization studies confirmed the presence of common amino acids in recovered sludge protein in significant quantities and heavy metal concentration was reduced after recovery process. The sodium dodecyl sulfate polyacrylamide gel electrophoresis analysis revealed the presence of both low and high molecular weight protein fractions in recovered sludge protein. After establishing the proof-of-concept in the use of recovered sludge protein as wood adhesive, the bonding mechanism of protein adhesives with cellulose substrate was further elucidated in a complementary protein-modification study involving soy protein isolate and its glycinin fractions. The results of this study validated the prevailing bonding theories by proving that surface wetting, protein structure, and type of wood play important role in determining final adhesive strength. Recovered sludge protein was also investigated for its compatibility to formulate hybrid adhesive blends with formaldehyde and bio-based polymers. Apart from chemical

  10. Molecular architecture of a complex between an adhesion protein from the malaria parasite and intracellular adhesion molecule 1.

    PubMed

    Brown, Alan; Turner, Louise; Christoffersen, Stig; Andrews, Katrina A; Szestak, Tadge; Zhao, Yuguang; Larsen, Sine; Craig, Alister G; Higgins, Matthew K

    2013-02-22

    The adhesion of Plasmodium falciparum-infected erythrocytes to human tissues or endothelium is central to the pathology caused by the parasite during malaria. It contributes to the avoidance of parasite clearance by the spleen and to the specific pathologies of cerebral and placental malaria. The PfEMP1 family of adhesive proteins is responsible for this sequestration by mediating interactions with diverse human ligands. In addition, as the primary targets of acquired, protective immunity, the PfEMP1s are potential vaccine candidates. PfEMP1s contain large extracellular ectodomains made from CIDR (cysteine-rich interdomain regions) and DBL (Duffy-binding-like) domains and show extensive variation in sequence, size, and domain organization. Here we use biophysical methods to characterize the entire ∼300-kDa ectodomain from IT4VAR13, a protein that interacts with the host receptor, intercellular adhesion molecule-1 (ICAM-1). We show through small angle x-ray scattering that IT4VAR13 is rigid, elongated, and monomeric. We also show that it interacts with ICAM-1 through the DBLβ domain alone, forming a 1:1 complex. These studies provide a first low resolution structural view of a PfEMP1 ectodomain in complex with its ligand. They show that it combines a modular domain arrangement consisting of individual ligand binding domains, with a defined higher order architecture that exposes the ICAM-1 binding surface to allow adhesion. PMID:23297413

  11. Experimental strategies for the identification and characterization of adhesive proteins in animals: a review.

    PubMed

    Hennebert, Elise; Maldonado, Barbara; Ladurner, Peter; Flammang, Patrick; Santos, Romana

    2015-02-01

    Adhesive secretions occur in both aquatic and terrestrial animals, in which they perform diverse functions. Biological adhesives can therefore be remarkably complex and involve a large range of components with different functions and interactions. However, being mainly protein based, biological adhesives can be characterized by classical molecular methods. This review compiles experimental strategies that were successfully used to identify, characterize and obtain the full-length sequence of adhesive proteins from nine biological models: echinoderms, barnacles, tubeworms, mussels, sticklebacks, slugs, velvet worms, spiders and ticks. A brief description and practical examples are given for a variety of tools used to study adhesive molecules at different levels from genes to secreted proteins. In most studies, proteins, extracted from secreted materials or from adhesive organs, are analysed for the presence of post-translational modifications and submitted to peptide sequencing. The peptide sequences are then used directly for a BLAST search in genomic or transcriptomic databases, or to design degenerate primers to perform RT-PCR, both allowing the recovery of the sequence of the cDNA coding for the investigated protein. These sequences can then be used for functional validation and recombinant production. In recent years, the dual proteomic and transcriptomic approach has emerged as the best way leading to the identification of novel adhesive proteins and retrieval of their complete sequences. PMID:25657842

  12. Experimental strategies for the identification and characterization of adhesive proteins in animals: a review

    PubMed Central

    Hennebert, Elise; Maldonado, Barbara; Ladurner, Peter; Flammang, Patrick; Santos, Romana

    2015-01-01

    Adhesive secretions occur in both aquatic and terrestrial animals, in which they perform diverse functions. Biological adhesives can therefore be remarkably complex and involve a large range of components with different functions and interactions. However, being mainly protein based, biological adhesives can be characterized by classical molecular methods. This review compiles experimental strategies that were successfully used to identify, characterize and obtain the full-length sequence of adhesive proteins from nine biological models: echinoderms, barnacles, tubeworms, mussels, sticklebacks, slugs, velvet worms, spiders and ticks. A brief description and practical examples are given for a variety of tools used to study adhesive molecules at different levels from genes to secreted proteins. In most studies, proteins, extracted from secreted materials or from adhesive organs, are analysed for the presence of post-translational modifications and submitted to peptide sequencing. The peptide sequences are then used directly for a BLAST search in genomic or transcriptomic databases, or to design degenerate primers to perform RT-PCR, both allowing the recovery of the sequence of the cDNA coding for the investigated protein. These sequences can then be used for functional validation and recombinant production. In recent years, the dual proteomic and transcriptomic approach has emerged as the best way leading to the identification of novel adhesive proteins and retrieval of their complete sequences. PMID:25657842

  13. Embedded proteins and sacrificial bonds provide the strong adhesive properties of gastroliths

    NASA Astrophysics Data System (ADS)

    Thormann, Esben; MizunoPresent Address: Nihon L'Oreal, Research; Innovation Center, 3-2-1 Sakado, Takatsu, Kawasaki, Kanagawa, Japan., Hiroyasu; Jansson, Kjell; Hedin, Niklas; Fernández, M. Soledad; Arias, José Luis; Rutland, Mark W.; PaiPresent Address: CenterFunctional Nanomaterials, Brookhaven National Laboratory, 735 Brookhaven Avenue, Upton, New York 11973., Ranjith Krishna; Bergström, Lennart

    2012-06-01

    The adhesive properties of gastroliths from a freshwater crayfish (Cherax quadricarinatus) were quantified by colloidal probe atomic force microscopy (AFM) between heavily demineralized gastrolith microparticles and gastrolith substrates of different composition. Combined AFM and transmission electron microscopy studies demonstrated that the sequential detachment and large adhesion energies that characterise the adhesive behaviour of a native gastrolith substrate are dominated by sacrificial bonds between chitin fibres and between chitin fibres and CaCO3. The sacrificial bonds were shown to be strongly related to the gastrolith proteins and when the majority of these proteins were removed by ethylenediaminetetraacetic acid (EDTA), the sequential detachment disappeared and the adhesive energy was reduced by more than two orders of magnitude.The adhesive properties of gastroliths from a freshwater crayfish (Cherax quadricarinatus) were quantified by colloidal probe atomic force microscopy (AFM) between heavily demineralized gastrolith microparticles and gastrolith substrates of different composition. Combined AFM and transmission electron microscopy studies demonstrated that the sequential detachment and large adhesion energies that characterise the adhesive behaviour of a native gastrolith substrate are dominated by sacrificial bonds between chitin fibres and between chitin fibres and CaCO3. The sacrificial bonds were shown to be strongly related to the gastrolith proteins and when the majority of these proteins were removed by ethylenediaminetetraacetic acid (EDTA), the sequential detachment disappeared and the adhesive energy was reduced by more than two orders of magnitude. Electronic supplementary information (ESI) available. See DOI: 10.1039/c2nr30536d

  14. The inhibition of cell proliferation using silencing of N-cadherin gene by siRNA process in human melanoma cell lines.

    PubMed

    Ciołczyk-Wierzbicka, D; Gil, D; Laidler, P

    2012-01-01

    Malignant melanoma is a disease with high mortality rate caused by rapid metastasis. Cell motility is physically and biochemically restricted by cadherin-mediated cell interactions and signalling pathways, and alterations in cadherin expression strongly correlate with E to N-cadherin switch as well as the metastasis and progression of tumours. Contrary to E-cadherin, N-cadherin plays an important role in stimulating processes of cell division, migration, differentiation and death. In this study we investigated the role of N-cadherin in proliferation and AKT, ERK, beta-catenin signalling pathway in human melanoma cells: WM793(VGP), WM115(VGP) from the primary tumor site, as well as Lu1205(lung) and WM266-4(skin) from metastatic sites. N-cadherin, pAKT(S473), β-catenin, pERK1/2(T202/Y204), cyclin D1, cyclin D3, cyclin-dependent kinases CDK4, CDK6, and p15, p16, p21, p27 inhibitors expression was determined by western blot analysis. The study on proliferation of cells was performed with the use of BrdU incorporation and crystal violet staining assays. Knock-out of N-cadherin gene expression by siRNA process reduced the expression of: pAKT(S473), pERK1/2(T202/Y204), betacatenin, cyclin D1, cyclin D3, cyclin-dependent kinases CDK4, CDK6 while increasing expression of cell cycle inhibitors p21 and p27, and significantly decreased cell proliferation (50-70%). The collected data indicate that N-cadherin mediates the effect of cell cycle in G1 phase by AKT, β-catenin, and ERK signalling pathway. These results suggest that increased expression of N-cadherin significantly contributes to the increased invasive potential of melanoma cells. Silencing of N-cadherin arrests cell growth at G1 phase and inhibits the entry into S-phase which is of great importance as to its possible future use in cancer treatment. PMID:22300088

  15. Specific Adhesion of Lipid Membranes Can Simultaneously Produce Two Types of Lipid and Protein Heterogeneities

    NASA Astrophysics Data System (ADS)

    Shindell, Orrin; Micah, Natalie; Ritzer, Max; Gordon, Vernita

    2015-03-01

    Living cells adhere to one another and their environment. Adhesion is associated with re-organization of the lipid and protein components of the cell membrane. The resulting heterogeneities are functional structures involved in biological processes. We use artificial lipid membranes that contain a single type of binding protein. Before adhesion, the lipid, protein, and dye components in the membrane are well-mixed and constitute a single disordered-liquid phase (Ld) . After adhesion, two distinct types of heterogeneities coexist in the adhesion zone: a central domain of ordered lipid phase that excludes both binding proteins and membrane dye, and a peripheral domain of disordered lipid phase that is densely packed with adhesion proteins and enriched in membrane dye relative to the non-adhered portion of the vesicle. Thus, we show that adhesion that is mediated by only one type of protein can organize the lipid and protein components of the membranes into heterogeneities that resemble those found in biology, for example the immune synapse.

  16. Cloning and expression of recombinant adhesive protein MEFP-2 of the blue mussel, Mytilus edulis

    DOEpatents

    Silverman, Heather G.; Roberto, Francisco F.

    2006-02-07

    The present invention includes a Mytilus edulis cDNA having a nucleotide sequence that encodes for the Mytilus edulis foot protein-2 (Mefp-2), an example of a mollusk foot protein. Mefp-2 is an integral component of the blue mussels' adhesive protein complex, which allows the mussel to attach to objects underwater. The isolation, purification and sequencing of the Mefp-2 gene will allow researchers to produce Mefp-2 protein using genetic engineering techniques. The discovery of Mefp-2 gene sequences will also allow scientists to better understand how the blue mussel creates its waterproof adhesive protein complex.

  17. Cloning and expression of recombinant adhesive protein Mefp-1 of the blue mussel, Mytilus edulis

    DOEpatents

    Silverman, Heather G.; Roberto, Francisco F.

    2006-01-17

    The present invention comprises a Mytilus edulis cDNA sequenc having a nucleotide sequence that encodes for the Mytilus edulis foot protein-1 (Mefp-1), an example of a mollusk foot protein. Mefp-1 is an integral component of the blue mussels' adhesive protein complex, which allows the mussel to attach to objects underwater. The isolation, purification and sequencing of the Mefp-1 gene will allow researchers to produce Mefp-1 protein using genetic engineering techniques. The discovery of Mefp-1 gene sequence will also allow scientists to better understand how the blue mussel creates its waterproof adhesive protein complex.

  18. Redundant control of migration and adhesion by ERM proteins in vascular smooth muscle cells

    SciTech Connect

    Baeyens, Nicolas; Latrache, Iman; Yerna, Xavier; Noppe, Gauthier; Horman, Sandrine; Morel, Nicole

    2013-11-22

    Highlights: •The three ERM proteins are expressed in vascular smooth muscle cell. •ERM depletion inhibited PDGF-evoked migration redundantly. •ERM depletion increased cell adhesion redundantly. •ERM depletion did not affect PDGF-evoked Ca signal, Rac1 activation, proliferation. •ERM proteins control PDGF-induced migration by regulating adhesion. -- Abstract: Ezrin, radixin, and moesin possess a very similar structure with a C-terminal actin-binding domain and a N-terminal FERM interacting domain. They are known to be involved in cytoskeleton organization in several cell types but their function in vascular smooth muscle cells (VSMC) is still unknown. The aim of this study was to investigate the role of ERM proteins in cell migration induced by PDGF, a growth factor involved in pathophysiological processes like angiogenesis or atherosclerosis. We used primary cultured VSMC obtained from rat aorta, which express the three ERM proteins. Simultaneous depletion of the three ERM proteins with specific siRNAs abolished the effects of PDGF on cell architecture and migration and markedly increased cell adhesion and focal adhesion size, while these parameters were only slightly affected by depletion of ezrin, radixin or moesin alone. Rac1 activation, cell proliferation, and Ca{sup 2+} signal in response to PDGF were unaffected by ERM depletion. These results indicate that ERM proteins exert a redundant control on PDGF-induced VSMC migration by regulating focal adhesion turn-over and cell adhesion to substrate.

  19. Role of surface layer collagen binding protein from indigenous Lactobacillus plantarum 91 in adhesion and its anti-adhesion potential against gut pathogen.

    PubMed

    Yadav, Ashok Kumar; Tyagi, Ashish; Kaushik, Jai Kumar; Saklani, Asha Chandola; Grover, Sunita; Batish, Virender Kumar

    2013-12-14

    Human feacal isolates were ascertain as genus Lactobacillus using specific primer LbLMA1/R16-1 and further identified as Lactobacillus plantarum with species specific primers Lpl-3/Lpl-2. 25 L. plantarum strains were further assessed for hydrophobicity following the microbial adhesion to hydrocarbons (MATH) method and colonization potentials based on their adherence to immobilized human collagen type-1. Surface proteins were isolated from selected L. plantarum 91(Lp91) strain. The purified collagen binding protein (Cbp) protein was assessed for its anti-adhesion activity against enteric Escherichia coli 0157:H7 pathogen on immobilized collagen. Four L. plantarum strains displayed high degree of hydrophobicity and significant adhesion to collagen. A 72 kDa protein was purified which reduced 59.71% adhesion of E. coli 0157:H7 on immobilized collagen as compared to control well during adhesion assay. Cbp protein is the major influencing factor in inhibition of E. coli 0157:H7 adhesion with extracellular matrix (ECM) components. Hydrophobicity and adhesion potential are closely linked attributes precipitating in better colonization potential of the lactobacillus strains. Cbp is substantiated as a crucial surface protein contributing in adhesion of lactobacillus strains. The study can very well be the platform for commercialization of indigenous probiotic strain once their functional attributes are clinically explored. PMID:23890721

  20. Adhesion of Mussel Foot Protein Mefp-5 to Mica: An Underwater Superglue†

    PubMed Central

    Danner, Eric W.; Kan, Yajing; Hammer, Malte U.; Israelachvili, Jacob N.; Waite, J. Herbert

    2012-01-01

    Mussels have a remarkable ability to attach their holdfast, or byssus, opportunistically to a variety of substrata that are wet, saline, corroded, and/or fouled by biofilms. Mytilus edulis foot protein-5 (Mefp-5) is one of several proteins in the byssal adhesive plaque of the mussel M. edulis. The high content of 3,4 dihydroxyphenylalanine (Dopa) (~30 mol%) and its localization near the plaque-substrate interface have often prompted speculation that Mefp-5 plays a key role in adhesion. Using the surface forces apparatus, we show that on mica surfaces Mefp-5 achieves an adhesion energy approaching Ead = ~− 14 mJ/m2. This exceeds the adhesion energy of another interfacial protein, Mefp-3, by a factor of 4–5 and is greater than the adhesion between highly oriented monolayers of biotin and streptavidin. The adhesion to mica is notable for its dependence on Dopa, which is most stable under reducing conditions and acidic pH. Mefp-5 also exhibits strong protein-protein interactions with itself as well as with Mefp-3 from M. edulis. PMID:22873939

  1. Effect of adhesion proteins and surface chemistry on the procoagulant state of adherent platelets

    NASA Astrophysics Data System (ADS)

    Grunkemeier, John Mark

    Poor hemocompatibility of a blood contacting device can lead to blood clotting, reduced blood flow, and depletion of platelets from the blood. Improved understanding of the processes by which blood-material contact leads to these responses could result in more hemocompatible materials. Platelets accelerate blood clotting by adhesion, aggregation, secretion of proteins and agonists and acceleration of thrombin generation. Platelets are said to be "procoagulant" after phosphatidylserine residues flip from the cytosolic to the extracellular face of the lipid bilayer. This then allows for the assembly of the prothrombinase complex (Xa, Va and calcium) on the platelet membrane, which can rapidly convert prothrombin to thrombin. In this study, three different methods confirmed that adhesion causes platelets to become procoagulant: shortening of clotting times of recalcified plasma, binding of FITC-annexin V, and generation of thrombin in the presence of Va, Xa and prothrombin by adherent platelets. Adherent platelets were 10--23 times more activated than bulk phase unactivated platelets and 10--24 times less activated than bulk phase platelets activated by calcium ionophore. The role of adsorbed fibrinogen, vWF, mixtures of fibrinogen and vWF, fibronectin, whole and dilute plasma, and plasma deficient in adhesion proteins in stimulating platelet procoagulant activity was investigated. The results of these experiments suggested that adhesion proteins affect procoagulant activation to varying degrees and that surfaces preadsorbed with mixtures of adhesion proteins are more activating that surfaces preadsorbed with single adhesion proteins. The hypothesis that materials that affect tightness of binding of adsorbed adhesion proteins affect platelet procoagulant activity was investigated. These studies showed that increasing fluorine content of RFGD polymerized films caused reduced platelet adhesion, but increased procoagulant activity, possibly due to their ability to adsorb

  2. Strong underwater adhesives made by self-assembling multi-protein nanofibres

    NASA Astrophysics Data System (ADS)

    Zhong, Chao; Gurry, Thomas; Cheng, Allen A.; Downey, Jordan; Deng, Zhengtao; Stultz, Collin M.; Lu, Timothy K.

    2014-10-01

    Many natural underwater adhesives harness hierarchically assembled amyloid nanostructures to achieve strong and robust interfacial adhesion under dynamic and turbulent environments. Despite recent advances, our understanding of the molecular design, self-assembly and structure-function relationships of these natural amyloid fibres remains limited. Thus, designing biomimetic amyloid-based adhesives remains challenging. Here, we report strong and multi-functional underwater adhesives obtained from fusing mussel foot proteins (Mfps) of Mytilus galloprovincialis with CsgA proteins, the major subunit of Escherichia coli amyloid curli fibres. These hybrid molecular materials hierarchically self-assemble into higher-order structures, in which, according to molecular dynamics simulations, disordered adhesive Mfp domains are exposed on the exterior of amyloid cores formed by CsgA. Our fibres have an underwater adhesion energy approaching 20.9 mJ m-2, which is 1.5 times greater than the maximum of bio-inspired and bio-derived protein-based underwater adhesives reported thus far. Moreover, they outperform Mfps or curli fibres taken on their own and exhibit better tolerance to auto-oxidation than Mfps at pH ≥ 7.0.

  3. Adhesive Proteins of Stalked and Acorn Barnacles Display Homology with Low Sequence Similarities

    PubMed Central

    Jonker, Jaimie-Leigh; Abram, Florence; Pires, Elisabete; Varela Coelho, Ana; Grunwald, Ingo; Power, Anne Marie

    2014-01-01

    Barnacle adhesion underwater is an important phenomenon to understand for the prevention of biofouling and potential biotechnological innovations, yet so far, identifying what makes barnacle glue proteins ‘sticky’ has proved elusive. Examination of a broad range of species within the barnacles may be instructive to identify conserved adhesive domains. We add to extensive information from the acorn barnacles (order Sessilia) by providing the first protein analysis of a stalked barnacle adhesive, Lepas anatifera (order Lepadiformes). It was possible to separate the L. anatifera adhesive into at least 10 protein bands using SDS-PAGE. Intense bands were present at approximately 30, 70, 90 and 110 kilodaltons (kDa). Mass spectrometry for protein identification was followed by de novo sequencing which detected 52 peptides of 7–16 amino acids in length. None of the peptides matched published or unpublished transcriptome sequences, but some amino acid sequence similarity was apparent between L. anatifera and closely-related Dosima fascicularis. Antibodies against two acorn barnacle proteins (ab-cp-52k and ab-cp-68k) showed cross-reactivity in the adhesive glands of L. anatifera. We also analysed the similarity of adhesive proteins across several barnacle taxa, including Pollicipes pollicipes (a stalked barnacle in the order Scalpelliformes). Sequence alignment of published expressed sequence tags clearly indicated that P. pollicipes possesses homologues for the 19 kDa and 100 kDa proteins in acorn barnacles. Homology aside, sequence similarity in amino acid and gene sequences tended to decline as taxonomic distance increased, with minimum similarities of 18–26%, depending on the gene. The results indicate that some adhesive proteins (e.g. 100 kDa) are more conserved within barnacles than others (20 kDa). PMID:25295513

  4. Desmoplastic melanoma: expression of epithelial-mesenchymal transition-related proteins.

    PubMed

    Garrido, Maria Concepción; Requena, Luis; Kutzner, Heinz; Ortiz, Pablo; Pérez-Gómez, Beatriz; Rodriguez-Peralto, José-Luis

    2014-03-01

    Desmoplastic melanoma (DM) is a rare variant of melanoma. Most frequently, it seems as clinically ambiguous and histologically characterized by a poorly demarcated neoplasm composed of a proliferation of spindle melanocytes dispersed in a prominent collagenous stroma. It often represents a diagnostic challenge, delaying its detection. We analyzed the expression profile of 29 (28 "pure" and 1 "combined") DM. These data were compared with a series of 62 primary vertical growth phase nondesmoplastic melanomas (NDMs) using a set of proteins including melanocytic markers (S-100 protein and melan-A) and epithelial-mesenchymal transition (EMT)-related proteins (E-cadherin, N-cadherin, SPARC, WT1, and PKCα). The S-100 protein confirmed the melanocytic origin of the DM (positive in 96%). The significant positive expression of N-cadherin, SPARC, and WT1 in DM (61%, 82%, and 71%) compared with NDM (28%, 43%, and 47%; P < 0.05) and a lower expression of E-cadherin in DM (14%) compared with NDM (61%) support specific adhesive and migratory properties of DM tumor cells. The study was carried out with tissue microarrays that partly limited the study of the tumor sections. This study demonstrates, for the first time, a prominent expression of epithelial-mesenchymal transition-related proteins in DMs and tries to be one more step in refining its knowledge and leading to a better understanding of its biological and clinical behaviors. PMID:23974224

  5. Embedded proteins and sacrificial bonds provide the strong adhesive properties of gastroliths.

    PubMed

    Thormann, Esben; Mizuno, Hiroyasu; Jansson, Kjell; Hedin, Niklas; Fernández, M Soledad; Arias, José Luis; Rutland, Mark W; Pai, Ranjith Krishna; Bergström, Lennart

    2012-07-01

    The adhesive properties of gastroliths from a freshwater crayfish (Cherax quadricarinatus) were quantified by colloidal probe atomic force microscopy (AFM) between heavily demineralized gastrolith microparticles and gastrolith substrates of different composition. Combined AFM and transmission electron microscopy studies demonstrated that the sequential detachment and large adhesion energies that characterise the adhesive behaviour of a native gastrolith substrate are dominated by sacrificial bonds between chitin fibres and between chitin fibres and CaCO(3). The sacrificial bonds were shown to be strongly related to the gastrolith proteins and when the majority of these proteins were removed by ethylenediaminetetraacetic acid (EDTA), the sequential detachment disappeared and the adhesive energy was reduced by more than two orders of magnitude. PMID:22653376

  6. Adhesion mechanism in a DOPA-deficient foot protein from green mussels†

    PubMed Central

    Hwang, Dong Soo; Zeng, Hongbo; Lu, Qingye; Israelachvili, Jacob; Waite, J. Herbert

    2012-01-01

    The holdfast or byssus of Asian green mussels, Perna viridis, contains a foot protein, pvfp-1, that differs in two respects from all other known adhesive mussel foot proteins (mfp): (1) instead of the hallmark L-3,4-dihydroxyphenylalanine (DOPA) residues in mfp-1, for example, pvfp-1 contains C2-mannosyl-7-hydroxytryptophan (Man7OHTrp). (2) In addition, pvfp-1 chains are not monomeric like mfp-1 but trimerized by collagen and coiled-coil domains near the carboxy terminus after a typical domain of tandemly repeated decapeptides. Here, the contribution of these peculiarities to adhesion was examined using a surface forces apparatus (SFA). Unlike previously studied mfp-1s, pvfp-1 showed significant adhesion to mica and, in symmetric pvfp-1 films, substantial cohesive interactions were present at pH 5.5. The role of Man7OHTrp in adhesion is not clear, and a DOPA-like role for Man7OHTrp in metal complexation (e.g., Cu2+, Fe3+) was not observed. Instead, cation–π interactions with low desolvation penalty between Man7OHTrp and lysyl side chains and conformational changes (raveling and unraveling of collagen helix and coiled-coil domains) are the best explanations for the strong adhesion between pvfp-1 monomolecular films. The strong adhesion mechanism induced by cation–π interactions and conformational changes in pvfp-1 provides new insights for the development of biomimetic underwater adhesives. PMID:23105946

  7. Anionic deep cavitands enable the adhesion of unmodified proteins at a membrane bilayer.

    PubMed

    Ghang, Yoo-Jin; Perez, Lizeth; Morgan, Melissa A; Si, Fang; Hamdy, Omar M; Beecher, Consuelo N; Larive, Cynthia K; Julian, Ryan R; Zhong, Wenwan; Cheng, Quan; Hooley, Richard J

    2014-12-28

    An anionic self-folding deep cavitand is capable of immobilizing unmodified proteins and enzymes at a supported lipid bilayer interface, providing a simple, soft bioreactive surface that allows enzymatic function under mild conditions. The adhesion is based on complementary charge interactions, and the hosts are capable of binding enzymes such as trypsin at the bilayer interface: the catalytic activity is retained upon adhesion, allowing selective reactions to be performed at the membrane surface. PMID:25366572

  8. Modulation of cationicity of chitosan for tuning mesenchymal stem cell adhesion, proliferation, and differentiation.

    PubMed

    He, Jing; Wu, Fang; Wang, Dong; Yao, Ruijuan; Wu, Yao; Wu, Fang

    2015-01-01

    The aim of this study was to modulate the cationicity of chitosan to influence the mesenchymal stem cell (MSC) responses in terms of cell adhesion, proliferation, and differentiation. The authors prepared water-soluble carboxymethyl chitosan hydrogels using genipin as the crosslinking agent. The chitosan cationicity was modulated by varying the genipin content from 0.5 to 10 wt. %. The results indicated that the cationicity exerted a striking modulation effect on various MSC responses. The increase of the genipin content, i.e., decrease of the free amino group content (cationicity), overall promoted the MSC adhesion, cytoskeleton organization, proliferation, and differentiation into the osteogenic lineage. A surprising cell alignment effect was also observed on chitosan samples with high genipin concentrations (>2.5%). The chitosan sample with the highest genipin concentrations (10%) exhibited the best MSC proliferation and highest protein expression levels toward osteogenic lineages. The genipin content also showed a strong modulation effect on MSC condensation, and cell-cell and cell-matrix interactions, as suggested by the expressions of the sry related HMG box9 (Sox9), intercellular adhesion molecule 1, and N-Cadherin. Overall, the authors have demonstrated that modulation of cationicity (amino content) of chitosan is an effective and simple approach to tuning various MSC responses, including adhesion, proliferation, differentiation, as well as cell-cell interactions. Such findings might have important implications in biomaterial design for various biomedical applications. PMID:26433366

  9. Use of adhesion-defective mutants of Staphylococcus aureus to define the role of specific plasma proteins in promoting bacterial adhesion to canine arteriovenous shunts.

    PubMed Central

    Vaudaux, P E; François, P; Proctor, R A; McDevitt, D; Foster, T J; Albrecht, R M; Lew, D P; Wabers, H; Cooper, S L

    1995-01-01

    We used an ex vivo canine arteriovenous shunt model, previously developed to study plasma protein adsorption and thrombogenesis on polymeric biomaterials, to define the role of host proteins in promoting adhesion of Staphylococcus aureus. Either polyethylene or polyvinyl chloride tubings were exposed to canine blood for 5, 15, or 60 min at a flow rate of 300 ml/min and then were flushed in phosphate-buffered saline (PBS), cut into 1.5-cm segments, and stored at -70 degrees C. After thawing, each segment was preincubated in 0.5% albumin in PBS to prevent nonspecific staphylococcal attachment to surfaces that were not exposed to blood. Each segment was then incubated with 4 x 10(6) CFU of [3H]thymidine-labelled S. aureus per ml for 60 min at 37 degrees C in an in vitro adhesion assay. Two site-specific mutants of S. aureus were tested: one specifically defective in adhesion to surface-bound fibronectin (FnAd-def) and the other defective in adhesion to fibrinogen (FgAD-def) [corrected]. Compared with their respective parental strains, the FgAd-def, but not the FnAd-def, mutant of S. aureus showed a strong (> 80%) decrease in attachment to ex vivo tubings. The adhesion of each strain of S. aureus onto polyethylene was consistently more than twofold higher than the adhesion onto polyvinyl chloride segments exposed to flowing blood for 5 or 15 min, but adhesion became similar to that on polyvinyl chloride after 60 min of exposure. In conclusion, the specific adhesion-defective mutants of S. aureus suggested that fibrinogen was the most active adhesion-promoting protein in a short-term blood-material interaction. The experimental approach described in this study should prove useful for screening materials thought to be resistant to protein-mediated staphylococcal adhesion and colonization. PMID:7822026

  10. Cell adhesion-dependent inactivation of a soluble protein kinase during fertilization in Chlamydomonas.

    PubMed Central

    Zhang, Y; Luo, Y; Emmett, K; Snell, W J

    1996-01-01

    Within seconds after the flagella of mt+ and mt- Chlamydomonas gametes adhere during fertilization, their flagellar adenylyl cyclase is activated several fold and preparation for cell fusion is initiated. Our previous studies indicated that early events in this pathway, including control of adenylyl cyclase, are regulated by phosphorylation and dephosphorylation. Here, we describe a soluble, flagellar protein kinase activity that is regulated by flagellar adhesion. A 48-kDa, soluble flagellar protein was consistently phosphorylated in an in vitro assay in flagella isolated from nonadhering mt+ and mt- gametes, but not in flagella isolated from mt+ and mt- gametes that had been adhering for 1 min. Although the 48-kDa protein was present in the flagella isolated from adhering gametes, we demonstrate that its protein kinase was inactivated by flagellar adhesion. Immunoblot analysis and inhibitor studies indicate that the 48-kDa protein in nonadhering gametes is phosphorylated by a protein tyrosine kinase. In vivo experiments showing that the protein tyrosine phosphatase inhibitor sodium orthovanadate inhibits fertilization suggest that protein dephosphorylation may be required for signal transduction. The 48-kDa protein and its protein kinase may be among the first elements of a novel signalling pathway that couples interaction of flagellar adhesion molecules to gamete activation. Images PMID:8730096

  11. Adhesion properties of Lactobacillus rhamnosus mucus-binding factor to mucin and extracellular matrix proteins.

    PubMed

    Nishiyama, Keita; Nakamata, Koichi; Ueno, Shintaro; Terao, Akari; Aryantini, Ni Putu Desy; Sujaya, I Nengah; Fukuda, Kenji; Urashima, Tadasu; Yamamoto, Yuji; Mukai, Takao

    2015-01-01

    We previously described potential probiotic Lactobacillus rhamnosus strains, isolated from fermented mare milk produced in Sumbawa Island, Indonesia, which showed high adhesion to porcine colonic mucin (PCM) and extracellular matrix (ECM) proteins. Recently, mucus-binding factor (MBF) was found in the GG strain of L. rhamnosus as a mucin-binding protein. In this study, we assessed the ability of recombinant MBF protein from the FSMM22 strain, one of the isolates of L. rhamnosus from fermented Sumbawa mare milk, to adhere to PCM and ECM proteins by overlay dot blot and Biacore assays. MBF bound to PCM, laminin, collagen IV, and fibronectin with submicromolar dissociation constants. Adhesion of the FSMM22 mbf mutant strain to PCM and ECM proteins was significantly less than that of the wild-type strain. Collectively, these results suggested that MBF contribute to L. rhamnosus host colonization via mucin and ECM protein binding. PMID:25351253

  12. Cadherin2 (N-cadherin) plays an essential role in zebrafish cardiovascular development

    PubMed Central

    Bagatto, Brian; Francl, Jessie; Liu, Bei; Liu, Qin

    2006-01-01

    Background Cadherins are cell surface adhesion molecules that play important roles in development of vertebrate tissues and organs. We studied cadherin2 expression in developing zebrafish heart using in situ hybridization and immunocytochemical methods, and we found that cadherin2 was strongly expressed by the myocardium of the embryonic zebrafish. To gain insight into cadherin2 role in the formation and function of the heart, we analyzed cardiac differentiation and performance in a cadherin2 mutant, glass onion (glo). Results We found that the cadherin2 mutant had enlarged pericardial cavity, disorganized atrium and ventricle, and reduced expression of a ventricular specific marker vmhc. Individual myocardiocytes in the glo mutant embryos became round shaped and loosely aggregated. In vivo measurements of cardiac performance revealed that the mutant heart had significantly reduced heart rate, stroke volume and cardiac output compared to control embryos. Formation of the embryonic vascular system in the glo mutants was also affected. Conclusion Our results suggest that cadherin2 plays an essential role in zebrafish cardiovascular development. Although the exact mechanisms remain unknown as to the formation of the enlarged pericardium and reduced peripheral blood flow, it is clear that myocardiocyte differentiation and physiological cardiovascular performance is impaired when cadherin2 function is disrupted. PMID:16719917

  13. Secreted Frizzled-related protein 1 (sFRP1) regulates spermatid adhesion in the testis via dephosphorylation of focal adhesion kinase and the nectin-3 adhesion protein complex

    PubMed Central

    Wong, Elissa W. P.; Lee, Will M.; Cheng, C. Yan

    2013-01-01

    Development of spermatozoa in adult mammalian testis during spermatogenesis involves extensive cell migration and differentiation. Spermatogonia that reside at the basal compartment of the seminiferous epithelium differentiate into more advanced germ cell types that migrate toward the apical compartment until elongated spermatids are released into the tubule lumen during spermiation. Apical ectoplasmic specialization (ES; a testis-specific anchoring junction) is the only cell junction that anchors and maintains the polarity of elongating/elongated spermatids (step 8–19 spermatids) in the epithelium. Little is known regarding the signaling pathways that trigger the disassembly of the apical ES at spermiation. Here, we show that secreted Frizzled-related protein 1 (sFRP1), a putative tumor suppressor gene that is frequently down-regulated in multiple carcinomas, is a crucial regulatory protein for spermiation. The expression of sFRP1 is tightly regulated in adult rat testis to control spermatid adhesion and sperm release at spermiation. Down-regulation of sFRP1 during testicular development was found to coincide with the onset of the first wave of spermiation at approximately age 45 d postpartum, implying that sFRP1 might be correlated with elongated spermatid adhesion conferred by the apical ES before spermiation. Indeed, administration of sFRP1 recombinant protein to the testis in vivo delayed spermiation, which was accompanied by down-regulation of phosphorylated (p)-focal adhesion kinase (FAK)-Tyr397 and retention of nectin-3 adhesion protein at the apical ES. To further investigate the functional relationship between p-FAK-Tyr397 and localization of nectin-3, we overexpressed sFRP1 using lentiviral vectors in the Sertoli-germ cell coculture system. Consistent with the in vivo findings, overexpression of sFRP1 induced down-regulation of p-FAK-Tyr397, leading to a decline in phosphorylation of nectin-3. In summary, this report highlights the critical role of s

  14. Single Adhesive Nanofibers from a Live Diatom Have the Signature Fingerprint of Modular Proteins

    PubMed Central

    Dugdale, T. M.; Dagastine, R.; Chiovitti, A.; Mulvaney, P.; Wetherbee, R.

    2005-01-01

    The adhesive and mechanical properties of a cell-substratum adhesive secreted by live diatom cells were examined in situ using atomic force microscopy. The resulting force curves have a regular saw-tooth pattern, the characteristic fingerprint of modular proteins, and when bridged between tip and surface can repeatedly be stretched and relaxed resulting in precisely overlaying saw-tooth curves (up to ∼600 successive cycles). The average rupture force of the peaks is 0.794 ± 0.007 (mean ± SE) nN at a loading rate of 0.8 μm/s and the average persistence length is 0.026 ± <0.001 (mean ± SE) nm (fit using the worm-like chain model). We propose that we are pulling on single adhesive nanofibers, each a cohesive unit composed of a set number of modular proteins aligned in register. Furthermore, we can observe and differentiate when up to three adhesive nanofibers are pulled based upon multimodal distributions of force and persistence length. The high force required for bond rupture, high extensibility (∼1.2 μm), and the accurate and rapid refolding upon relaxation, together provide strong and flexible properties ideally suited for the cell-substratum adhesion of this fouling diatom and allow us to understand the mechanism responsible for the strength of adhesion. PMID:16169972

  15. N-cadherin is Key to Expression of the Nucleus Pulposus Cell Phenotype under Selective Substrate Culture Conditions

    PubMed Central

    Hwang, Priscilla Y; Jing, Liufang; Chen, Jun; Lim, Foon-Lian; Tang, Ruhang; Choi, Hyowon; Cheung, Kenneth M; Risbud, Makarand V; Gersbach, Charles A; Guilak, Farshid; Leung, Victor Y; Setton, Lori A

    2016-01-01

    Nucleus pulposus (NP) cells of the intervertebral disc are essential for synthesizing extracellular matrix that contributes to disc health and mechanical function. NP cells have a unique morphology and molecular expression pattern derived from their notochordal origin, and reside in N-cadherin (CDH2) positive cell clusters in vivo. With disc degeneration, NP cells undergo morphologic and phenotypic changes including loss of CDH2 expression and ability to form cell clusters. Here, we investigate the role of CDH2 positive cell clusters in preserving healthy, biosynthetically active NP cells. Using a laminin-functionalized hydrogel system designed to mimic features of the native NP microenvironment, we demonstrate NP cell phenotype and morphology is preserved only when NP cells form CDH2 positive cell clusters. Knockdown (CRISPRi) or blocking CDH2 expression in vitro and in vivo results in loss of a healthy NP cell. Findings also reveal that degenerate human NP cells that are CDH2 negative can be promoted to re-express CDH2 and healthy, juvenile NP matrix synthesis patterns by promoting cell clustering for controlled microenvironment conditions. This work also identifies CDH2 interactions with β-catenin-regulated signaling as one mechanism by which CDH2-mediated cell interactions can control NP cell phenotype and biosynthesis towards maintenance of healthy intervertebral disc tissues. PMID:27292569

  16. N-cadherin is Key to Expression of the Nucleus Pulposus Cell Phenotype under Selective Substrate Culture Conditions.

    PubMed

    Hwang, Priscilla Y; Jing, Liufang; Chen, Jun; Lim, Foon-Lian; Tang, Ruhang; Choi, Hyowon; Cheung, Kenneth M; Risbud, Makarand V; Gersbach, Charles A; Guilak, Farshid; Leung, Victor Y; Setton, Lori A

    2016-01-01

    Nucleus pulposus (NP) cells of the intervertebral disc are essential for synthesizing extracellular matrix that contributes to disc health and mechanical function. NP cells have a unique morphology and molecular expression pattern derived from their notochordal origin, and reside in N-cadherin (CDH2) positive cell clusters in vivo. With disc degeneration, NP cells undergo morphologic and phenotypic changes including loss of CDH2 expression and ability to form cell clusters. Here, we investigate the role of CDH2 positive cell clusters in preserving healthy, biosynthetically active NP cells. Using a laminin-functionalized hydrogel system designed to mimic features of the native NP microenvironment, we demonstrate NP cell phenotype and morphology is preserved only when NP cells form CDH2 positive cell clusters. Knockdown (CRISPRi) or blocking CDH2 expression in vitro and in vivo results in loss of a healthy NP cell. Findings also reveal that degenerate human NP cells that are CDH2 negative can be promoted to re-express CDH2 and healthy, juvenile NP matrix synthesis patterns by promoting cell clustering for controlled microenvironment conditions. This work also identifies CDH2 interactions with β-catenin-regulated signaling as one mechanism by which CDH2-mediated cell interactions can control NP cell phenotype and biosynthesis towards maintenance of healthy intervertebral disc tissues. PMID:27292569

  17. Modification of human platelet adhesion on biomaterial surfaces by protein preadsorption under static and flow conditions.

    PubMed

    Otto, Mike; Franzen, Arno; Hansen, Torsten; Kirkpatrick, Charles James

    2004-01-01

    Biomaterial-induced thrombosis remains one of the main complications of vascular implant devices. Preadsorbed proteins on the biomaterial/blood interface will modify the adhesion and activation of platelets (PTLs) during the initial contact-phase. Our results clearly show that PTL-adherence on biomaterials is influenced not only by protein preadsorption, but also by flow conditions. The covalent coating of TCPS and glass by phosphorylcholine (PC) induces a significant decrease of PTL adhesion but leads to a slight, but nevertheless significant activation of PTL, which was detected by the induction of P-selectin expression using FACS analysis. Methodologically, the visualization of PTL adhesion gave more reliable results for measurement of PTL adhesion than the cell-enzyme immunoassay (EIA) for P-selectin. Human citrated plasma caused an inhibition of PTL. It is probable, that the contained sodium citrate may inhibit PTL adhesion by its calcium ion-binding capacity. The flow experiment as dynamic system is in our view absolutely essential for the evaluation of biomaterials for vascular prosthesis, and is in accordance with the international standards. The results of the experiments also suggest that investigations under static and flow conditions are needed to determine the influence of protein adsorption on mixed blood cell populations, for example, on PTL and PMN mixtures/co-cultures in order to achieve a better simulation of the in vivo situation. PMID:15338589

  18. A focal adhesion protein-based mechanochemical checkpoint regulates cleft progression during branching morphogenesis

    PubMed Central

    Daley, William P.; Kohn, Joshua M.; Larsen, Melinda

    2011-01-01

    Cleft formation is the initial step of branching morphogenesis in many organs. We previously demonstrated that ROCK 1 regulates a non-muscle myosin II-dependent mechanochemical checkpoint to transition initiated clefts to progressing clefts in developing submandibular salivary glands. Here, we report that ROCK-mediated integrin activation and subsequent formation of focal adhesion complexes comprise this mechanochemical checkpoint. Inhibition of ROCK1 and non-muscle myosin II activity decreased integrin β1 activation in the cleft region and interfered with localization and activation of focal adhesion complex proteins, such as focal adhesion kinase (FAK). Inhibition of FAK activity also prevented cleft progression, by disrupting recruitment of the focal adhesion proteins talin and vinculin and subsequent fibronectin assembly in the cleft region while decreasing ERK1/2 activation. These results demonstrate that inside-out integrin signaling leading to a localized recruitment of active FAK-containing focal adhesion protein complexes generates a mechanochemical checkpoint that facilitates progression of branching morphogenesis. PMID:22016182

  19. Flavonoids inhibit cytokine-induced endothelial cell adhesion protein gene expression.

    PubMed Central

    Gerritsen, M. E.; Carley, W. W.; Ranges, G. E.; Shen, C. P.; Phan, S. A.; Ligon, G. F.; Perry, C. A.

    1995-01-01

    Treatment of human endothelial cells with cytokines such as interleukin-1, tumor necrosis factor-alpha (TNF-alpha) or interferon-gamma induces the expression of specific leukocyte adhesion molecules on the endothelial cell surface. Interfering with either leukocyte adhesion or adhesion protein upregulation is an important therapeutic target as evidenced by the potent anti-inflammatory actions of neutralizing antibodies to these ligands in various animal models and in patients. In the present study we report that cotreatment of human endothelial cells with certain hydroxyflavones and flavanols blocks cytokine-induced ICAM-1, VCAM-1, and E-selectin expression on human endothelial cells. One of the most potent flavones, apigenin, exhibited a dose- and time-dependent, reversible effect on adhesion protein expression as well as inhibiting adhesion protein upregulation at the transcriptional level. Apigenin also inhibited IL-1 alpha-induced prostaglandin synthesis and TNF-alpha-induced IL-6 and IL-8 production, suggesting that the hydroxyflavones may act as general inhibitors of cytokine-induced gene expression. Although apigenin did not inhibit TNF-alpha-induced nuclear translocation of NF-kappa B(p50(NFKB1)/p65(RelA)) we found this flavonoid did inhibit TNF-alpha induced beta-galactosidase activity in SW480 cells stably transfected with a beta-galactosidase reporter construct driven by four NF-kappa B elements, suggesting an action on NF-kappa B transcriptional activation. Adhesion of leukocytes to cytokine-treated endothelial cells was blocked in endothelial cells cotreated with apigenin. Finally, apigenin demonstrated potent anti-inflammatory activity in carrageenan induced rat paw edema and delayed type hypersensitivity in the mouse. We conclude that flavonoids offer important therapeutic potential for the treatment of a variety of inflammatory diseases involving an increase in leukocyte adhesion and trafficking. Images Figure 7 Figure 8 Figure 11 PMID:7543732

  20. Corneal Cell Adhesion to Contact Lens Hydrogel Materials Enhanced via Tear Film Protein Deposition

    PubMed Central

    Elkins, Claire M.; Qi, Qin M.; Fuller, Gerald G.

    2014-01-01

    Tear film protein deposition on contact lens hydrogels has been well characterized from the perspective of bacterial adhesion and viability. However, the effect of protein deposition on lens interactions with the corneal epithelium remains largely unexplored. The current study employs a live cell rheometer to quantify human corneal epithelial cell adhesion to soft contact lenses fouled with the tear film protein lysozyme. PureVision balafilcon A and AirOptix lotrafilcon B lenses were soaked for five days in either phosphate buffered saline (PBS), borate buffered saline (BBS), or Sensitive Eyes Plus Saline Solution (Sensitive Eyes), either pure or in the presence of lysozyme. Treated contact lenses were then contacted to a live monolayer of corneal epithelial cells for two hours, after which the contact lens was sheared laterally. The apparent cell monolayer relaxation modulus was then used to quantify the extent of cell adhesion to the contact lens surface. For both lens types, lysozyme increased corneal cell adhesion to the contact lens, with the apparent cell monolayer relaxation modulus increasing up to an order of magnitude in the presence of protein. The magnitude of this increase depended on the identity of the soaking solution: lenses soaked in borate-buffered solutions (BBS, Sensitive Eyes) exhibited a much greater increase in cell attachment upon protein addition than those soaked in PBS. Significantly, all measurements were conducted while subjecting the cells to moderate surface pressures and shear rates, similar to those experienced by corneal cells in vivo. PMID:25144576

  1. N-Cadherin, ADAM-10 and Aquaporin 1 expression in lung tissue exposed to fluoro-edenite fibers: an immunohistochemical study.

    PubMed

    Musumeci, Giuseppe; Loreto, Carla; Szychlinska, Marta Anna; Imbesi, Rosa; Rapisarda, Venerando; Aiello, Flavia Concetta; Castorina, Sergio; Castrogiovanni, Paola

    2015-08-01

    Fluoro-edenite (FE) fibers are similar to other amphibole asbestos fibers. The scientific relevance of FE is due to its ability to lead to chronic inflammation and carcinogenesis in lung tissue shown after its inhalation. These fibers stimulate aberrant host cell proliferation and induce the release of cytokines, growth factors, reactive oxygen and nitrite species, which results in DNA damage. In previous studies, we showed that FE induces functional modifications in sheep and human lung fibroblasts and alveolar epithelial cells, where the overexpression of several molecules probably involved in pathological cellular mechanisms induced by FE exposition have been detected. However, the mechanisms of cellular and molecular toxicity and the cellular response to FE fibers are still not well known. N-cadherin, ADAM-10 and AQP1 are molecules involved in carcinogenesis and in inflammatory process. In this study we analyzed, through immunohistochemistry, their expression in the lung tissue of sheep exposed to FE. Our results showed different patterns of immunolabeling for N-cadherin, ADAM-10 and AQP1. N-cadherin and ADAM-10 were more expressed in FE exposed lung tissue, when compared with the control. On the contrary, AQP1 was more expressed in non exposed lung tissue. These results suggest that N-Cadherin, ADAM-10 and AQP1 are probably involved in different pathological processes induced by FE fiber exposition. The aim of the study was to better understand the mechanisms of cellular and molecular toxicity and of cellular response to FE fibers in order to identify, in the future, a possible therapeutic intervention in cases of FE-associated pathogenesis. PMID:25757887

  2. The role of serum proteins in Staphylococcus aureus adhesion to ethylene glycol coated surfaces.

    PubMed

    Schuster, Swen; Yu, Wenqi; Nega, Mulugeta; Chu, Ya-Yun; Zorn, Stefan; Zhang, Fajun; Götz, Friedrich; Schreiber, Frank

    2014-11-01

    Bacterial adhesion on implants is a first step in the development of chronic foreign body associated infections. Finding strategies to minimize bacterial adhesion may contribute to minimize such infections. It is known that surfaces with oligo-ethylene-glycol (EG3OMe) or poly-ethylene-glycol (PEG2k) terminations decrease unspecific protein adsorption and bacterial adhesion. However, little is known about the influence of serum and its components on bacterial adhesion. We therefore prepared two coatings on gold surface with HS-(CH2)11EG3OMe (EG3OMe) and PEG2k-thiol and studied the role of bovine serum albumin (BSA), γ-globulins, and serum on Staphylococcus aureus adhesion. While BSA and lysozyme showed no adherence even when applied at very high concentrations (100 mg/ml), γ-globulins adsorbed already from 10 mg/ml on. The adsorption of γ-globulins was, however, significantly decreased when it was mixed with BSA in a ratio of 3:1, as it is in the serum. Pretreatment of EG3OMe and PEG2k coatings with γ-globulins or serum strongly promoted adherence of S. aureus when resuspended in buffer, suggesting that γ-globulins play a pivotal role in promoting S. aureus adhesion by its IgG binding proteins; the finding that a spa-deletion mutant, lacking the IgG binding protein A, showed decreased adherence corroborated this. Similarly, when S. aureus was pretreated with serum or γ-globulins its adherence was also significantly decreased. Our findings show that particularly γ-globulins bind to the coated surfaces thus mediating adherence of S. aureus via its protein A. As pretreatment of S. aureus with serum or γ-globulins significantly decreased adherence, treatment of patients with γ-globulins before implant surgery might lower the risk of implant-associated infections. PMID:24980510

  3. Reduced bacterial adhesion to hydrocephalus shunt catheters mediated by cerebrospinal fluid proteins.

    PubMed Central

    Brydon, H L; Bayston, R; Hayward, R; Harkness, W

    1996-01-01

    BACKGROUND--Prosthetic infections are a major problem, requiring complex and lengthy management. The role of blood proteins in the pathogenesis of implant infection has been investigated, but research into the role of CSF protein in shunt infections has not been undertaken, even though a high CSF protein has been assumed to increase the risk of such infections. METHODS--New shunt catheters were exposed to either CSF or individual protein solutions, and the numbers of radiolabelled staphylococci that adhered to them were compared with controls that had been exposed to saline only. RESULTS--A significant reduction in bacteria adhering to the test catheter was found in each instance. Furthermore, the CSF with the highest protein content, from a patient with intraventricular haemorrhage, had the greatest inhibitory effect on bacterial adhesion. The effect of the solutions on the hydrophobicity of the silicone rubber was also investigated. The silicone rubber was more hydrophilic, and bacterial adhesion was less, with solutions containing a higher protein content, and these findings were in keeping with the current theories on the mechanism of bacterial adhesion to polymers. CONCLUSIONS--A high CSF protein content does not predispose to the development of shunt infections. PMID:8648336

  4. Low-cost Soybean Protein Products as Extenders in Plywood Adhesives

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Soybean flour and meal were evaluated as alternate protein extenders in plywood adhesives. This research is part of our laboratory’s efforts to develop new uses for the proteinaceous co-products from soybean and cereal processing. Ground soybean meal was tested as replacement for wheat flour in glu...

  5. Effect of Milk Proteins on Adhesion of Bacteria to Stainless Steel Surfaces

    PubMed Central

    Barnes, L.-M.; Lo, M. F.; Adams, M. R.; Chamberlain, A. H. L.

    1999-01-01

    Stainless steel coupons were treated with skim milk and subsequently challenged with individual bacterial suspensions of Staphylococcus aureus, Pseudomonas fragi, Escherichia coli, Listeria monocytogenes, and Serratia marcescens. The numbers of attached bacteria were determined by direct epifluorescence microscopy and compared with the attachment levels on clean stainless steel with two different surface finishes. Skim milk was found to reduce adhesion of S. aureus, L. monocytogenes, and S. marcescens. P. fragi and E. coli attached in very small numbers to the clear surfaces, making the effect of any adsorbed protein layer difficult to assess. Individual milk proteins α-casein, β-casein, κ-casein, and α-lactalbumin were also found to reduce the adhesion of S. aureus and L. monocytogenes. The adhesion of bacteria to samples treated with milk dilutions up to 0.001% was investigated. X-ray photoelectron spectroscopy was used to determine the proportion of nitrogen in the adsorbed films. Attached bacterial numbers were inversely related to the relative atomic percentage of nitrogen on the surface. A comparison of two types of stainless steel surface, a 2B and a no. 8 mirror finish, indicated that the difference in these levels of surface roughness did not greatly affect bacterial attachment, and reduction in adhesion to a milk-treated surface was still observed. Cross-linking of adsorbed proteins partially reversed the inhibition of bacterial attachment, indicating that protein chain mobility and steric exclusion may be important in this phenomenon. PMID:10508087

  6. Low-Cost Soybean Protein Products as Extenders in Plywood Adhesives

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Soybean flour and meal were evaluated as alternate protein extenders in plywood adhesives. This research is part of our laboratory’s efforts to develop new uses for the proteinaceous co-products from soybean and cereal processing. Ground soybean meal was tested as replacement for wheat flour in gl...

  7. Protein Kinase C beta Mediates CD40 Ligand-Induced Adhesion of Monocytes to Endothelial Cells

    PubMed Central

    Wu, Zeyu; Zhao, Gang; Peng, Lin; Du, Jialin; Wang, Sanming; Huang, Yijie; Ou, Jinrui; Jian, Zhixiang

    2013-01-01

    Accumulating evidence supports the early involvement of monocyte/macrophage recruitment to activated endothelial cells by leukocyte adhesion molecules during atherogenesis. CD40 and its ligand CD40L are highly expressed in vascular endothelial cells, but its impact on monocyte adhesion and the related molecular mechanisms are not fully understood. The present study was designed to evaluate the direct effect of CD40L on monocytic cell adhesion and gain mechanistic insight into the signaling coupling CD40L function to the proinflammatory response. Exposure of cultured human aortic endothelial cells (HAECs) to clinically relevant concentrations of CD40L (20 to 80 ng/mL) dose-dependently increased human monocytic THP-1 cells to adhere to them under static condition. CD40L treatment induced the expression of vascular cell adhesion molecule-1 (VCAM-1) mRNA and protein expression in HAECs. Furthermore, exposure of HAECs to CD40L robustly increased the activation of protein kinase C beta (PKCβ) in ECs. A selective inhibitor of PKCβ prevented the rise in VCAM-1 and THP-1 cell adhesion to ECs. Moreover, stimulation of ECs to CD40L induced nuclear factor-κB (NF-κB) activation. PKCβ inhibition abolished CD40L-induced NF-κB activation, and NF-κB inhibition reduced expression of VCAM-1, each resulting in reduced THP-1 cell adhesion. Our findings provide the evidence that CD40L increases VCAM-1 expression in ECs by activating PKCβ and NF-κB, suggesting a novel mechanism for EC activation. Finally, administration of CD40L resulted in PKCβ activation, increased VCAM-1 expression and activated monocytes adhesiveness to HAECs, processes attenuated by PKCβ inhibitor. Therefore, CD40L may contribute directly to atherogenesis by activating ECs and recruiting monocytes to them. PMID:24039784

  8. Retrograde Fluxes of Focal Adhesion Proteins in Response to Cell Migration and Mechanical Signals

    PubMed Central

    Guo, Wei-hui

    2007-01-01

    Recent studies suggest that mechanical signals mediated by the extracellular matrix play an essential role in various physiological and pathological processes; yet, how cells respond to mechanical stimuli remains elusive. Using live cell fluorescence imaging, we found that actin filaments, in association with a number of focal adhesion proteins, including zyxin and vasodilator-stimulated phosphoprotein, undergo retrograde fluxes at focal adhesions in the lamella region. This flux is inversely related to cell migration, such that it is amplified in fibroblasts immobilized on micropatterned islands. In addition, the flux is regulated by mechanical signals, including stretching forces applied to flexible substrates and substrate stiffness. Conditions favoring the flux share the common feature of causing large retrograde displacements of the interior actin cytoskeleton relative to the substrate anchorage site, which may function as a switch translating mechanical input into chemical signals, such as tyrosine phosphorylation. In turn, the stimulation of actin flux at focal adhesions may function as part of a feedback mechanism, regulating structural assembly and force production in relation to cell migration and mechanical load. The retrograde transport of associated focal adhesion proteins may play additional roles in delivering signals from focal adhesions to the interior of the cell. PMID:17804814

  9. Adhesion of MRC-5 and A549 cells on poly(dimethylsiloxane) surface modified by proteins.

    PubMed

    Zuchowska, Agnieszka; Kwiatkowski, Piotr; Jastrzebska, Elzbieta; Chudy, Michal; Dybko, Artur; Brzozka, Zbigniew

    2016-02-01

    PDMS is a very popular material used for fabrication of Lab-on-a-Chip systems for biological applications. Although PDMS has numerous advantages, it is a highly hydrophobic material, which inhibits adhesion and proliferation of the cells. PDMS surface modifications are used to enrich growth of the cells. However, due to the fact that each cell type has specific adhesion, it is necessary to optimize the parameters of these modifications. In this paper, we present an investigation of normal (MRC-5) and carcinoma (A549) human lung cell adhesion and proliferation on modified PDMS surfaces. We have chosen these cell types because often they are used as models for basic cancer research. To the best of our knowledge, this is the first presentation of this type of investigation. The combination of a gas-phase processing (oxygen plasma or ultraviolet irradiation) and wet chemical methods based on proteins' adsorption was used in our experiments. Different proteins such as poly-l-lysine, fibronectin, laminin, gelatin, and collagen were incubated with the activated PDMS samples. To compare with other works, here, we also examined how ratio of prepolymer to curing agent (5:1, 10:1, and 20:1) influences PDMS hydrophilicity during further modifications. The highest adhesion of the tested cells was observed for the usage of collagen, regardless of PDMS ratio. However, the MRC-5 cell line demonstrated better adhesion than A549 cells. This is probably due to the difference in their morphology and type (normal/cancer). PMID:26311334

  10. Thrombolytic protein from cobra venom with anti-adhesive properties.

    PubMed

    Chanda, Chandrasekhar; Sarkar, Angshuman; Chakrabarty, Dibakar

    2016-01-15

    A metalloproteinase anticoagulant toxin of molecular weight 66 kDa has been purified from the venom of Indian monocled cobra (Naja kaouthia). This toxin named as NKV 66 cleaved fibrinogen in a dose and time dependent manner. The digestion process was specific to Aα chain and cleaved fibrinogen to peptide fragments. NKV 66 completely liquefied the fibrin clots developed in vitro in 18 h. Plasma recalcification time and thrombin time were significantly prolonged following treatment of plasma with NKV 66. NKV 66 significantly inhibited ADP and collagen induced platelet aggregation in a dose dependent manner. It showed disintegrin like activity on A549 cells cultured in vitro. About 40% inhibition of adherence of A549 cells to matrix was observed following NKV 66 treatment also NKV 66 treated A549 cells were drastically inhibited from passing through the matrix in cell invasion assays in vitro, suggesting anti-adhesive properties of NKV 66. PMID:26558696

  11. Identification of proteins associated with adhesive prints from Holothuria dofleinii Cuvierian tubules.

    PubMed

    Peng, Yong Y; Glattauer, Veronica; Skewes, Timothy D; McDevitt, Andrew; Elvin, Christopher M; Werkmeister, Jerome A; Graham, Lloyd D; Ramshaw, John A M

    2014-12-01

    Cuvierian tubules are expelled as a defence mechanism against predators by various species within the family Holothuridae. When the tubules are expelled, they become sticky almost immediately and ensnare the predator. The mechanism of this rapid adhesion is not clear, but proteins on the surface of the expelled tubules are widely believed to be involved. This study has examined such proteins from Holothuria dofleinii, sourced from adhesive prints left on glass after the removal of adhered tubules. Gel electrophoresis showed that seven strongly staining protein bands were consistently present in all samples, with molecular masses ranging from 89 to 17 kDa. N-terminal sequence data was obtained from two bands, while others seemed blocked. Tandem mass spectrometry-based sequencing of tryptic peptides derived from individual protein bands indicated that the proteins were unlikely to be homopolymers. PCR primers designed using the peptide sequences enabled us to amplify, clone and sequence cDNA segments relating to four gel bands; for each, the predicted translation product contained other peptide sequences observed for that band that had not been used in primer design. Database searches using the peptide and cDNA-encoded sequences suggest that two of the seven proteins are novel and one is a C-type lectin, while-surprisingly-at least three of the other four are closely related to enzymes associated with the pentose phosphate cycle and glycolysis. We discuss precedents in which lectins and metabolic enzymes are involved in attachment and adhesion phenomena. PMID:25086572

  12. Heat shock protein 60 acts as a receptor for the Listeria adhesion protein in Caco-2 cells.

    PubMed

    Wampler, Jennifer L; Kim, Kwang-Pyo; Jaradat, Ziad; Bhunia, Arun K

    2004-02-01

    The 104-kDa Listeria adhesion protein (LAP) in Listeria monocytogenes is involved in binding to various mammalian cell lines. However, the receptor that interacts with LAP in eukaryotic cells is unknown. In this study, scanning immunoelectron microscopy qualitatively demonstrated greater binding capacity of wild-type (WT) L. monocytogenes strain (F4244) than a LAP-deficient mutant strain (KB208) to Caco-2 cells. The goal of this study was identification of the host cell receptor for LAP. Using a Western blot ligand overlay assay, we identified a protein of 58 kDa to be the putative receptor for LAP from Caco-2 cells. N-terminal sequencing and subsequent database search identified this protein as heat shock protein 60 (Hsp60). Modified immunoseparation with protein A-Sepharose beads bound to the LAP-specific monoclonal antibody H7 (MAb-H7) and a sequential incubation with LAP preparation and Caco-2 lysate confirmed the receptor to be the same 58-kDa protein. Western blot analysis with anti-Hsp60 MAb of whole-cell adhesion between Caco-2 and WT also revealed the receptor protein to be a 58-kDa protein, thus corroborating the identification of Hsp60 as a host cell receptor for LAP. Furthermore, the anti-Hsp60 antibody also caused approximately 74% reduction in binding of L. monocytogenes WT to Caco-2 cells, whereas a control antibody, C11E9, had no effect on binding. The adhesion mechanism of L. monocytogenes to eukaryotic cells is a complex process, and identification of Hsp60 as a receptor for LAP adds to the list of previously discovered ligand-receptor modules that are essential to achieve successful adhesion. PMID:14742538

  13. Rapidly light-activated surgical protein glue inspired by mussel adhesion and insect structural crosslinking.

    PubMed

    Jeon, Eun Young; Hwang, Byeong Hee; Yang, Yun Jung; Kim, Bum Jin; Choi, Bong-Hyuk; Jung, Gyu Yong; Cha, Hyung Joon

    2015-10-01

    Currently approved surgical tissue glues do not satisfy the requirements for ideal bioadhesives due to limited adhesion in wet conditions and severe cytotoxicity. Herein, we report a new light-activated, mussel protein-based bioadhesive (LAMBA) inspired by mussel adhesion and insect dityrosine crosslinking chemistry. LAMBA exhibited substantially stronger bulk wet tissue adhesion than commercially available fibrin glue and good biocompatibility in both in vitro and in vivo studies. Besides, the easily tunable, light-activated crosslinking enabled an effective on-demand wound closure and facilitated wound healing. Based on these outstanding properties, LAMBA holds great potential as an ideal surgical tissue glue for diverse medical applications, including sutureless wound closures of skin and internal organs. PMID:26197411

  14. Influence of Aae Autotransporter Protein on Adhesion and Biofilm Formation by Aggregatibacter actinomycetemcomitans.

    PubMed

    Nunes, Ana Carla Robatto; Longo, Priscila Larcher; Mayer, Marcia Pinto Alves

    2016-01-01

    The periodontopathogen Aggregatibacter actinomycetemcomitans colonizes oral cavity by binding to and invading epithelial cells as well as by participating in biofilms formed on hard surfaces. Aae, an autotransporter protein, is implicated in bacterial adhesion to epithelial cells. Due to the multiple functions of bacterial autotransporter proteins, this study aimed to evaluate the role of aae in A. actinomycetemcomitans ability to adhere to both saliva-coated hydroxyapatite (SHA) and biofilm. An aae null mutant was constructed. Its hydrophobic properties as well as its ability to adhere to epithelial cells, SHA and to form biofilm were evaluated and compared with the parental strain, A. actinomycetemcomitans VT1169. The aae null mutant showed reduced hydrophobicity, as well as decreased binding to SHA and biofilm formation compared to the parental strain. These data suggest that aae mediates A. actinomycetemcomitans adhesion to epithelial cells and may be involved in biofilm formation and interaction with adsorbed salivary proteins. PMID:27224556

  15. Mechanical and water soaking properties of medium density fiberboard with wood fiber and soybean protein adhesive.

    PubMed

    Li, Xin; Li, Yonghui; Zhong, Zhikai; Wang, Donghai; Ratto, Jo A; Sheng, Kuichuan; Sun, Xiuzhi Susan

    2009-07-01

    Soybean protein is a renewable and abundant material that offers an alternative to formaldehyde-based resins. In this study, soybean protein was modified with sodium dodecyl sulfate (SDS) as an adhesive for wood fiber medium density fiberboard (MDF) preparation. Second-order response surface regression models were used to study the effects and interactions of initial moisture content (IMC) of coated wood fiber, press time (PT) and temperature on mechanical and water soaking properties of MDF. Results showed that IMC of coated fiber was the dominant influencing factor. Mechanical and soaking properties improved as IMC increased and reached their highest point at an IMC of 35%. Press time and temperature also had a significant effect on mechanical and water soaking properties of MDF. Second-order regression results showed that there were strong relationships between mechanical and soaking properties of MDF and processing parameters. Properties of MDF made using soybean protein adhesive are similar to those of commercial board. PMID:19329303

  16. Adhesive strength and curing rate of marine mussel protein extracts on porcine small intestinal submucosa*

    PubMed Central

    Ninan, Lal; Stroshine, R L; Wilker, J.J.; Shi, Riyi

    2008-01-01

    An adhesive protein extracted from marine mussel (Mytilus edulis) was used to bond strips of connective tissue for the purpose of evaluating the use of curing agents to improve adhesive curing. Specifically, mussel adhesive protein solution (MAPS, 0.5 mM dihydroxyphenylalanine) was applied, with or without the curing agents, to the ends of two overlapping strips of porcine small intestinal submucosa. The bond strength of this lap joint was determined after curing for 1 h at room temperature (25°C). The strength of joints formed using only MAPS or with only the ethyl, butyl or octyl cyanoacrylate adhesives were determined. Although joints bonded using ethyl cyanoacrylate were strongest, those using MAPS were stronger than those using butyl and octyl cyanoacrylates. The addition of 25 mM solutions of the transition metal ions V5+, Fe3+ and Cr6+, which are all oxidants, increased the bond strength of the MAPS joints. The V5+ gave the strongest bonds and the Fe3+ the second strongest. In subsequent tests with V5+ and Fe3+ solutions, the bond strength increased with V5+ concentration, but it did not increase with Fe3+ concentration. Addition of 250 mM V5+ gave a very strong bond. PMID:17434815

  17. Ubiquitous distribution of salts and proteins in spider glue enhances spider silk adhesion

    NASA Astrophysics Data System (ADS)

    Amarpuri, Gaurav; Chaurasia, Vishal; Jain, Dharamdeep; Blackledge, Todd A.; Dhinojwala, Ali

    2015-03-01

    Modern orb-weaving spiders use micron-sized glue droplets on their viscid silk to retain prey in webs. A combination of low molecular weight salts and proteins makes the glue viscoelastic and humidity responsive in a way not easily achieved by synthetic adhesives. Optically, the glue droplet shows a heterogeneous structure, but the spatial arrangement of its chemical components is poorly understood. Here, we use optical and confocal Raman microscopy to show that salts and proteins are present ubiquitously throughout the droplet. The distribution of adhesive proteins in the peripheral region explains the superior prey capture performance of orb webs as it enables the entire surface area of the glue droplet to act as a site for prey capture. The presence of salts throughout the droplet explains the recent Solid-State NMR results that show salts directly facilitate protein mobility. Understanding the function of individual glue components and the role of the droplet's macro-structure can help in designing better synthetic adhesives for humid environments.

  18. Ubiquitous distribution of salts and proteins in spider glue enhances spider silk adhesion

    PubMed Central

    Amarpuri, Gaurav; Chaurasia, Vishal; Jain, Dharamdeep; Blackledge, Todd A.; Dhinojwala, Ali

    2015-01-01

    Modern orb-weaving spiders use micron-sized glue droplets on their viscid silk to retain prey in webs. A combination of low molecular weight salts and proteins makes the glue viscoelastic and humidity responsive in a way not easily achieved by synthetic adhesives. Optically, the glue droplet shows a heterogeneous structure, but the spatial arrangement of its chemical components is poorly understood. Here, we use optical and confocal Raman microscopy to show that salts and proteins are present ubiquitously throughout the droplet. The distribution of adhesive proteins in the peripheral region explains the superior prey capture performance of orb webs as it enables the entire surface area of the glue droplet to act as a site for prey capture. The presence of salts throughout the droplet explains the recent Solid-State NMR results that show salts directly facilitate protein mobility. Understanding the function of individual glue components and the role of the droplet's macro-structure can help in designing better synthetic adhesives for humid environments. PMID:25761668

  19. Mussel adhesive protein provides cohesive matrix for collagen type-1α

    PubMed Central

    Martinez Rodriguez, Nadine R.; Das, Saurabh; Kaufman, Yair; Wei, Wei; Israelachvili, Jacob N.; Waite, J. Herbert

    2015-01-01

    Understanding the interactions between collagen and adhesive mussel foot proteins (mfps) can lead to improved medical and dental adhesives, particularly for collagen-rich tissues. Here we investigated interactions between collagen type-1, the most abundant loadbearing animal protein, and mussel foot protein-3 (mfp-3) using a quartz crystal microbalance and surface forces apparatus (SFA). Both hydrophilic and hydrophobic variants of mfp-3 were exploited to probe the nature of the interaction between the protein and collagen. Our chief findings are: 1) mfp-3 is an effective chaperone for tropocollagen adsorption to TiO2 and mica surfaces; 2) at pH 3, collagen addition between two mfp-3 films (Wc = 5.4 ± 0.2 mJ/m2) increased their cohesion by nearly 35%; 3) oxidation of Dopa in mfp-3 by periodate did not abolish the adhesion between collagen and mfp-3 films, and 4) collagen bridging between both hydrophilic and hydrophobic mfp-3 variant films is equally robust, suggesting that hydrophobic interactions play a minor role. Extensive H-bonding, π-cation and electrostatic interactions are more plausible to explain the reversible bridging of mfp-3 films by collagen. PMID:25770997

  20. Mussel adhesive protein provides cohesive matrix for collagen type-1α.

    PubMed

    Martinez Rodriguez, Nadine R; Das, Saurabh; Kaufman, Yair; Wei, Wei; Israelachvili, Jacob N; Waite, J Herbert

    2015-05-01

    Understanding the interactions between collagen and adhesive mussel foot proteins (mfps) can lead to improved medical and dental adhesives, particularly for collagen-rich tissues. Here we investigated interactions between collagen type-1, the most abundant load-bearing animal protein, and mussel foot protein-3 (mfp-3) using a quartz crystal microbalance and surface forces apparatus (SFA). Both hydrophilic and hydrophobic variants of mfp-3 were exploited to probe the nature of the interaction between the protein and collagen. Our chief findings are: 1) mfp-3 is an effective chaperone for tropocollagen adsorption to TiO2 and mica surfaces; 2) at pH 3, collagen addition between two mfp-3 films (Wc = 5.4 ± 0.2 mJ/m(2)) increased their cohesion by nearly 35%; 3) oxidation of Dopa in mfp-3 by periodate did not abolish the adhesion between collagen and mfp-3 films, and 4) collagen bridging between both hydrophilic and hydrophobic mfp-3 variant films is equally robust, suggesting that hydrophobic interactions play a minor role. Extensive H-bonding, π-cation and electrostatic interactions are more plausible to explain the reversible bridging of mfp-3 films by collagen. PMID:25770997

  1. Adhesion properties of a putative polymorphic fimbrial subunit protein from Bifidobacterium longum subsp. longum

    PubMed Central

    SUZUKI, Kenta; NISHIYAMA, Keita; MIYAJIMA, Hiroki; OSAWA, Ro; YAMAMOTO, Yuji; MUKAI, Takao

    2015-01-01

    In our previous study, we found that the open reading frame bl0675 in the genome of Bifidobacterium longum subsp. longum isolated from human feces encoded a novel putative fimbrial protein, was highly polymorphic, and had five variants (A, B, C, D, and E types). The aim of this study was to evaluate the affinity of these variants to porcine colonic mucins (PCMs). Protein-binding properties were examined using the recombinant BL0675 protein containing a C-terminal 6 × His tag (His-BL0675). Surface plasmon resonance analysis demonstrated that the His-BL0675 A type had strong affinity to PCMs (KD = 9.82 × 10−8 M), whereas the B, C, D, and E types exhibited little or no binding. In a competitive enzyme-linked immunosorbent assay, His-BL0675 A type binding was reduced by addition of mucin oligosaccharides, suggesting that the binding occurs via carbohydrate chains of PCMs. The localization of BL0675 to the B. longum subsp. longum cell surface was confirmed by western blot analysis using A type polyclonal antibodies. Bacterial adhesion of B. longum subsp. longum to PCMs was also blocked by A type-specific antibodies; however, its adhesion properties were strain specific. Our results suggest that the BL0675 variants significantly contribute to the adhesion of B. longum subsp. longum strains. The expression and the adhesive properties of this protein are affected by genetic polymorphisms and are specific for B. longum subsp. longum strains. However, further studies are required on the properties of binding of these putative fimbrial proteins to the human gastrointestinal tract. PMID:26858927

  2. Adhesion properties of a putative polymorphic fimbrial subunit protein from Bifidobacterium longum subsp. longum.

    PubMed

    Suzuki, Kenta; Nishiyama, Keita; Miyajima, Hiroki; Osawa, Ro; Yamamoto, Yuji; Mukai, Takao

    2016-01-01

    In our previous study, we found that the open reading frame bl0675 in the genome of Bifidobacterium longum subsp. longum isolated from human feces encoded a novel putative fimbrial protein, was highly polymorphic, and had five variants (A, B, C, D, and E types). The aim of this study was to evaluate the affinity of these variants to porcine colonic mucins (PCMs). Protein-binding properties were examined using the recombinant BL0675 protein containing a C-terminal 6 × His tag (His-BL0675). Surface plasmon resonance analysis demonstrated that the His-BL0675 A type had strong affinity to PCMs (KD = 9.82 × 10(-8) M), whereas the B, C, D, and E types exhibited little or no binding. In a competitive enzyme-linked immunosorbent assay, His-BL0675 A type binding was reduced by addition of mucin oligosaccharides, suggesting that the binding occurs via carbohydrate chains of PCMs. The localization of BL0675 to the B. longum subsp. longum cell surface was confirmed by western blot analysis using A type polyclonal antibodies. Bacterial adhesion of B. longum subsp. longum to PCMs was also blocked by A type-specific antibodies; however, its adhesion properties were strain specific. Our results suggest that the BL0675 variants significantly contribute to the adhesion of B. longum subsp. longum strains. The expression and the adhesive properties of this protein are affected by genetic polymorphisms and are specific for B. longum subsp. longum strains. However, further studies are required on the properties of binding of these putative fimbrial proteins to the human gastrointestinal tract. PMID:26858927

  3. Application of tung oil to improve adhesion strength and water resistance of cottonseed meal and protein adhesives on maple veneer

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Cottonseed meal-based products show promise in serving as environment-friendly wood adhesives. However, their practical utilization is currently limited due to low durability and water resistant properties. In this research, we tested the improvement of adhesion strength and water resistance of cott...

  4. MicroRNA-145-5p inhibits gastric cancer invasiveness through targeting N-cadherin and ZEB2 to suppress epithelial–mesenchymal transition

    PubMed Central

    Jiang, Shi-Bin; He, Xu-Jun; Xia, Ying-Jie; Hu, Wei-Jian; Luo, Jun-Gang; Zhang, Jun; Tao, Hou-Quan

    2016-01-01

    MicroRNA (miR)-145-5p has been reported to function as a suppressor of cancer and plays an important role in cancer invasiveness. Epithelial–mesenchymal transition (EMT) is an important process in cancer invasion and migration. However, the involvement of miR-145-5p in EMT in human gastric cancer (GC) remains unclear. In this study, we aimed to investigate the molecular mechanisms by which miR-145-5p regulates EMT in GC invasiveness. We used quantitative real-time polymerase chain reaction to investigate the miR-145-5p expression level in GC and matched normal tissues. The effects of miR-145-5p on GC cell invasion and migration abilities were evaluated using Transwell models. The relationships among miR-145-5p and zinc-finger E-box binding homeobox 2 (ZEB2), E-cadherin, and N-cadherin were analyzed by quantitative real-time polymerase chain reaction and Western blot analyses. miR-145-5p levels in primary GC tissues obtained from 60 patients were significantly downregulated, compared to those in paired normal tissues. Lauren classification, depth of tumor invasion, lymph node metastasis, lymphatic invasion, and tumor–node–metastasis stage were associated with miR-145-5p expression. miR-145-5p inhibits the expression of the candidate target gene ZEB2 to delay the invasion and migration of GC cells. ZEB2 acts as transcriptional repressor of E-cadherin, while miR-145-5p is known to suppress N-cadherin directly to regulate EMT. Therefore, we concluded that miR-145-5p may target N-cadherin and ZEB2 directly to influence EMT. PMID:27143926

  5. Protein Adhesion and Ion Substitution (on/in)to Minerals

    NASA Astrophysics Data System (ADS)

    Charlet, L.; Fernandez Martinez, A.; Chapron, Y.; Sahai, N.; Cuello, G.; Brendle, J.; Marichal, C.

    2008-12-01

    Arsenic and pathogenic prion protein-scrapie (PrPsc) are important contaminants which may soil and water for decades, unless they are removed by sorption. Two sorption mechanisms will be discussed, namely the organics (Prp and single aminoacid) adsorption on clay and the arsenic substitution in gypsum. The elucidation of these contrasted mechanisms will be shown to request complementary molecular-mechanical simulations with experimental spectroscopic investigations. As first example, structural studies performed at ILL/ESRF on As-doped gypsum (CaSO4 2H2O) using neutron and X-ray diffraction data and EXAFS were performed to determine how As fits into the bulk of gypsum structure. The combined Rietveld analysis of neutron and X-ray diffraction data shows an expansion of the unit cell volume proportional to the As concentration within the samples. to-sulfate substitution mechanisms were used as simulation starting hypotheses. DFT-based simulations (Mulliken analysis) were used to interpret charge distribution and to show that among the possible mechanisms, a sulphate substitution by either protonated, or fully deprotonated, arsenate ion, only the protonated arsenate substitution could best fit the EXAFS data. In the second example, we used Molecular Dynamics to understand the mechanism of strong binding of the pathogenic PrP peptide with clay mineral surfaces. We modeled only the infectious moiety, PrP92-138, of the whole PrPsc structure, with explicitly solvating water molecules in contact with the cleavage plane of pyrophillite, as a model for montmorillonite without any cationic substitution. Partial residual negative charges on the cleavage plane were balanced with K+ ions. The peptide anchored to the clay surface via up to 10 hydrogen bonds from lysine and histidine residues to oxygen atoms of the siloxane cavities, and a total adsorption energy of 3465 KJ.mol-1 was obtained. Our results were compared to the one obtained by chemical and thermal analysis, 23Na, 1H

  6. Lectin Receptor Kinases Participate in Protein-Protein Interactions to Mediate Plasma Membrane-Cell Wall Adhesions in Arabidopsis1

    PubMed Central

    Gouget, Anne; Senchou, Virginie; Govers, Francine; Sanson, Arnaud; Barre, Annick; Rougé, Pierre; Pont-Lezica, Rafael; Canut, Hervé

    2006-01-01

    Interactions between plant cell walls and plasma membranes are essential for cells to function properly, but the molecules that mediate the structural continuity between wall and membrane are unknown. Some of these interactions, which are visualized upon tissue plasmolysis in Arabidopsis (Arabidopsis thaliana), are disrupted by the RGD (arginine-glycine-aspartic acid) tripeptide sequence, a characteristic cell adhesion motif in mammals. In planta induced-O (IPI-O) is an RGD-containing protein from the plant pathogen Phytophthora infestans that can disrupt cell wall-plasma membrane adhesions through its RGD motif. To identify peptide sequences that specifically bind the RGD motif of the IPI-O protein and potentially play a role in receptor recognition, we screened a heptamer peptide library displayed in a filamentous phage and selected two peptides acting as inhibitors of the plasma membrane RGD-binding activity of Arabidopsis. Moreover, the two peptides also disrupted cell wall-plasma membrane adhesions. Sequence comparison of the RGD-binding peptides with the Arabidopsis proteome revealed 12 proteins containing amino acid sequences in their extracellular domains common with the two RGD-binding peptides. Eight belong to the receptor-like kinase family, four of which have a lectin-like extracellular domain. The lectin domain of one of these, At5g60300, recognized the RGD motif both in peptides and proteins. These results imply that lectin receptor kinases are involved in protein-protein interactions with RGD-containing proteins as potential ligands, and play a structural and signaling role at the plant cell surfaces. PMID:16361528

  7. Phosphorylation of the beta-subunit of CD11/CD18 integrins by protein kinase C correlates with leukocyte adhesion.

    PubMed

    Valmu, L; Autero, M; Siljander, P; Patarroyo, M; Gahmberg, C G

    1991-11-01

    Adhesion of activated leukocytes to cells is of critical functional importance. The adhesion is known to be mediated mainly by the CD11/CD18 integrins, also known as leukocytic cell adhesion molecules, or Leu-CAM. We have now studied the phosphorylation of Leu-CAM by protein kinase C and the correlation of phosphorylation with the generation of the adhesive phenotype among human peripheral blood mononuclear leukocytes during cell activation. We here show that a good correlation exists between the phosphorylation of the beta subunit of Leu-CAM (CD18), and the extent of cell-to-cell adhesion. The phosphorylated CD18 subunit was associated with both CD11a and CD11b. Purified protein kinase C was able to phosphorylate the beta subunit of isolated Leu-CAM in vitro. The phosphorylation occurred mainly on serine residues. PMID:1682156

  8. In vitro investigation of protein adsorption and platelet adhesion on inorganic biomaterial surfaces

    NASA Astrophysics Data System (ADS)

    Huang, Yan; Lü, Xiaoying; Jingwu, Ma; Huang, Nan

    2008-11-01

    The aim of this paper was to study the surface properties, protein adsorption and platelet adhesion behaviors of diamond-like carbon (DLC) and titanium (Ti) films. The surface energy and microstructures of these films were characterized by contact angle measurement and atomic force microscopy (AFM). A modified Coomassie brilliant blue (CBB) protein assay was used to study the amount of adsorbed proteins. Platelet adhesion was assessed by scanning electron microscopy (SEM). The AFM results show that the DLC film is smoother than Ti. Protein adsorption results from CBB protein assay show that the ratio of adsorbed albumin (Alb) to IgG ( RA/I) on DLC is larger than Ti, which coincide with the sequence of the ratio of interfacial tension between solid surface and Alb ( γS,Alb) to interfacial tension between surface and IgG ( γS,IgG) ( γS,Alb/ γS,IgG). The DLC film has a preferential adsorption for Alb. The results suggest that the ratio of γS,Alb/ γS,IgG may indicate an Alb/IgG affinity ratio of materials. More platelets adhere on Ti film than on DLC, which may correspond to the surface roughness of materials. The conclusion is the blood compatibility of DLC seems to be better than Ti.

  9. Mapping the dynamics and nanoscale organization of synaptic adhesion proteins using monomeric streptavidin

    PubMed Central

    Chamma, Ingrid; Letellier, Mathieu; Butler, Corey; Tessier, Béatrice; Lim, Kok-Hong; Gauthereau, Isabel; Choquet, Daniel; Sibarita, Jean-Baptiste; Park, Sheldon; Sainlos, Matthieu; Thoumine, Olivier

    2016-01-01

    The advent of super-resolution imaging (SRI) has created a need for optimized labelling strategies. We present a new method relying on fluorophore-conjugated monomeric streptavidin (mSA) to label membrane proteins carrying a short, enzymatically biotinylated tag, compatible with SRI techniques including uPAINT, STED and dSTORM. We demonstrate efficient and specific labelling of target proteins in confined intercellular and organotypic tissues, with reduced steric hindrance and no crosslinking compared with multivalent probes. We use mSA to decipher the dynamics and nanoscale organization of the synaptic adhesion molecules neurexin-1β, neuroligin-1 (Nlg1) and leucine-rich-repeat transmembrane protein 2 (LRRTM2) in a dual-colour configuration with GFP nanobody, and show that these proteins are diffusionally trapped at synapses where they form apposed trans-synaptic adhesive structures. Furthermore, Nlg1 is dynamic, disperse and sensitive to synaptic stimulation, whereas LRRTM2 is organized in compact and stable nanodomains. Thus, mSA is a versatile tool to image membrane proteins at high resolution in complex live environments, providing novel information about the nano-organization of biological structures. PMID:26979420

  10. International Union of Basic and Clinical Pharmacology. XCIV. Adhesion G protein-coupled receptors.

    PubMed

    Hamann, Jörg; Aust, Gabriela; Araç, Demet; Engel, Felix B; Formstone, Caroline; Fredriksson, Robert; Hall, Randy A; Harty, Breanne L; Kirchhoff, Christiane; Knapp, Barbara; Krishnan, Arunkumar; Liebscher, Ines; Lin, Hsi-Hsien; Martinelli, David C; Monk, Kelly R; Peeters, Miriam C; Piao, Xianhua; Prömel, Simone; Schöneberg, Torsten; Schwartz, Thue W; Singer, Kathleen; Stacey, Martin; Ushkaryov, Yuri A; Vallon, Mario; Wolfrum, Uwe; Wright, Mathew W; Xu, Lei; Langenhan, Tobias; Schiöth, Helgi B

    2015-01-01

    The Adhesion family forms a large branch of the pharmacologically important superfamily of G protein-coupled receptors (GPCRs). As Adhesion GPCRs increasingly receive attention from a wide spectrum of biomedical fields, the Adhesion GPCR Consortium, together with the International Union of Basic and Clinical Pharmacology Committee on Receptor Nomenclature and Drug Classification, proposes a unified nomenclature for Adhesion GPCRs. The new names have ADGR as common dominator followed by a letter and a number to denote each subfamily and subtype, respectively. The new names, with old and alternative names within parentheses, are: ADGRA1 (GPR123), ADGRA2 (GPR124), ADGRA3 (GPR125), ADGRB1 (BAI1), ADGRB2 (BAI2), ADGRB3 (BAI3), ADGRC1 (CELSR1), ADGRC2 (CELSR2), ADGRC3 (CELSR3), ADGRD1 (GPR133), ADGRD2 (GPR144), ADGRE1 (EMR1, F4/80), ADGRE2 (EMR2), ADGRE3 (EMR3), ADGRE4 (EMR4), ADGRE5 (CD97), ADGRF1 (GPR110), ADGRF2 (GPR111), ADGRF3 (GPR113), ADGRF4 (GPR115), ADGRF5 (GPR116, Ig-Hepta), ADGRG1 (GPR56), ADGRG2 (GPR64, HE6), ADGRG3 (GPR97), ADGRG4 (GPR112), ADGRG5 (GPR114), ADGRG6 (GPR126), ADGRG7 (GPR128), ADGRL1 (latrophilin-1, CIRL-1, CL1), ADGRL2 (latrophilin-2, CIRL-2, CL2), ADGRL3 (latrophilin-3, CIRL-3, CL3), ADGRL4 (ELTD1, ETL), and ADGRV1 (VLGR1, GPR98). This review covers all major biologic aspects of Adhesion GPCRs, including evolutionary origins, interaction partners, signaling, expression, physiologic functions, and therapeutic potential. PMID:25713288

  11. In Vivo Detection of Vascular Adhesion Protein-1 in Experimental Inflammation

    PubMed Central

    Jaakkola, Kimmo; Nikula, Tuomo; Holopainen, Riikka; Vähäsilta, Tommi; Matikainen, Marja-Terttu; Laukkanen, Marja-Leena; Huupponen, Risto; Halkola, Lauri; Nieminen, Lauri; Hiltunen, Jukka; Parviainen, Sakari; Clark, Michael R.; Knuuti, Juhani; Savunen, Timo; Kääpä, Pekka; Voipio-Pulkki, Liisa Maria; Jalkanen, Sirpa

    2000-01-01

    Vascular adhesion protein-1 (VAP-1) is an inflammation-inducible endothelial glycoprotein which mediates leukocyte-endothelial cell interactions. To study the pathogenetic significance of VAP-1 in inflammatory disorders, an in vivo immunodetection method was used to detect the regulation of luminally expressed VAP-1 in experimental skin and joint inflammation in the pig and dog. Moreover, VAP-1 was studied as a potential target to localize inflammation by radioimmunoscintigraphy. Up-regulation of VAP-1 in experimental dermatitis and arthritis could be visualized by specifically targeted immunoscintigraphy. Moreover, the translocation of VAP-1 to the functional position on the endothelial surface was only seen in inflamed tissues. These results suggest that VAP-1 is both an optimal candidate for anti-adhesive therapy and a potential target molecule for imaging inflammation. PMID:10934150

  12. Redox Capacity of an Extracellular Matrix Protein Associated with Adhesion in Mytilus californianus.

    PubMed

    Nicklisch, Sascha C T; Spahn, Jamie E; Zhou, Hongjun; Gruian, Cristina M; Waite, J Herbert

    2016-04-01

    Adhesive mussel foot proteins (Mfps) rely in part on DOPA (3,4-dihydroxyphenyl-l-alanine) side chains to mediate attachment to mineral surfaces underwater. Oxidation of DOPA to Dopaquinone (Q) effectively abolishes the adsorption of Mfps to these surfaces. The thiol-rich mussel foot protein-6 (Mfp-6) rescues adhesion compromised by adventitious DOPA oxidation by reducing Q back to DOPA. The redox chemistry and kinetics of foot-extracted Mfp-6 were investigated by using a nonspecific chromogenic probe to equilibrate with the redox pool. Foot-extracted Mfp-6 has a reducing capacity of ~17 e(-) per protein; half of this comes from the cysteine residues, whereas the other half comes from other constituents, probably a cohort of four or five nonadhesive, redox-active DOPA residues in Mfp-6 with an anodic peak potential ~500 mV lower than that for oxidation of cysteine to cystine. At higher pH, DOPA redox reversibility is lost possibly due to Q scavenging by Cys thiolates. Analysis by one- and two-dimensional proton nuclear magnetic resonance identified a pronounced β-sheet structure with a hydrophobic core in foot-extracted Mfp-6 protein. The structure endows redox-active side chains in Mfp-6, i.e., cysteine and DOPA, with significant reducing power over a broad pH range, and this power is measurably diminished in recombinant Mfp-6. PMID:26998552

  13. Partial characterization of a human submandibular/sublingual salivary adhesion-promoting protein.

    PubMed

    Akintoye, S O; Dasso, M; Hay, D I; Ganeshkumar, N; Spielman, A I

    2002-05-01

    Human submandibular/sublingual saliva contains a protein that promotes adhesion of Streptococcus mutans JBP serotype-c to spheroidal hydroxyapatite in vitro. A high molecular-weight (250,000-300,000 Da) adhesion-promoting protein (APP) was purified by Trisacryl 2000 M gel-filtration chromatography and gel electroelution before it was partially characterized. Lectin blotting identified that the terminal carbohydrates include N-acetyl glucosamine-beta 1-4-N-acetylglucosamine, galactose and galactose-beta 1-3-N-acetyl galactosamine. Antibodies to APP demonstrated no difference in the immunoreactive pattern of APP from saliva of caries-active or caries-resistant individuals belonging to four different ethnic groups: Asian, African-American, Hispanic or Caucasian. No immunological similarities to salivary mucins or parotid agglutinins were detected by Western blotting using immuno-cross-reactivity as a criterion. APP appears to be a unique protein found in submandibular/sublingual saliva. Understanding such a protein could help prevent S. mutans attachment to the enamel surface. PMID:12015214

  14. Antiadhesive properties of arabinogalactan protein from ribes nigrum seeds against bacterial adhesion of Helicobacter pylori.

    PubMed

    Messing, Jutta; Niehues, Michael; Shevtsova, Anna; Borén, Thomas; Hensel, Andreas

    2014-01-01

    Fruit extracts from black currants (Ribes nigrum L.) are traditionally used for treatment of gastritis based on seed polysaccharides that inhibit the adhesion of Helicobacter pylori to stomach cells. For detailed investigations an arabinogalactan protein (F2) was isolated from seeds and characterized concerning molecular weight, carbohydrate, amino acid composition, linkage, configuration and reaction with β-glucosyl Yariv. Functional testing of F2 was performed by semiquantitative in situ adhesion assay on sections of human gastric mucosa and by quantitative in vitro adhesion assay with FITC-labled H. pylori strain J99 and human stomach AGS cells. Bacterial adhesins affected were identified by overlay assay with immobilized ligands. ¹²⁵I-radiolabeled F2 served for binding studies to H. pylori and interaction experiments with BabA and SabA. F2 had no cytotoxic effects against H. pylori and AGS cells; but inhibited bacterial binding to human gastric cells. F2 inhibited the binding of BabA and fibronectin-binding adhesin to its specific ligands. Radiolabeled F2 bound non-specifically to different strains of H. pylori; and to BabA deficient mutant. F2 did not lead to subsequent feedback regulation or increased expression of adhesins or virulence factors. From these data the non-specific interactions between F2 and the H. pylori lead to moderate antiadhesive effects. PMID:24662083

  15. Serum protein layers on parylene-C and silicon oxide: Effect on cell adhesion

    PubMed Central

    Delivopoulos, Evangelos; Ouberai, Myriam M.; Coffey, Paul D.; Swann, Marcus J.; Shakesheff, Kevin M.; Welland, Mark E.

    2015-01-01

    Among the range of materials used in bioengineering, parylene-C has been used in combination with silicon oxide and in presence of the serum proteins, in cell patterning. However, the structural properties of adsorbed serum proteins on these substrates still remain elusive. In this study, we use an optical biosensing technique to decipher the properties of fibronectin (Fn) and serum albumin adsorbed on parylene-C and silicon oxide substrates. Our results show the formation of layers with distinct structural and adhesive properties. Thin, dense layers are formed on parylene-C, whereas thicker, more diffuse layers are formed on silicon oxide. These results suggest that Fn acquires a compact structure on parylene-C and a more extended structure on silicon oxide. Nonetheless, parylene-C and silicon oxide substrates coated with Fn host cell populations that exhibit focal adhesion complexes and good cell attachment. Albumin adopts a deformed structure on parylene-C and a globular structure on silicon oxide, and does not support significant cell attachment on either surface. Interestingly, the co-incubation of Fn and albumin at the ratio found in serum, results in the preferential adsorption of albumin on parylene-C and Fn on silicon oxide. This finding is supported by the exclusive formation of focal adhesion complexes in differentiated mouse embryonic stem cells (CGR8), cultured on Fn/albumin coated silicon oxide, but not on parylene-C. The detailed information provided in this study on the distinct properties of layers of serum proteins on substrates such as parylene-C and silicon oxide is highly significant in developing methods for cell patterning. PMID:25555155

  16. Cadherin adhesion depends on a salt bridge at the N-terminus.

    PubMed

    Harrison, Oliver J; Corps, Elaine M; Kilshaw, Peter J

    2005-09-15

    There is now considerable evidence that cell adhesion by cadherins requires a strand exchange process in which the second amino acid at the N-terminus of the cadherin molecule, Trp2, docks into a hydrophobic pocket in the domain fold of the opposing cadherin. Here we show that strand exchange depends on a salt bridge formed between the N-terminal amino group of one cadherin molecule and the acidic side chain of Glu89 of the other. Prevention of this bond in N-cadherin by introducing the mutation Glu89Ala or by extending the N-terminus with additional amino acids strongly inhibited strand exchange. But when the two modifications were present in opposing cadherin molecules respectively, they acted in a complementary manner, lowering activation energy for strand exchange and greatly increasing the strength of the adhesive interaction. N-cadherin that retained an uncleaved prodomain or lacked Trp2 adhered strongly to the Glu89Ala mutant but not to wild-type molecules. Similarly, N-cadherin in which the hydrophobic acceptor pocket was blocked by an isoleucine side chain adhered to a partner that had an extended N-terminus. We explain these results in terms of the free energy changes that accompany strand exchange. Our findings provide new insight into the mechanism of adhesion and demonstrate the feasibility of greatly increasing cadherin affinity. PMID:16118243

  17. Wiskott–Aldrich syndrome protein is involved in αIIbβ3-mediated cell adhesion

    PubMed Central

    Tsuboi, Shigeru; Nonoyama, Shigeaki; Ochs, Hans D

    2006-01-01

    The Wiskott–Aldrich syndrome (WAS) is an X-chromosome-linked immunodeficiency disorder. The most common symptom seen in WAS patients is bleeding. One of the main causes of bleeding is defective platelet aggregation. The causative gene of WAS encodes WAS protein (WASP). Here, we show that WASP binds to the calcium- and integrin-binding protein (CIB) in platelets. CIB was originally identified as a protein binding to the αIIb cytoplasmic tail of platelet integrin αIIbβ3, which has a primary role in platelet aggregation. We also show that the WASP–CIB complex is important in αIIbβ3-mediated cell adhesion, and that in patients mutant forms of WASP are expressed at reduced levels or show lower affinities for CIB than wild-type WASP. Our results indicate that impaired complex formation between mutant WASPs and CIB reduces αIIbβ3-mediated cell adhesion and causes defective platelet aggregation, resulting in bleeding. PMID:16582881

  18. Amigo Adhesion Protein Regulates Development of Neural Circuits in Zebrafish Brain*

    PubMed Central

    Zhao, Xiang; Kuja-Panula, Juha; Sundvik, Maria; Chen, Yu-Chia; Aho, Vilma; Peltola, Marjaana A.; Porkka-Heiskanen, Tarja; Panula, Pertti; Rauvala, Heikki

    2014-01-01

    The Amigo protein family consists of three transmembrane proteins characterized by six leucine-rich repeat domains and one immunoglobulin-like domain in their extracellular moieties. Previous in vitro studies have suggested a role as homophilic adhesion molecules in brain neurons, but the in vivo functions remain unknown. Here we have cloned all three zebrafish amigos and show that amigo1 is the predominant family member expressed during nervous system development in zebrafish. Knockdown of amigo1 expression using morpholino oligonucleotides impairs the formation of fasciculated tracts in early fiber scaffolds of brain. A similar defect in fiber tract development is caused by mRNA-mediated expression of the Amigo1 ectodomain that inhibits adhesion mediated by the full-length protein. Analysis of differentiated neural circuits reveals defects in the catecholaminergic system. At the behavioral level, the disturbed formation of neural circuitry is reflected in enhanced locomotor activity and in the inability of the larvae to perform normal escape responses. We suggest that Amigo1 is essential for the development of neural circuits of zebrafish, where its mechanism involves homophilic interactions within the developing fiber tracts and regulation of the Kv2.1 potassium channel to form functional neural circuitry that controls locomotion. PMID:24904058

  19. Characterizing the modification of surface proteins with poly(ethylene glycol) to interrupt platelet adhesion

    PubMed Central

    Xu, Haiyan; Kaar, Joel L.; Russell, Alan J.; Wagner, William R.

    2010-01-01

    Surface protein modification with poly(ethylene glycol) (PEG) can inhibit acute thrombosis on damaged vascular and biomaterial surfaces by blocking surface protein–platelet interactions. However, the feasibility of employing protein reactive PEGs to limit intravascular and biomaterial thrombosis in vivo is contingent upon rapid and extensive surface protein modification. To characterize the factors controlling this potential therapeutic approach, the model protein bovine serum albumin was adsorbed onto polyurethane surfaces and modified with PEG-carboxymethyl succinimidyl ester (PEG-NHS), PEG-isocyanate (PEG-ISO), or PEG-diisocyanate (PEG-DISO) in aqueous buffer at varying concentrations and contact times. It was found that up to 5 PEGs could be attached per albumin molecule within one min and that adsorbed albumin PEGylation approached maximal levels by 6 min. The lability of reactive PEGs in aqueous buffer reduced total protein modification by 50% when the PEG solution was incubated for 7 min prior to application. For fibrinogen PEGylation (performed in the solution phase), PEG-NHS was more reactive than PEG-ISO or PEG-DISO. The γ peptide of fibrinogen, which contains several key platelet-binding motifs, was highly modified. A marked reduction in platelet adhesion was observed on fibrinogen-adsorbed polyurethane treated with PEG-NHS or PEG-DISO. Relative differences in platelet adhesion on PEG-NHS and PEG-DISO modified surfaces could be attributed to differences in reactivity towards fibrinogen and the size of the polymer backbone. Taken together, these findings provide insight and guidance for applying protein reactive PEGs for the interruption of acute thrombotic deposition. PMID:16457880

  20. Paradigms lost-an emerging role for over-expression of tight junction adhesion proteins in cancer pathogenesis.

    PubMed

    Leech, Astrid O; Cruz, Rodrigo G B; Hill, Arnold D K; Hopkins, Ann M

    2015-08-01

    Tight junctions (TJ) are multi-protein complexes located at the apicalmost tip of the lateral membrane in polarised epithelial and endothelial cells. Their principal function is in mediating intercellular adhesion and polarity. Accordingly, it has long been a paradigm that loss of TJ proteins and consequent deficits in cell-cell adhesion are required for tumour cell dissemination in the early stages of the invasive/metastatic cascade. However it is becoming increasingly apparent that TJ proteins play important roles in not just adhesion but also intracellular signalling events, activation of which can contribute to, or even drive, tumour progression and metastasis. In this review, we shall therefore highlight cases wherein the gain of TJ proteins has been associated with signals promoting tumour progression. We will also discuss the potential of overexpressed TJ proteins to act as therapeutic targets in cancer treatment. The overall purpose of this review is not to disprove the fact that loss of TJ-based adhesion contributes to the progression of several cancers, but rather to introduce the growing body of evidence that gain of TJ proteins may have adhesion-independent consequences for promoting progression in other cancers. PMID:26366401

  1. Focal adhesion kinase regulates expression of thioredoxin-interacting protein (TXNIP) in cancer cells.

    PubMed

    Ho, Baotran; Huang, Grace; Golubovskaya, Vita M

    2014-01-01

    Focal Adhesion Kinase (FAK) plays an important role in cancer cell survival. Previous microarray gene profiling study detected inverse regulation between expression of thioredoxin-interacting protein (TXNIP) and FAK, where down-regulation of FAK by siRNA in MCF-7 cells caused up-regulation of TXNIP mRNA level, and in contrast up-regulation of doxycyclin- induced FAK caused repression of TXNIP. In the present report, we show that overexpression of FAK in MCF-7 cells repressed TXNIP promoter activity. Treatment of MCF-7 cells with 1alpha, 25-dihydroxyvitamin D3 (1,25D) down-regulated endogenous FAK and up-regulated TXNIP protein level, and treatment with 5-FU decreased FAK protein expression and up-regulated TXNIP protein expression in 293 cells. Moreover, silencing of FAK with siRNA increased TXNIP protein expression, while overexpression of FAK inhibited TXNIP protein expression in 293 cells. In addition, treatment of DBTRG glioblastoma cells with FAK inhibitor Y15 increased TXNIP mRNA, decreased cancer cell viability and increased apoptosis. These results for the first time demonstrate FAK-regulated TXNIP expression which is important for apoptotic, survival and oxidative stress signaling pathways in cancer cells. PMID:23387972

  2. Nanospherical arabinogalactan proteins are a key component of the high-strength adhesive secreted by English ivy.

    PubMed

    Huang, Yujian; Wang, Yongzhong; Tan, Li; Sun, Leming; Petrosino, Jennifer; Cui, Mei-Zhen; Hao, Feng; Zhang, Mingjun

    2016-06-01

    Over 130 y have passed since Charles Darwin first discovered that the adventitious roots of English ivy (Hedera helix) exude a yellowish mucilage that promotes the capacity of this plant to climb vertical surfaces. Unfortunately, little progress has been made in elucidating the adhesion mechanisms underlying this high-strength adhesive. In the previous studies, spherical nanoparticles were observed in the viscous exudate. Here we show that these nanoparticles are predominantly composed of arabinogalactan proteins (AGPs), a superfamily of hydroxyproline-rich glycoproteins present in the extracellular spaces of plant cells. The spheroidal shape of the AGP-rich ivy nanoparticles results in a low viscosity of the ivy adhesive, and thus a favorable wetting behavior on the surface of substrates. Meanwhile, calcium-driven electrostatic interactions among carboxyl groups of the AGPs and the pectic acids give rise to the cross-linking of the exuded adhesive substances, favor subsequent curing (hardening) via formation of an adhesive film, and eventually promote the generation of mechanical interlocking between the adventitious roots of English ivy and the surface of substrates. Inspired by these molecular events, a reconstructed ivy-mimetic adhesive composite was developed by integrating purified AGP-rich ivy nanoparticles with pectic polysaccharides and calcium ions. Information gained from the subsequent tensile tests, in turn, substantiated the proposed adhesion mechanisms underlying the ivy-derived adhesive. Given that AGPs and pectic polysaccharides are also observed in bioadhesives exuded by other climbing plants, the adhesion mechanisms revealed by English ivy may forward the progress toward understanding the general principles underlying diverse botanic adhesives. PMID:27217558

  3. Nanospherical arabinogalactan proteins are a key component of the high-strength adhesive secreted by English ivy

    NASA Astrophysics Data System (ADS)

    Huang, Yujian; Wang, Yongzhong; Tan, Li; Sun, Leming; Petrosino, Jennifer; Cui, Mei-Zhen; Hao, Feng; Zhang, Mingjun

    2016-06-01

    Over 130 y have passed since Charles Darwin first discovered that the adventitious roots of English ivy (Hedera helix) exude a yellowish mucilage that promotes the capacity of this plant to climb vertical surfaces. Unfortunately, little progress has been made in elucidating the adhesion mechanisms underlying this high-strength adhesive. In the previous studies, spherical nanoparticles were observed in the viscous exudate. Here we show that these nanoparticles are predominantly composed of arabinogalactan proteins (AGPs), a superfamily of hydroxyproline-rich glycoproteins present in the extracellular spaces of plant cells. The spheroidal shape of the AGP-rich ivy nanoparticles results in a low viscosity of the ivy adhesive, and thus a favorable wetting behavior on the surface of substrates. Meanwhile, calcium-driven electrostatic interactions among carboxyl groups of the AGPs and the pectic acids give rise to the cross-linking of the exuded adhesive substances, favor subsequent curing (hardening) via formation of an adhesive film, and eventually promote the generation of mechanical interlocking between the adventitious roots of English ivy and the surface of substrates. Inspired by these molecular events, a reconstructed ivy-mimetic adhesive composite was developed by integrating purified AGP-rich ivy nanoparticles with pectic polysaccharides and calcium ions. Information gained from the subsequent tensile tests, in turn, substantiated the proposed adhesion mechanisms underlying the ivy-derived adhesive. Given that AGPs and pectic polysaccharides are also observed in bioadhesives exuded by other climbing plants, the adhesion mechanisms revealed by English ivy may forward the progress toward understanding the general principles underlying diverse botanic adhesives.

  4. Understanding Marine Mussel Adhesion

    SciTech Connect

    H. G. Silverman; F. F. Roberto

    2007-12-01

    In addition to identifying the proteins that have a role in underwater adhesion by marine mussels, research efforts have focused on identifying the genes responsible for the adhesive proteins, environmental factors that may influence protein production, and strategies for producing natural adhesives similar to the native mussel adhesive proteins. The production-scale availability of recombinant mussel adhesive proteins will enable researchers to formulate adhesives that are waterimpervious and ecologically safe and can bind materials ranging from glass, plastics, metals, and wood to materials, such as bone or teeth, biological organisms, and other chemicals or molecules. Unfortunately, as of yet scientists have been unable to duplicate the processes that marine mussels use to create adhesive structures. This study provides a background on adhesive proteins identified in the blue mussel, Mytilus edulis, and introduces our research interests and discusses the future for continued research related to mussel adhesion.

  5. Understanding marine mussel adhesion.

    PubMed

    Silverman, Heather G; Roberto, Francisco F

    2007-01-01

    In addition to identifying the proteins that have a role in underwater adhesion by marine mussels, research efforts have focused on identifying the genes responsible for the adhesive proteins, environmental factors that may influence protein production, and strategies for producing natural adhesives similar to the native mussel adhesive proteins. The production-scale availability of recombinant mussel adhesive proteins will enable researchers to formulate adhesives that are water-impervious and ecologically safe and can bind materials ranging from glass, plastics, metals, and wood to materials, such as bone or teeth, biological organisms, and other chemicals or molecules. Unfortunately, as of yet scientists have been unable to duplicate the processes that marine mussels use to create adhesive structures. This study provides a background on adhesive proteins identified in the blue mussel, Mytilus edulis, and introduces our research interests and discusses the future for continued research related to mussel adhesion. PMID:17990038

  6. Understanding Marine Mussel Adhesion

    PubMed Central

    Roberto, Francisco F.

    2007-01-01

    In addition to identifying the proteins that have a role in underwater adhesion by marine mussels, research efforts have focused on identifying the genes responsible for the adhesive proteins, environmental factors that may influence protein production, and strategies for producing natural adhesives similar to the native mussel adhesive proteins. The production-scale availability of recombinant mussel adhesive proteins will enable researchers to formulate adhesives that are water-impervious and ecologically safe and can bind materials ranging from glass, plastics, metals, and wood to materials, such as bone or teeth, biological organisms, and other chemicals or molecules. Unfortunately, as of yet scientists have been unable to duplicate the processes that marine mussels use to create adhesive structures. This study provides a background on adhesive proteins identified in the blue mussel, Mytilus edulis, and introduces our research interests and discusses the future for continued research related to mussel adhesion. PMID:17990038

  7. Inhibition of fibroblast adhesion by covalently immobilized protein repellent polymer coatings studied by single cell force spectroscopy.

    PubMed

    Aliuos, Pooyan; Sen, Aromita; Reich, Uta; Dempwolf, Wibke; Warnecke, Athanasia; Hadler, Christoph; Lenarz, Thomas; Menzel, Henning; Reuter, Guenter

    2014-01-01

    Cochlea implants (CI) restore the hearing in patients with sensorineural hearing loss by electrical stimulation of the auditory nerve via an electrode array. The increase of the impedance at the electrode-tissue interface due to a postoperative connective tissue encapsulation leads to higher power consumption of the implants. Therefore, reduced adhesion and proliferation of connective tissue cells around the CI electrode array is of great clinical interest. The adhesion of cells to substrate surfaces is mediated by extracellular matrix (ECM) proteins. Protein repellent polymers (PRP) are able to inhibit unspecific protein adsorption. Thus, a reduction of cell adhesion might be achieved by coating the electrode carriers with PRPs. The aim of this study was to investigate the effects of two different PRPs, poly(dimethylacrylamide) (PDMAA) and poly(2-ethyloxazoline) (PEtOx), on the strength and the temporal dynamics of the initial adhesion of fibroblasts. Polymers were immobilized onto glass plates by a photochemical grafting onto method. Water contact angle measurements proved hydrophilic surface properties of both PDMAA and PEtOx (45 ± 1° and 44 ± 1°, respectively). The adhesion strength of NIH3T3 fibroblasts after 5, 30, and 180 s of interaction with surfaces was investigated by using single cell force spectroscopy. In comparison to glass surfaces, both polymers reduced the adhesion of fibroblasts significantly at all different interaction times and lower dynamic rates of adhesion were observed. Thus, both PDMAA and PEtOx represented antiadhesive properties and can be used as implant coatings to reduce the unspecific ECM-mediated adhesion of fibroblasts to surfaces. PMID:23596088

  8. A standardized bamboo leaf extract inhibits monocyte adhesion to endothelial cells by modulating vascular cell adhesion protein-1

    PubMed Central

    Choi, Sunga; Park, Myoung Soo; Lee, Yu Ran; Lee, Young Chul; Kim, Tae Woo; Do, Seon-Gil; Kim, Dong Seon

    2013-01-01

    Bamboo leaves (Phyllostachys pubescens Mazel ex J. Houz (Poacea)) have a long history of food and medical applications in Asia, including Japan and Korea. They have been used as a traditional medicine for centuries. We investigated the mechanism of anti-inflammatory activity of a bamboo leaf extract (BLE) on tumor necrosis factor-alpha (TNF-α)-induced monocyte adhesion in human umbilical vein endothelial cells (HUVECs). Exposure of HUVECs to BLE did not inhibit cell viability or cause morphological changes at concentrations ranging from 1 µg/ml to 1 mg/ml. Treatment with 0.1 mg/ml BLE caused 63% inhibition of monocyte adhesion in TNF-α-activated HUVECs, which was associated with 38.4% suppression of vascular cell adhesion molecule-1 expression. Furthermore, TNF-α-induced reactive oxygen species generation was decreased to 47.9% in BLE treated TNF-α-activated HUVECs. BLE (0.05 mg/ml) also caused about 50% inhibition of interleukin-6 secretion from lipopolysaccharide-stimulated monocyte. The results indicate that BLE may be clinically useful as an anti-inflammatory or anti-oxidant for human cardiovascular disease including atherosclerosis. PMID:23422838

  9. Protein-mediated adhesion of the dissimilatory Fe(III)-reducing bacterium Shewanella alga BrY to hydrous ferric oxide

    SciTech Connect

    Caccavo, F. Jr.

    1999-11-01

    The rate and extent of bacterial Fe(III) mineral reduction are governed by molecular-scale interactions between the bacterial cell surface and the mineral surface. These interactions are poorly understood. This study examined the role of surface proteins in the adhesion of Shewanella alga BrY to hydrous ferric oxide (HFO). Enzymatic degradation of cell surface polysaccharides had no effect on cell adhesion to HFO. The proteolytic enzymes Streptomyces griseus protease and chymotrypsin inhibited the adhesion of S. alga BrY cells to HFO through catalytic degradation of surface proteins. Trypsin inhibited S. alga BrY adhesion solely through surface-coating effects. Protease and chymotrypsin also mediated desorption of adhered S. alga BrY cells from HFO while trypsin did not mediate cell desorption. Protease removed a single peptide band that represented a protein with an apparent molecular mass of 50 kDa. Chymotrypsin removed two peptide bands that represented proteins with apparent molecular masses of 60 and 31 kDa. These proteins represent putative HGO adhesion molecules. A. alga BrY adhesion was inhibited by up to 46% when cells were cultured at sub-MICs of chloramphenicol, suggesting that protein synthesis is necessary for adhesion. Proteins extracted from the surface of S. alga BrY cells inhibited adhesion to HFO by up to 41%. A number of these proteins bound specifically to HFO, suggesting that a complex system of surface proteins mediates S. alga BrY adhesion to HFO.

  10. The proline-rich focal adhesion and microfilament protein VASP is a ligand for profilins.

    PubMed Central

    Reinhard, M; Giehl, K; Abel, K; Haffner, C; Jarchau, T; Hoppe, V; Jockusch, B M; Walter, U

    1995-01-01

    Profilins are small proteins that form complexes with G-actin and phosphoinositides and are therefore considered to link the microfilament system to signal transduction pathways. In addition, they bind to poly-L-proline, but the biological significance of this interaction is not yet known. The recent molecular cloning of the vasodilator-stimulated phosphoprotein (VASP), an established in vivo substrate of cAMP- and cGMP-dependent protein kinases, revealed the presence of a proline-rich domain which prompted us to investigate a possible interaction with profilins. VASP is a microfilament and focal adhesion associated protein which is also concentrated in highly dynamic regions of the cell cortex. Here, we demonstrate that VASP is a natural proline-rich profilin ligand. Human platelet VASP bound directly to purified profilins from human platelets, calf thymus and birch pollen. Moreover, VASP and a novel protein were specifically extracted from total cell lysates by profilin affinity chromatography and subsequently eluted either with poly-L-proline or a peptide corresponding to a proline-rich VASP motif. Finally, the subcellular distributions of VASP and profilin suggest that both proteins also interact within living cells. Our data support the hypothesis that profilin and VASP act in concert to convey signal transduction to actin filament formation. Images PMID:7737110

  11. Recombinant mussel adhesive protein fp-5 (MAP fp-5) as a bulk bioadhesive and surface coating material.

    PubMed

    Choi, Yoo Seong; Kang, Dong Gyun; Lim, Seonghye; Yang, Yun Jung; Kim, Chang Sup; Cha, Hyung Joon

    2011-08-01

    Mussel adhesive proteins (MAPs) attach to all types of inorganic and organic surfaces, even in wet environments. MAP of type 5 (fp-5), in particular, has been considered as a key adhesive material. However, the low availability of fp-5 has hampered its biochemical characterization and practical applications. Here, soluble recombinant fp-5 is mass-produced in Escherichia coli. Tyrosinase-modified recombinant fp-5 showed ∼1.11 MPa adhesive shear strength, which is the first report of a bulk-scale adhesive force measurement for purified recombinant of natural MAP type. Surface coatings were also performed through simple dip-coating of various objects. In addition, complex coacervate using recombinant fp-5 and hyaluronic acid was prepared as an efficient adhesive formulation, which greatly improved the bulk adhesive strength. Collectively, it is expected that this work will enhance basic understanding of mussel adhesion and that recombinant fp-5 can be successfully used as a realistic bulk-scale bioadhesive and an efficient surface coating material. PMID:21770718

  12. Peptide-presenting two-dimensional protein matrix on supported lipid bilayers: an efficient platform for cell adhesion.

    PubMed

    Bérat, Rémi; Rémy-Zolghadry, Murielle; Gounou, Céline; Manigand, Claude; Tan, Sisareuth; Saltó, Carmen; Arenas, Ernest; Bordenave, Laurence; Brisson, Alain R

    2007-12-01

    Understanding and controlling cell adhesion to biomaterials and synthetic materials are important issues in basic research and applied sciences. Supported lipid bilayers (SLBs) functionalized with cell adhesion peptides linked to lipid molecules are popular platforms of cell adhesion. In this paper, an alternative approach of peptide presentation is presented in which peptides are stereo-selectively linked to proteins self-assembling in a rigid two-dimensional (2D) matrix on SLBs. Annexin-A5 (Anx5) was used as prototype protein for its known properties of forming stable and rigid 2D matrices on lipid surfaces. Two types of Anx5-peptide complexes, containing either a RGD or an IKVAV sequence, were synthesized. The authors show that both Anx5-peptide complexes present the same properties of binding and 2D organization on lipid surfaces as Anx5, when investigated by quartz crystal microbalance with dissipation monitoring, atomic force microscopy, and transmission electron microscopy techniques. Anx5-RGD and Anx5-IKVAV 2D matrices were found to promote specific adhesion of human saphenous vein endothelial cells and mouse embryonic stem cells, respectively. The influence of the surface density of exposed peptides on cell adhesion was investigated, showing that cells attach to Anx5-peptide matrices when the average distance between peptides is smaller than about 60 nm. This cell adhesion platform provides control of the orientation and density of cell ligands, opening interesting possibilities for future applications. PMID:20408654

  13. Vasoconstrictor-induced endocytic recycling regulates focal adhesion protein localization and function in vascular smooth muscle

    PubMed Central

    Poythress, Ransom H.; Gallant, Cynthia; Vetterkind, Susanne

    2013-01-01

    Turnover of focal adhesions (FAs) is known to be critical for cell migration and adhesion of proliferative vascular smooth muscle (VSM) cells. However, it is often assumed that FAs in nonmigratory, differentiated VSM (dVSM) cells embedded in the wall of healthy blood vessels are stable structures. Recent work has demonstrated agonist-induced actin polymerization and Src-dependent FA phosphorylation in dVSM cells, suggesting that agonist-induced FA remodeling occurs. However, the mechanisms and extent of FA remodeling are largely unknown in dVSM. Here we show, for the first time, that a distinct subpopulation of dVSM FA proteins, but not the entire FA, remodels in response to the α-agonist phenylephrine. Vasodilator-stimulated phosphoprotein and zyxin displayed the largest redistributions, while β-integrin and FA kinase showed undetectable redistribution. Vinculin, metavinculin, Src, Crk-associated substrate, and paxillin displayed intermediate degrees of redistribution. Redistributions into membrane fractions were especially prominent, suggesting endosomal mechanisms. Deconvolution microscopy, quantitative colocalization analysis, and Duolink proximity ligation assays revealed that phenylephrine increases the association of FA proteins with early endosomal markers Rab5 and early endosomal antigen 1. Endosomal disruption with the small-molecule inhibitor primaquine inhibits agonist-induced redistribution of FA proteins, confirming endosomal recycling. FA recycling was also inhibited by cytochalasin D, latrunculin B, and colchicine, indicating that the redistribution is actin- and microtubule-dependent. Furthermore, inhibition of endosomal recycling causes a significant inhibition of the rate of development of agonist-induced dVSM contractions. Thus these studies are consistent with the concept that FAs in dVSM cells, embedded in the wall of the aorta, remodel during the action of a vasoconstrictor. PMID:23703522

  14. Adhesion and fusion efficiencies of human immunodeficiency virus type 1 (HIV-1) surface proteins

    NASA Astrophysics Data System (ADS)

    Dobrowsky, Terrence M.; Rabi, S. Alireza; Nedellec, Rebecca; Daniels, Brian R.; Mullins, James I.; Mosier, Donald E.; Siliciano, Robert F.; Wirtz, Denis

    2013-10-01

    In about half of patients infected with HIV-1 subtype B, viral populations shift from utilizing the transmembrane protein CCR5 to CXCR4, as well as or instead of CCR5, during late stage progression of the disease. How the relative adhesion efficiency and fusion competency of the viral Env proteins relate to infection during this transition is not well understood. Using a virus-cell fusion assay and live-cell single-molecule force spectroscopy, we compare the entry competency of viral clones to tensile strengths of the individual Env-receptor bonds of Env proteins obtained from a HIV-1 infected patient prior to and during coreceptor switching. The results suggest that the genetic determinants of viral entry were predominantly enriched in the C3, HR1 and CD regions rather than V3. Env proteins can better mediate entry into cells after coreceptor switch; this effective entry capacity does not correlate with the bond strengths between viral Env and cellular receptors.

  15. Fabrication of three-dimensional multi-protein microstructures for cell migration and adhesion enhancement

    PubMed Central

    Da Sie, Yong; Li, Yi-Cheng; Chang, Nan-Shan; Campagnola, Paul J.; Chen, Shean-Jen

    2015-01-01

    In this study, three-dimensional (3D) multi-component microstructures were precisely fabricated via multiphoton excited photochemistry using a femtosecond laser direct-writing system with proposed repetition positioning and vector scanning techniques. Extracellular matrix (ECM) proteins, such as fibronectin (FN), are difficult to stack and form 3D structures larger than several-hundred microns in height due to the nature of their protein structure. Herein, to fabricate complex 3D microstructures with FN, a 3D scaffold was designed and formed from bovine serum albumin (BSA), after which human FN was inserted at specific locations on the BSA scaffold; in this manner, the fabricated ECM microstructure can guide cells in a 3D environment. A human breast cancer cell line, MDA-MB-231, was used to investigate the behavior of cell migration and adhesion on the fabricated human FN and BSA protein structures. Experimental results indicate that many cells are not able to attach or climb on a 3D structure’s inclined plane without FN support; hence, the influence of cell growth in a 3D context with FN should being taken into consideration. This 3D multi-protein fabrication technique holds potential for cell studies in designed complex 3D ECM scaffolds. PMID:25780738

  16. Syntenin-1 and Ezrin Proteins Link Activated Leukocyte Cell Adhesion Molecule to the Actin Cytoskeleton*

    PubMed Central

    Tudor, Cicerone; te Riet, Joost; Eich, Christina; Harkes, Rolf; Smisdom, Nick; Bouhuijzen Wenger, Jessica; Ameloot, Marcel; Holt, Matthew; Kanger, Johannes S.; Figdor, Carl G.; Cambi, Alessandra; Subramaniam, Vinod

    2014-01-01

    Activated leukocyte cell adhesion molecule (ALCAM) is a type I transmembrane protein member of the immunoglobulin superfamily of cell adhesion molecules. Involved in important pathophysiological processes such as the immune response, cancer metastasis, and neuronal development, ALCAM undergoes both homotypic interactions with other ALCAM molecules and heterotypic interactions with the surface receptor CD6 expressed at the T cell surface. Despite biochemical and biophysical evidence of a dynamic association between ALCAM and the actin cytoskeleton, no detailed information is available about how this association occurs at the molecular level. Here, we exploit a combination of complementary microscopy techniques, including FRET detected by fluorescence lifetime imaging microscopy and single-cell force spectroscopy, and we demonstrate the existence of a preformed ligand-independent supramolecular complex where ALCAM stably interacts with actin by binding to syntenin-1 and ezrin. Interaction with the ligand CD6 further enhances these multiple interactions. Altogether, our results propose a novel biophysical framework to understand the stabilizing role of the ALCAM supramolecular complex engaged to CD6 during dendritic cell-T cell interactions and provide novel information on the molecular players involved in the formation and signaling of the immunological synapse at the dendritic cell side. PMID:24662291

  17. Evaluation of Serum Vascular Adhesion Protein-1 as a Potential Biomarker in Thyroid Cancer

    PubMed Central

    Zhao, Pengxin; Zhang, Kaili

    2016-01-01

    Vascular adhesion protein-1 (VAP-1) is a glycoprotein that mediates tissue-selective lymphocyte adhesion. The prognostic value of VAP-1 has been determined in gastric cancer. The aim of this study was to evaluate the changes and the predictive value of serum VAP-1 in patients with thyroid cancer. A total of 126 patients with thyroid nodules and 53 healthy controls participated in this study. The patients were further divided into subgroup 1 (69 cases with benign thyroid nodules) and subgroup 2 (57 cases with thyroid cancer). Serum VAP-1 was measured by time-resolved immunofluorometric assay. Diagnostic value of presurgical VAP-1 for thyroid cancer was conducted by receiver operating characteristic (ROC) curves. Serum levels of VAP-1 were significantly lower in thyroid cancer group than in healthy control and benign thyroid nodule groups. VAP-1 concentrations negatively correlated with serum thyroglobulin (Tg) levels in thyroid cancer patients (r = −0.81; p < 0.001). The optimum cut-off value of VAP-1 was 456.6 ng/mL with a 77.4% specificity and 66.7% sensitivity for thyroid cancer diagnosis. Serum VAP-1 decreased in thyroid cancer patients and VAP-1 could be a potential useful adjunct biomarker in the diagnosis of thyroid cancer. PMID:27446209

  18. Anterior Gradient Protein-2 Is a Regulator of Cellular Adhesion in Prostate Cancer

    PubMed Central

    Chanda, Diptiman; Lee, Joo Hyoung; Sawant, Anandi; Hensel, Jonathan A.; Isayeva, Tatyana; Reilly, Stephanie D.; Siegal, Gene P.; Smith, Claire; Grizzle, William; Singh, Raj; Ponnazhagan, Selvarangan

    2014-01-01

    Anterior Gradient Protein (AGR-2) is reported to be over-expressed in many epithelial cancers and promotes metastasis. A clear-cut mechanism for its observed function(s) has not been previously identified. We found significant upregulation of AGR-2 expression in a bone metastatic prostate cancer cell line, PC3, following culturing in bone marrow-conditioned medium. Substantial AGR-2 expression was also confirmed in prostate cancer tissue specimens in patients with bone lesions. By developing stable clones of PC3 cells with varying levels of AGR-2 expression, we identified that abrogation of AGR-2 significantly reduced cellular attachment to fibronectin, collagen I, collagen IV, laminin I and fibrinogen. Loss of cellular adhesion was associated with sharp decrease in the expression of α4, α5, αV, β3 and β4 integrins. Failure to undergo apoptosis following detachment is a hallmark of epithelial cancer metastasis. The AGR-2-silenced PC3 cells showed higher resistance to Tumor necrosis factor-related apoptosis- inducing ligand (TRAIL) induced apoptosis in vitro. This observation was also supported by significantly reduced Caspase-3 expression in AGR-2-silenced PC3 cells, which is a key effector of both extrinsic and intrinsic death signaling pathways. These data suggest that AGR-2 influence prostate cancer metastasis by regulation of cellular adhesion and apoptosis. PMID:24587138

  19. Characterization of the protein fraction of the temporary adhesive secreted by the tube feet of the sea star Asterias rubens.

    PubMed

    Hennebert, Elise; Wattiez, Ruddy; Waite, J Herbert; Flammang, Patrick

    2012-01-01

    Sea stars are able to make firm but temporary attachments to various substrata by secretions released by their tube feet. After tube foot detachment, the adhesive secretions remain on the substratum as a footprint. Proteins presumably play a key role in sea star adhesion, as evidenced by the removal of footprints from surfaces after a treatment with trypsin. However, until now, characterisation was hampered by their high insolubility. In this study, a non-hydrolytic method was used to render most of the proteins constituting the adhesive footprints soluble. After analysis by SDS-PAGE, the proteins separated into about 25 bands, which ranged from 25 to 450 kDa in apparent molecular weight. Using mass spectrometry and a homology-database search, it was shown that several of the proteins are known intracellular proteins, presumably resulting from contamination of footprint material with tube foot epidermal cells. However, 11 protein bands, comprising the most abundant proteins, were not identified and might correspond to novel adhesive proteins. They were named 'Sea star footprint proteins' (Sfps). Tandem mass spectrometry analysis of the protein bands yielded 43 de novo-generated peptide sequences. Most of them were shared by several, if not all, Sfps. Polyclonal antibodies were raised against one of the peptides (HEASGEYYR from Sfp-115) and were used in immunoblotting. They specifically labelled Sfp-115 and other bands with lower apparent molecular weights. The different results suggest that all Sfps might belong to a single family of related proteins sharing similar motifs or, alternatively, they are the products of polymerization and/or degradation processes. PMID:22439774

  20. Novel Pyridazinone Inhibitors for Vascular Adhesion Protein-1 (VAP-1): Old target – New Inhibition Mode

    PubMed Central

    Bligt-Lindén, Eva; Pihlavisto, Marjo; Szatmári, István; Otwinowski, Zbyszek; Smith, David J.; Lázár, László; Fülöp, Ferenc; Salminen, Tiina A.

    2014-01-01

    Vascular adhesion protein-1 (VAP-1) is a primary amine oxidase and a drug target for inflammatory and vascular diseases. Despite extensive attempts to develop potent, specific and reversible inhibitors of its enzyme activity, the task has proven challenging. Here we report the synthesis, inhibitory activity and molecular binding mode of novel pyridazinone inhibitors, which show specificity for VAP-1 over monoamine and diamine oxidases. The crystal structures of three inhibitor-VAP-1 complexes show that these compounds bind reversibly into a unique binding site in the active site channel. Though they are good inhibitors of human VAP-1, they do not inhibit rodent VAP-1 well. To investigate this further, we used homology modeling and structural comparison to identify amino acid differences, which explain the species-specific binding properties. Our results prove the potency and specificity of these new inhibitors and the detailed characterization of their binding mode is of importance for further development of VAP-1 inhibitors. PMID:24304424

  1. Investigation of alginate binding to germanium and polystyrene substrata conditioned with mussel adhesive protein

    SciTech Connect

    Suci, P.A.; Geesey, G.G.

    1995-06-15

    Binding of alginate from Macrocystis pyrifera (kelp) to germanium and polystyrene substrata conditioned with mussel adhesive protein (MAP) from Mytilis edulis, to germanium substrata conditioned with bovine serum albumin (BSA) and polylysine, and to germanium substrata coated with aminopropyltriethoxysilane (APS) was investigated using attenuated total reflection Fourier transform infrared spectrometry. Binding of alginate to MAP appears to be proportional to surface coverage for levels tested. Distinct spectral features appear in the region associated with pyranose ring vibrations upon binding of alginate to MAP, polylysine, and APS, indicating that lysine residues play a prominent role in promoting irreversible adsorption with perturbation of pyranose ring atoms. BSA does not appear to enhance alginate adsorption over that observed on clean germanium and no new spectral features appear as a result of binding. The level of irreversible binding of alginate to germanium and polystyrene substrata conditioned with MAP is similar.

  2. Adhesion G protein-coupled receptors in nervous system development and disease.

    PubMed

    Langenhan, Tobias; Piao, Xianhua; Monk, Kelly R

    2016-09-01

    Members of the adhesion G protein-coupled receptor (aGPCR) class have emerged as crucial regulators of nervous system development, with important implications for human health and disease. In this Review, we discuss the current understanding of aGPCR functions during key steps in neural development, including cortical patterning, dendrite and synapse formation, and myelination. We focus on aGPCR modulation of cell-cell and cell-matrix interactions and signalling to control these varied aspects of neural development, and we discuss how impaired aGPCR function leads to neurological disease. We further highlight the emerging hypothesis that aGPCRs can be mechanically activated and the implications of this property in the nervous system. PMID:27466150

  3. Pleiotrophin disrupts calcium-dependent homophilic cell–cell adhesion and initiates an epithelial–mesenchymal transition

    PubMed Central

    Perez-Pinera, P.; Alcantara, S.; Dimitrov, T.; Vega, J. A.; Deuel, T. F.

    2006-01-01

    Regulation of the levels of tyrosine phosphorylation is essential to maintain the functions of proteins in different signaling pathways and other cellular systems, but how the steady-state levels of tyrosine phosphorylation are coordinated in different cellular systems to initiate complex cellular functions remains a formidable challenge. The receptor protein tyrosine phosphatase (RPTP)β/ζ is a transmembrane tyrosine phosphatase whose substrates include proteins important in intracellular and transmembrane protein-signaling pathways, cytoskeletal structure, cell–cell adhesion, endocytosis, and chromatin remodeling. Pleiotrophin (PTN the protein and Ptn the gene) is a ligand for RPTPβ/ζ; PTN inactivates RPTPβ/ζ, leaving unchecked the continued endogenous activity of tyrosine kinases that increase phosphorylation of the substrates of RPTPβ/ζ at sites dephosphorylated by RPTPβ/ζ in cells not stimulated by PTN. Thus, through the regulation of the tyrosine phosphatase activity of RPTPβ/ζ, the PTN/RPTPβ/ζ signaling pathway coordinately regulates the levels of tyrosine phosphorylation of proteins in many cellular systems. We now demonstrate that PTN disrupts cytoskeletal protein complexes, ablates calcium-dependent homophilic cell–cell adhesion, stimulates ubiquitination and degradation of N-cadherin, reorganizes the actin cytoskeleton, and induces a morphological epithelial–mesenchymal transition (EMT) in PTN-stimulated U373 cells. The data suggest that increased tyrosine phosphorylation of the different substrates of RPTPβ/ζ in PTN-stimulated cells alone is sufficient to coordinately stimulate the different functions needed for an EMT; it is possible that PTN initiates an EMT in cells at sites where PTN is expressed in development and in malignant cells that inappropriately express Ptn. PMID:17098867

  4. Focal adhesion kinases and calcium/calmodulin-dependent protein kinases regulate protein tyrosine phosphorylation in stallion sperm.

    PubMed

    González-Fernández, Lauro; Macías-García, Beatriz; Loux, Shavahn C; Varner, Dickson D; Hinrichs, Katrin

    2013-06-01

    Protein tyrosine phosphorylation (PY) is a hallmark of sperm capacitation. In stallion sperm, calcium inhibits PY at pH <7.8, mediated by calmodulin. To explore the mechanism of that inhibition, we incubated stallion sperm in media without added calcium, with calcium, or with calcium plus the calmodulin inhibitor W-7 (Ca/W-7 treatment). Treatment with inhibitors of calcium/calmodulin-dependent kinases, protein kinase A (PRKA), or Src family kinases suppressed the PY induced by the absence of added calcium, but not that induced by the Ca/W-7 treatment, indicating that PY in the absence of added calcium occurred via the canonical PRKA pathway, but that PY in the Ca/W-7 treatment did not. This suggested that when calmodulin was inhibited, calcium stimulated PY via a noncanonical pathway. Incubation with PF-431396, an inhibitor of focal adhesion kinases (FAKs), a family of calcium-induced protein tyrosine kinases, inhibited the PY induced both by the absence of added calcium and by the Ca/W-7 treatment. Western blotting demonstrated that both FAK family members, protein tyrosine kinases 2 and 2B, were phosphorylated in the absence of added calcium and in the Ca/W-7 treatment, but not in the presence of calcium without calmodulin inhibitors. Inhibition of FAK proteins inhibited PY in stallion sperm incubated under capacitating conditions (in the presence of calcium, bovine serum albumin, and bicarbonate at pH >7.8). These results show for the first time a role for calcium/calmodulin-dependent kinases in PRKA-dependent sperm PY; a non-PRKA-dependent pathway regulating sperm PY; and the apparent involvement of the FAK family of protein tyrosine kinases downstream in both pathways. PMID:23595906

  5. Effect of dispersion method and CNT loading on the quality and performance of nanocomposite soy protein/CNTs adhesive for wood application

    NASA Astrophysics Data System (ADS)

    Afolabi, Ayo Samuel; Oluwafolakemi Sadare, Olawumi; Olawale Daramola, Michael

    2016-09-01

    In this article the effect of dispersion method and carbon nanotubes (CNTs) loading on the quality and performance of a nanocomposite adhesive is reported. The nanocomposite soy protein isolate adhesive was successfully developed by incorporating CNTs into the soy protein isolate (SPI) for enhanced bond strength and water resistance. Dispersion methods, namely mechanical (shear) mixing and mechanical/sonication were employed to aid good dispersion and interfacial interaction between soy protein matrix and the carbon nanofillers during the preparation of the adhesive. The concentration of the CNT was varied from 0.1–0.7 wt% in the nanocomposite adhesive. The morphology and the surface chemistry of the adhesives were checked with SEM and FTIR, respectively. The shear strength of the developed adhesives was investigated according to European standard (EN-204) for interior wood application on a tensile testing machine. The morphological structure of the nanocomposite adhesive obtained from SEM images showed homogeneous dispersion of CNTs in SPI using the two dispersion methods; shear mixing and sonication/shear mixing. Fourier transform infrared spectra showed chemical functionalities and successful interaction between CNTs and SPI adhesive. Thermogravimetric profile of the adhesive samples showed that the newly developed nanocomposite adhesive was thermally stable at a temperature up to about 600 °C at a higher percentage loading of 0.5 wt% CNTs. The result showed that sonication method of dispersion of CNTs into the SPI adhesive had a higher shear strength compared to the mechanical method of dispersion both at dry and wet state.

  6. Diamagnetic levitation causes changes in the morphology, cytoskeleton, and focal adhesion proteins expression in osteocytes.

    PubMed

    Qian, A R; Wang, L; Gao, X; Zhang, W; Hu, L F; Han, J; Li, J B; Di, S M; Shang, Peng

    2012-01-01

    Diamagnetic levitation technology is a novel simulated weightless technique and has recently been applied in life-science research. We have developed a superconducting magnet platform with large gradient high magnetic field (LG-HMF), which can provide three apparent gravity levels, namely, μg (diamagnetic levitation), 1g, and 2g for diamagnetic materials. In this study, the effects of LG-HMF on the activity, morphology, and cytoskeleton (actin filament, microtubules, and vimentin intermediate filaments) in osteocyte - like cell line MLO-Y4 were detected by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) methods, hematoxylin-eosin (HE) staining, and laser scanning confocal microscopy (LSCM), respectively. The changes induced by LG-HMF in distribution and expression of focal adhesion (FA) proteins, including vinculin, paxillin, and talin in MLO-Y4 were determined by LSCM and Western blotting. The results showed that LG-HMF produced by superconducting magnet had no lethal effects on MLO-Y4. Compared to control, diamagnetic levitation (μg) affected MLO-Y4 morphology, nucleus size, cytoskeleton architecture, and FA proteins distribution and expression. The study indicates that osteocytes are sensitive to altered gravity and FA proteins (vinculin, paxillin, and talin) may be involved in osteocyte mechanosensation. The diamagnetic levitation may be a novel ground-based space-gravity simulator and can be used for biological experiment at cellular level. PMID:21216704

  7. Medium-density particleboards from modified rice husks and soybean protein concentrate-based adhesives.

    PubMed

    Ciannamea, Emiliano M; Stefani, Pablo M; Ruseckaite, Roxana A

    2010-01-01

    The main goal of this work was to evaluate the technical feasibility of using rice husk (RH) as wood substitute in the production of environmentally sound medium-density particleboards using adhesives from soybean protein concentrate (SPC). Chemical modification of rice husk with sodium hydroxide and sodium hydroxide followed by hydrogen peroxide (bleaching) were undertaken to evaluate the effect of such treatments on the composition and topology of rice husk and the performance of produced panels. Both treatments were efficient in partially eliminating hemicelluloses, lignin and silica from RH, as evidenced by thermo-gravimetric analysis (TGA). Scanning electron microscopy observations suggested that alkaline treatment resulted in a more damaged RH substrate than bleaching. The dependence of mechanical properties (modulus of rupture, modulus of elasticity, and internal bond) and the physical properties (water absorption and thickness swelling) on chemical treatments performed on both, rice husk and SPC was studied. Bleached-rice husk particleboards bonded with alkaline-treated soybean protein concentrate displayed the best set of final properties. Particleboards with this formulation met the minimum requirements of internal bond, modulus of elasticity and modulus of rupture recommended by the US Standard ANSI/A208.1 specifications for M1, MS and M2-grade medium-density particleboards, but failed to achieve the thickness swelling value recommended for general use panels. This limitation of soybean protein concentrate-bonded rice husk particleboards was counterbalanced by the advantage of being formaldehyde-free which makes them a suitable alternative for indoor applications. PMID:19766482

  8. [AMP-activated protein kinase activation regulates adhesion of monocytes to vascular endothelial cells and the underlying mechanism].

    PubMed

    Bai, Hong-Bo; Wang, Yun; Zhang, Yu-Hua; Zhang, Yuan

    2016-02-25

    The present study was aimed to explore the effect of AMP-activated protein kinase (AMPK) on monocyte adhesion to vascular endothelial cells and underlying molecular mechanism. Tumor necrosis factor α (TNFα)-activated human aortic endothelial cells (HAECs) were treated with different concentrations of AMPK agonist 5-Aminoimidazole-4-carboxamide-1-β-D-ribonucleotide (AICAR) or AMPK inhibitor compound C. And other HAECs were overexpressed with constitutive active or dominant negative AMPK protein and then treated with TNFα. The rates of monocytes adhering to endothelial cells were detected by fluorescent staining. Intercellular cell adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1) mRNA levels and protein secretions were detected by quantitative PCR and ELISA, respectively. Acetylation of NF-κB p65 at lysine 221 site was assessed by Western blot. NF-κB p65 DNA binding activity was analyzed by an ELISA-based method. By using small interfering RNA based strategy, p300 expression in HAECs was down-regulated and then cells were incubated with TNFα. NF-κB p65 DNA binding activity, ICAM-1 and VCAM-1 expressions and adhesion rates were detected, respectively. The activity of p300 was also detected by ELISA. The results showed that AICAR treatment significantly reduced monocyte-endothelial adhesion rate, as well as ICAM-1 and VCAM-1 mRNA levels and protein secretions, in TNFα-activated HAECs. Moreover, transfection of constitutive active AMPKα but not dominant negative AMPKα strongly diminished TNFα-induced upregulation of ICAM-1 and VCAM-1 mRNA expressions and secretions, as well as monocyte-endothelial adhesion. Furthermore, AMPK activation decreased TNFα-mediated acetylation of NF-κB p65 at Lys221 site and reduced NF-κB p65 DNA binding activity. Silencing p300 by siRNA significantly abolished the effect of TNFα- induced adhesion molecules expression and monocyte-endothelial adhesion. Blocking AMPK activation by compound C almost

  9. The Terminal A Domain of the Fibrillar Accumulation-Associated Protein (Aap) of Staphylococcus epidermidis Mediates Adhesion to Human Corneocytes▿

    PubMed Central

    Macintosh, Robin L.; Brittan, Jane L.; Bhattacharya, Ritwika; Jenkinson, Howard F.; Derrick, Jeremy; Upton, Mathew; Handley, Pauline S.

    2009-01-01

    The opportunistic pathogen Staphylococcus epidermidis colonizes indwelling medical devices by biofilm formation but is primarily a skin resident. In many S. epidermidis strains biofilm formation is mediated by a cell wall-anchored protein, the accumulation-associated protein (Aap). Here, we investigate the role of Aap in skin adhesion. Aap is an LPXTG protein with a domain architecture including a terminal A domain and a B-repeat region. S. epidermidis NCTC 11047 expresses Aap as localized, lateral tufts of fibrils on one subpopulation of cells (Fib+), whereas a second subpopulation does not express these fibrils of Aap (Fib−). Flow cytometry showed that 72% of NCTC 11047 cells expressed Aap and that 28% of cells did not. Aap is involved in the adhesion of Fib+ cells to squamous epithelial cells from the hand (corneocytes), as the recombinant A-domain protein partially blocked binding to corneocytes. To confirm the role of the Aap A domain in corneocyte attachment, Aap was expressed on the surface of Lactococcus lactis MG1363 as sparsely distributed, peritrichous fibrils. The expression of Aap increased corneocyte adhesion 20-fold compared to L. lactis carrying Aap without an A domain. S. epidermidis isolates from catheters, artificial joints, skin, and the nose also used the A domain of Aap to adhere to corneocytes, emphasizing the role of Aap in skin adhesion. In addition, L. lactis expressing Aap with different numbers of B repeats revealed a positive correlation between the number of B repeats and adhesion to corneocytes, suggesting an additional function for the B region in enhancing A-domain-dependent attachment to skin. Therefore, in addition to its established role in biofilm formation, Aap can also promote adhesion to corneocytes and is likely to be an important adhesin in S. epidermidis skin colonization. PMID:19749046

  10. A protein interaction map for cell-cell adhesion regulators identifies DUSP23 as a novel phosphatase for β-catenin

    PubMed Central

    Gallegos, Lisa Leon; Ng, Mei Rosa; Sowa, Mathew E.; Selfors, Laura M.; White, Anne; Zervantonakis, Ioannis K.; Singh, Pragya; Dhakal, Sabin; Harper, J. Wade; Brugge, Joan S.

    2016-01-01

    Cell-cell adhesion is central to morphogenesis and maintenance of epithelial cell state. We previously identified 27 candidate cell-cell adhesion regulatory proteins (CCARPs) whose down-regulation disrupts epithelial cell-cell adhesion during collective migration. Using a protein interaction mapping strategy, we found that 18 CCARPs link to core components of adherens junctions or desmosomes. We further mapped linkages between the CCARPs and other known cell-cell adhesion proteins, including hits from recent screens uncovering novel components of E-cadherin adhesions. Mechanistic studies of one novel CCARP which links to multiple cell-cell adhesion proteins, the phosphatase DUSP23, revealed that it promotes dephosphorylation of β-catenin at Tyr 142 and enhances the interaction between α- and β-catenin. DUSP23 knockdown specifically diminished adhesion to E-cadherin without altering adhesion to fibronectin matrix proteins. Furthermore, DUSP23 knockdown produced “zipper-like” cell-cell adhesions, caused defects in transmission of polarization cues, and reduced coordination during collective migration. Thus, this study identifies multiple novel connections between proteins that regulate cell-cell interactions and provides evidence for a previously unrecognized role for DUSP23 in regulating E-cadherin adherens junctions through promoting the dephosphorylation of β-catenin. PMID:27255161

  11. Dihydromunduletone Is a Small-Molecule Selective Adhesion G Protein-Coupled Receptor Antagonist.

    PubMed

    Stoveken, Hannah M; Bahr, Laura L; Anders, M W; Wojtovich, Andrew P; Smrcka, Alan V; Tall, Gregory G

    2016-09-01

    Adhesion G protein-coupled receptors (aGPCRs) have emerging roles in development and tissue maintenance and is the most prevalent GPCR subclass mutated in human cancers, but to date, no drugs have been developed to target them in any disease. aGPCR extracellular domains contain a conserved subdomain that mediates self-cleavage proximal to the start of the 7-transmembrane domain (7TM). The two receptor protomers, extracellular domain and amino terminal fragment (NTF), and the 7TM or C-terminal fragment remain noncovalently bound at the plasma membrane in a low-activity state. We recently demonstrated that NTF dissociation liberates the 7TM N-terminal stalk, which acts as a tethered-peptide agonist permitting receptor-dependent heterotrimeric G protein activation. In many cases, natural aGPCR ligands are extracellular matrix proteins that dissociate the NTF to reveal the tethered agonist. Given the perceived difficulty in modifying extracellular matrix proteins to create aGPCR probes, we developed a serum response element (SRE)-luciferase-based screening approach to identify GPR56/ADGRG1 small-molecule inhibitors. A 2000-compound library comprising known drugs and natural products was screened for GPR56-dependent SRE activation inhibitors that did not inhibit constitutively active Gα13-dependent SRE activation. Dihydromunduletone (DHM), a rotenoid derivative, was validated using cell-free aGPCR/heterotrimeric G protein guanosine 5'-3-O-(thio)triphosphate binding reconstitution assays. DHM inhibited GPR56 and GPR114/ADGRG5, which have similar tethered agonists, but not the aGPCR GPR110/ADGRF1, M3 muscarinic acetylcholine, or β2 adrenergic GPCRs. DHM inhibited tethered peptide agonist-stimulated and synthetic peptide agonist-stimulated GPR56 but did not inhibit basal activity, demonstrating that it antagonizes the peptide agonist. DHM is a novel aGPCR antagonist and potentially useful chemical probe that may be developed as a future aGPCR therapeutic. PMID:27338081

  12. Osteoblast-specific factor 2: cloning of a putative bone adhesion protein with homology with the insect protein fasciclin I.

    PubMed Central

    Takeshita, S; Kikuno, R; Tezuka, K; Amann, E

    1993-01-01

    A cDNA library prepared from the mouse osteoblastic cell line MC3T3-E1 was screened for the presence of specifically expressed genes by employing a combined subtraction hybridization/differential screening approach. A cDNA was identified and sequenced which encodes a protein designated osteoblast-specific factor 2 (OSF-2) comprising 811 amino acids. OSF-2 has a typical signal sequence, followed by a cysteine-rich domain, a fourfold repeated domain and a C-terminal domain. The protein lacks a typical transmembrane region. The fourfold repeated domain of OSF-2 shows homology with the insect protein fasciclin I. RNA analyses revealed that OSF-2 is expressed in bone and to a lesser extent in lung, but not in other tissues. Mouse OSF-2 cDNA was subsequently used as a probe to clone the human counterpart. Mouse and human OSF-2 show a high amino acid sequence conservation except for the signal sequence and two regions in the C-terminal domain in which 'in-frame' insertions or deletions are observed, implying alternative splicing events. On the basis of the amino acid sequence homology with fasciclin I, we suggest that OSF-2 functions as a homophilic adhesion molecule in bone formation. Images Figure 3 Figure 4 Figure 5 Figure 6 PMID:8363580

  13. Cell adhesion and growth enabled by biomimetic oligopeptide modification of a polydopamine-poly(ethylene oxide) protein repulsive surface.

    PubMed

    Musilkova, Jana; Kotelnikov, Ilya; Novotna, Katarina; Pop-Georgievski, Ognen; Rypacek, Frantisek; Bacakova, Lucie; Proks, Vladimir

    2015-11-01

    Protein-repulsive surfaces modified with ligands for cell adhesion receptors have been widely developed for controlling the cell adhesion and growth in tissue engineering. However, the question of matrix production and deposition by cells on these surfaces has rarely been addressed. In this study, protein-repulsive polydopamine-poly(ethylene oxide) (PDA-PEO) surfaces were functionalized with an RGD-containing peptide (RGD), with a collagen-derived peptide binding fibronectin (Col), or by a combination of these peptides (RGD + Col, ratio 1:1) in concentrations of 90 fmol/cm(2) and 700 fmol/cm(2) for each peptide type. When seeded with vascular endothelial CPAE cells, the PDA-PEO surfaces proved to be completely non-adhesive for cells. On surfaces with lower peptide concentrations and from days 1 to 3 after seeding, cell adhesion and growth was restored practically only on the RGD-modified surface. However, from days 3 to 7, cell adhesion and growth was improved on surfaces modified with Col and with RGD + Col. At higher peptide concentrations, the cell adhesion and growth was markedly improved on all peptide-modified surfaces in both culture intervals. However, the collagen-derived peptide did not increase the expression of fibronectin in the cells. The deposition of fibronectin on the material surface was generally very low and similar on all peptide-modified surfaces. Nevertheless, the RGD + Col surfaces exhibited the highest cell adhesion stability under a dynamic load, which correlated with the highest expression of talin and vinculin in the cells on these surfaces. A combination of RGD + Col therefore seems to be the most promising for surface modification of biomaterials, e.g. vascular prostheses. PMID:26449443

  14. Adsorption and adhesion of common serum proteins to nanotextured gallium nitride

    NASA Astrophysics Data System (ADS)

    Bain, Lauren E.; Hoffmann, Marc P.; Bryan, Isaac; Collazo, Ramón; Ivanisevic, Albena

    2015-01-01

    , particularly when considering biological systems. It is well known that thin films and nanostructures feature different optical, electrical, and mechanical properties from their bulk composites; however, interactions taking place at the interface between nanomaterials and their surroundings are less understood. Here, we explore interactions between common serum proteins - serum albumin, fibrinogen, and immunoglobulin G - and a nanotextured gallium nitride surface. Atomic force microscopy with a carboxyl-terminated colloid tip is used to probe the `activity' of proteins adsorbed onto the surface, including both the accessibility of the terminal amine to the tip as well as the potential for protein extension. By evaluating the frequency of tip-protein interactions, we can establish differences in protein behaviour on the basis of both the surface roughness as well as morphology, providing an assessment of the role of surface texture in dictating protein-surface interactions. Unidirectional surface features - either the half-unit cell steppes of as-grown GaN or those produced by mechanical polishing - appear to promote protein accessibility, with a higher frequency of protein extension events taking place on these surfaces when compared with less ordered surface features. Development of a full understanding of the factors influencing surface-biomolecule interactions can pave the way for specific surface modification to tailor the bio-material interface, offering a new path for device optimization. Electronic supplementary information (ESI) available: Additional figures demonstrating the adhesion force magnitude (Fig. S1) and lateral steppe surface topography (Fig. S2). See DOI: 10.1039/c4nr06353h

  15. Adsorption of parotid saliva proteins and adhesion of Streptococcus mutans ATCC 21752 to dental fiber-reinforced composites.

    PubMed

    Tanner, Johanna; Carlén, Anette; Söderling, Eva; Vallittu, Pekka K

    2003-07-15

    The use of fiber-reinforced composites (FRC) in dentistry has increased during recent years. In marginal areas of crowns and removable partial dentures the fibers may become exposed and come into contact with oral tissues, saliva, and microbes. To date, few articles have been published on oral microbial adhesion to FRCs. The aim of this study was to compare different FRCs, their components, and conventional restorative materials with respect to S. mutans ATCC 21752 adhesion and adsorption of specific S. mutans binding proteins. Surface roughness of the materials was also determined. Four different FRCs, a restorative composite, and a high-leucite ceramic material were studied. Polyethylene FRC was found to be significantly rougher than all other materials. Aramid FRC also showed higher surface roughness in comparison with all materials but polyethylene FRC. Without a saliva pellicle, adhesion of S. mutans coincided with surface roughness and polyethylene and aramid FRC promoted S. mutans adhesion better than the other smoother materials. In the presence of salivary pellicle, ceramic and polyethylene FRC bound more bacteria than the other materials studied. Higher quantities of S. mutans binding proteins in the pellicles may in part account for the higher S. mutans adhesion to saliva-coated ceramic and polyethylene FRC. PMID:12808599

  16. Scaffold-forming and Adhesive Contributions of Synthetic Laminin-binding Proteins to Basement Membrane Assembly.

    PubMed

    McKee, Karen K; Capizzi, Stephanie; Yurchenco, Peter D

    2009-03-27

    Laminins that possess three short arms contribute to basement membrane assembly by anchoring to cell surfaces, polymerizing, and binding to nidogen and collagen IV. Although laminins containing the alpha4 and alpha5 subunits are expressed in alpha2-deficient congenital muscular dystrophy, they may be ineffective substitutes because they bind weakly to cell surfaces and/or because they lack the third arm needed for polymerization. We asked whether linker proteins engineered to bind to deficient laminins that provide such missing activities would promote basement membrane assembly in a Schwann cell model. A chimeric fusion protein (alphaLNNd) that adds a short arm terminus to laminin through the nidogen binding locus was generated and compared with the dystrophy-ameliorating protein miniagrin (mAgrin) that binds to the laminin coiled-coil dystroglycan and sulfatides. alphaLNNd was found to mediate laminin binding to collagen IV, to bind to galactosyl sulfatide, and to selectively convert alpha-short arm deletion-mutant laminins LmDeltaalphaLN and LmDeltaalphaLN-L4b into polymerizing laminins. This protein enabled polymerization-deficient laminin but not an adhesion-deficient laminin lacking LG domains (LmDeltaLG) to assemble an extracellular matrix on Schwann cell surfaces. mAgrin, on the other hand, enabled LmDeltaLG to form an extracellular matrix on cell surfaces without increasing accumulation of non-polymerizing laminins. These gain-of-function studies reveal distinct polymerization and anchorage contributions to basement membrane assembly in which the three different LN domains mediate the former, and the LG domains provide primary anchorage with secondary contributions from the alphaLN domain. These findings may be relevant for an understanding of the pathogenesis and treatment of laminin deficiency states. PMID:19189961

  17. Overexpression of vascular adhesion protein-1 is associated with poor prognosis of astrocytomas.

    PubMed

    Kostoro, Joanna; Chang, Shu-Jyuan; Clark Lai, Yen-Chang; Wu, Chun-Chieh; Chai, Chee-Yin; Kwan, Aij-Lie

    2016-06-01

    Vascular adhesion protein-1 (VAP-1) is one of the endothelial adhesion molecules that is believed to play a role in tumor progression and metastasis, supporting cancer cell extravasation. Very few studies have been performed on analyzing the contribution of VAP-1 in brain tumor. Astrocytomas are the most common type of brain tumors, which are classified by World Health Organization (WHO) into four grades according to the degree of malignancy. This study was designed to investigate VAP-1 expression level in different astrocytoma grades and its correlation with clinicopathological features as well as prognosis of astrocytoma patients. Eighty-seven patients with different grades of astrocytoma (WHO Grade I-Grade IV) were enrolled in this study. The expression of VAP-1 was assayed by immunohistochemistry. The correlation between VAP-1 expression and clinicopathological features was evaluated by Chi-square test, and overall survival was analyzed by Kaplan-Meier method. Cox regression analysis was applied to analyze the independent influence of each parameter on overall survival. The expression level of VAP-1 was significantly higher in diffuse astrocytoma than those of pilocytic astrocytoma (p < 0.0001). In the subgroup analysis, upregulated VAP-1 expression was frequently found in older age patients (≥50 years). The VAP-1 expression was found to be significantly correlated with the overall survival (p = 0.0002). There was a statistical correlation between VAP-1(high) tumors in diffuse astrocytoma and VAP-1(low) tumors in pilocytic astrocytoma (p < 0.0001). Multivariate Cox analysis indicated VAP-1 was an independent predictive marker for poorer prognosis (p = 0.0036). Therefore, VAP-1 could be a promising prognostic biomarker in astrocytoma. PMID:26935340

  18. Scanning-force techniques to monitor time-dependent changes in topography and adhesion force of proteins on surfaces.

    PubMed

    Mondon, M; Berger, S; Ziegler, C

    2003-04-01

    Scanning-force microscopy (SFM) investigations were conducted to probe the influences of the interactions of proteins with surfaces relevant in medicine. These interactions are an important feature in the area of biofilm formation. The adsorption of proteins leads to changes in topography, which was monitored for the build up of protein layers of hen egg-white lysozyme and bovine serum albumin (BSA) on mica in real time in phosphate-buffered aqueous solution over a time period of 10 min. Phase imaging was additionally applied to compare material contrasts and to evaluate this method for further application in this field. The adhesion forces that develop on a time scale below 20 s between a protein-modified SFM tip and titanium surfaces (TiO(2), TiAl6V4 and TiAl6Nb7) were investigated. The influences of the parameters loading force and interaction time between the protein and the surface were monitored as well as the influence of protein structure. The interaction time dependency of the adhesion force could be described with a kinetic model of two consecutive first-order reactions. For the maximal adhesion force a correlation to the ratio of the amino acids cysteine, proline and glycine has been proposed. PMID:12707750

  19. Early Growth Response Protein 1 Promotes Restenosis by Upregulating Intercellular Adhesion Molecule-1 in Vein Graft

    PubMed Central

    Zhang, Kui; Cao, Jian; Dong, Ran; Du, Jie

    2013-01-01

    Objectives. To verify the relationship between Egr-1 and vein graft restenosis and investigate the related mechanisms. Methods. Mouse vein graft models were established in Egr-1 knockout (KO) and wild-type (WT) mice. The vein grafts in the mice were taken for pathological examination and immunohistochemical analysis. The endothelial cells (ECs) were stimulated by using a computer-controlled cyclic stress unit. BrdU staining and PCR were used to detect ECs proliferation activity and Egr-1 and ICAM-1 mRNA expression, respectively. Western-blot analysis was also used to detect expression of Egr-1 and intercellular adhesion molecule-1 (ICAM-1) proteins. Results. The lumens of vein grafts in Egr-1 KO mice were wider than in WT mice. ECs proliferation after mechanical stretch stimulation was suppressed by Egr-1 knockout (P < 0.05). Both in vein grafts and ECs from WT mice after mechanical stretch stimulation, mRNA expression and protein of Egr-1 and ICAM-1 showed increases (P < 0.05). However, ICAM-1 expression was significantly suppressed in ECs from Egr-1 knockout mice (P < 0.05). Conclusions. Egr-1 may promote ECs proliferation and result in vein graft restenosis by upregulating the expression of ICAM-1. As a key factor of vein graft restenosis, it could be a target for the prevention of restenosis after CABG surgery. PMID:24386503

  20. Neural Cell Adhesion Protein CNTN1 Promotes the Metastatic Progression of Prostate Cancer.

    PubMed

    Yan, Judy; Ojo, Diane; Kapoor, Anil; Lin, Xiaozeng; Pinthus, Jehonathan H; Aziz, Tariq; Bismar, Tarek A; Wei, Fengxiang; Wong, Nicholas; De Melo, Jason; Cutz, Jean-Claude; Major, Pierre; Wood, Geoffrey; Peng, Hao; Tang, Damu

    2016-03-15

    Prostate cancer metastasis is the main cause of disease-related mortality. Elucidating the mechanisms underlying prostate cancer metastasis is critical for effective therapeutic intervention. In this study, we performed gene-expression profiling of prostate cancer stem-like cells (PCSC) derived from DU145 human prostate cancer cells to identify factors involved in metastatic progression. Our studies revealed contactin 1 (CNTN1), a neural cell adhesion protein, to be a prostate cancer-promoting factor. CNTN1 knockdown reduced PCSC-mediated tumor initiation, whereas CNTN1 overexpression enhanced prostate cancer cell invasion in vitro and promoted xenograft tumor formation and lung metastasis in vivo. In addition, CNTN1 overexpression in DU145 cells and corresponding xenograft tumors resulted in elevated AKT activation and reduced E-cadherin (CDH1) expression. CNTN1 expression was not readily detected in normal prostate glands, but was clearly evident on prostate cancer cells in primary tumors and lymph node and bone metastases. Tumors from 637 patients expressing CNTN1 were associated with prostate cancer progression and worse biochemical recurrence-free survival following radical prostatectomy (P < 0.05). Collectively, our findings demonstrate that CNTN1 promotes prostate cancer progression and metastasis, prompting further investigation into the mechanisms that enable neural proteins to become aberrantly expressed in non-neural malignancies. PMID:26795349

  1. Interfacial tension of complex coacervated mussel adhesive protein according to the Hofmeister series.

    PubMed

    Lim, Seonghye; Moon, Dustin; Kim, Hyo Jeong; Seo, Jeong Hyun; Kang, In Seok; Cha, Hyung Joon

    2014-02-01

    Complex coacervation is a liquid-liquid phase separation in a colloidal system of two oppositely charged polyelectrolytes or colloids. The interfacial tension of the coacervate phase is the key parameter for micelle formation and interactions with the encapsulating material. However, the relationship between interfacial tensions and various salt solutions is poorly understood in complex coacervation. In the present work, the complex coacervate dynamics of recombinant mussel adhesive protein (MAP) with hyaluronic acid (HA) were determined in the presence of Hofmeister series salt ions. Using measurements of absorbance, hydrodynamic diameter, capillary force, and receding contact angle in the bulk phase, the interfacial tensions of complex coacervated MAP/HA were determined to be 0.236, 0.256, and 0.287 mN/m in 250 mM NaHCOO, NaCl, and NaNO3 solutions, respectively. The sequences of interfacial tensions and contact angles of the complex coacervates in the presence of three sodium salts with different anions were found to follow the Hofmeister ordering. The tendency of interfacial tension between the coacervate and dilute phases in the presence of different types of Hofmeister salt ions could provide a better understanding of Hofmeister effects on complex coacervated materials based on the protein-polysaccharide system. This information can also be utilized for microencapsulation and adsorption by controlling intramolecular interactions. In addition, the injection molding dynamics of mussel byssus formation was potentially explained based on the measured interfacial tension of coacervated MAP. PMID:24490867

  2. [Expression and purification of an adhesive protein of rabbit Pasteurella multocida C51-3 and detection of its antigenicity].

    PubMed

    Nazierbieke, Wulumuhan; Yan, Fang; He, Cui; Zhang, Lei; Borrathybay, Entomack

    2008-08-01

    The cp36 gene encoding an adhesive protein was amplified by PCR from genomic DNA of rabbit P. multocida C51-3 strain, and cloned into the pMD18-T vector and then sequenced. The mature adhesive protein without a signal peptide of cpm36 gene was amplified by PCR from the recombinant plasmid pMD18-cp36, then cloned into the prokaryotic expression vector pQE30 to provide a recombinant plasmid pQE30-cpm36. The recombinant protein of CPM36 was produced in Escherichia coli M15 harboring the recombinant plasmid pQE30-cpm36 by IPTG induction, and the recombinant protein purified by the affinity chromatography with Ni(2+)-NTA resin. The sequence analyses showed that the ORF of cp36 gene was 1032 bp in length, and DNA homology of the cp36 genes between the C51-3 strain and the previously reported different serotype strains of P. multocida in GenBank was 76.9 to 100%. The SDS-PAGE analyses revealed a single fusion protein band with a molecular weight of 37 kD, and the Western blotting analysis demonstrated that the recombinant protein CPM36 and native 36 kD protein of C51-3 were recognized specifically by an antiserum against the recombinant protein, suggesting that the recombinant protein is an antigenic protein. PMID:18998549

  3. Proteinaceous determinants of surface colonization in bacteria: bacterial adhesion and biofilm formation from a protein secretion perspective

    PubMed Central

    Chagnot, Caroline; Zorgani, Mohamed A.; Astruc, Thierry; Desvaux, Mickaël

    2013-01-01

    Bacterial colonization of biotic or abiotic surfaces results from two quite distinct physiological processes, namely bacterial adhesion and biofilm formation. Broadly speaking, a biofilm is defined as the sessile development of microbial cells. Biofilm formation arises following bacterial adhesion but not all single bacterial cells adhering reversibly or irreversibly engage inexorably into a sessile mode of growth. Among molecular determinants promoting bacterial colonization, surface proteins are the most functionally diverse active components. To be present on the bacterial cell surface, though, a protein must be secreted in the first place. Considering the close association of secreted proteins with their cognate secretion systems, the secretome (which refers both to the secretion systems and their protein substrates) is a key concept to apprehend the protein secretion and related physiological functions. The protein secretion systems are here considered in light of the differences in the cell-envelope architecture between diderm-LPS (archetypal Gram-negative), monoderm (archetypal Gram-positive) and diderm-mycolate (archetypal acid-fast) bacteria. Besides, their cognate secreted proteins engaged in the bacterial colonization process are regarded from single protein to supramolecular protein structure as well as the non-classical protein secretion. This state-of-the-art on the complement of the secretome (the secretion systems and their cognate effectors) involved in the surface colonization process in diderm-LPS and monoderm bacteria paves the way for future research directions in the field. PMID:24133488

  4. Multiscale approaches to protein-mediated interactions between membranes—relating microscopic and macroscopic dynamics in radially growing adhesions

    NASA Astrophysics Data System (ADS)

    Bihr, Timo; Seifert, Udo; Smith, Ana-Sunčana

    2015-08-01

    Macromolecular complexation leading to coupling of two or more cellular membranes is a crucial step in a number of biological functions of the cell. While other mechanisms may also play a role, adhesion always involves the fluctuations of deformable membranes, the diffusion of proteins and the molecular binding and unbinding. Because these stochastic processes couple over a multitude of time and length scales, theoretical modeling of membrane adhesion has been a major challenge. Here we present an effective Monte Carlo scheme within which the effects of the membrane are integrated into local rates for molecular recognition. The latter step in the Monte Carlo approach enables us to simulate the nucleation and growth of adhesion domains within a system of the size of a cell for tens of seconds without loss of accuracy, as shown by comparison to 106 times more expensive Langevin simulations. To perform this validation, the Langevin approach was augmented to simulate diffusion of proteins explicitly, together with reaction kinetics and membrane dynamics. We use the Monte Carlo scheme to gain deeper insight to the experimentally observed radial growth of micron sized adhesion domains, and connect the effective rate with which the domain is growing to the underlying microscopic events. We thus demonstrate that our technique yields detailed information about protein transport and complexation in membranes, which is a fundamental step toward understanding even more complex membrane interactions in the cellular context.

  5. Human heat shock protein (Hsp) 90 interferes with Neisseria meningitidis adhesin A (NadA)-mediated adhesion and invasion.

    PubMed

    Montanari, Paolo; Bozza, Giuseppe; Capecchi, Barbara; Caproni, Elena; Barrile, Riccardo; Norais, Nathalie; Capitani, Mirco; Sallese, Michele; Cecchini, Paola; Ciucchi, Laura; Gao, Zhenai; Rappuoli, Rino; Pizza, Mariagrazia; Aricò, Beatrice; Merola, Marcello

    2012-03-01

    NadA (N eisseria meningitidisadhesin A), a meningococcal surface protein, mediates adhesion to and invasion of human cells, an activity in which host membrane proteins have been implicated. While investigating these host factors in human epithelial cells by affinity chromatography, we discovered an unanticipated interaction of NadA with heat shock protein (Hsp) 90, a molecular chaperone. The specific in vitro interaction of recombinant soluble NadA and Hsp90 was confirmed by co-immunoprecipitations, dot and far-Western blot. Intriguingly, ADP, but not ATP, was required for this association, and the Hsp90 inhibitor 17-AAG promoted complex formation. Hsp90 binding to an Escherichia coli strain used as carrier to express surface exposed NadA confirmed these results in live bacteria. We also examined RNA interference, plasmid-driven overexpression, addition of exogenous rHsp90 and 17-AAG inhibition in human epithelial cells to further elucidate the involvement of Hsp90 in NadA-mediated adhesion and invasion. Together, these data suggest an inverse correlation between the amount of host Hsp90 and the NadA adhesive/invasive phenotype. Confocal microscopy also demonstrated that meningococci interact with cellular Hsp90, a completely novel finding. Altogether our results show that variation of host Hsp90 expression or activity interferes with adhesive and invasive events driven by NadA. PMID:22066472

  6. Actions of translocator protein ligands on neutrophil adhesion and motility induced by G-protein coupled receptor signaling.

    PubMed

    de Lima, Camila Bento; Tamura, Eduardo K; Montero-Melendez, Trindad; Palermo-Neto, João; Perretti, Mauro; Markus, Regina P; Farsky, Sandra Helena Poliselli

    2012-01-13

    The 18 kDa translocator protein (TSPO) also known as the peripheral benzodiazepine receptor (PBR), mediates the transportation of cholesterol and anions from the outer to the inner mitochondrial membrane in different cells types. Although recent evidences indicate a potential role for TSPO in the development of inflammatory processes, the mechanisms involved have not been elucidated. The present study investigated the ability of the specific TSPO ligands, the isoquinoline carboxamide PK11195 and benzodiazepine Ro5-4864, on neutrophil recruitment promoted by the N-formylmethionyl-leucyl-phenylalanine peptide (fMLP), an agonist of G-protein coupled receptor (GPCR). Pre-treatment with Ro5-4864 abrograted fMLP-induced leukocyte-endothelial interactions in mesenteric postcapillary venules in vivo. Moreover, in vitro Ro5-4864 treatment prevented fMLP-induced: (i) L-selectin shedding and overexpression of PECAM-1 on the neutrophil cell surface; (ii) neutrophil chemotaxis and (iii) enhancement of intracellular calcium cations (iCa(+2)). Intriguingly, the two latter effects were augmented by cell treatment with PK11195. An allosteric agonist/antagonist relation may be suggested, as the effects of Ro5-4864 on fMLP-stimulated neutrophils were reverted by simultaneous treatment with PK11195. Taken together, these data highlight TSPO as a modulator of pathways of neutrophil adhesion and locomotion induced by GPCR, connecting TSPO actions and the onset of an innate inflammatory response. PMID:22209795

  7. Composites containing albumin protein or cyanoacrylate adhesives and biodegradable scaffolds: II. In vivo wound closure study in a rat model

    NASA Astrophysics Data System (ADS)

    McNally-Heintzelman, Karen M.; Heintzelman, Douglas L.; Duffy, Mark T.; Bloom, Jeffrey N.; Soller, Eric C.; Gilmour, Travis M.; Hoffman, Grant T.; Edward, Deepak

    2004-07-01

    Our Scaffold-Enhanced Biological Adhesive (SEBA) system was investigated as an alternative to sutures or adhesives alone for repair of wounds. Two scaffold materials were investigated: (i) a synthetic biodegradable material fabricated from poly(L-lactic-co-glycolic acid); and (ii) a biologic material, small intestinal submucosa, manufactured by Cook BioTech. Two adhesive materials were also investigated: (i) a biologic adhesive composed of 50%(w/v) bovine serum albumin solder and 0.5mg/ml indocyanine green dye mixed in deionized water, and activated with an 808-nm diode laser; and (ii) Ethicon"s Dermabond, a 2-octyl-cyanoacrylate. The tensile strength and time-to-failure of skin incisions repaired in vivo in a rat model were measured at seven days postoperative. Incisions closed by protein solder alone, by Dermabond alone, or by suture, were also tested for comparison. The tensile strength of repairs formed using the SEBA system were 50% to 65% stronger than repairs formed by suture or either adhesive alone, with significantly less variations within each experimental group (average standard deviations of 15% for SEBA versus 38% for suture and 28% for adhesive alone). In addition, the time-to-failure curves showed a longevity not previously seen with the suture or adhesive alone techniques. The SEBA system acts to keep the dermis in tight apposition during the critical early phase of wound healing when tissue gaps are bridged by scar and granulation tissue. It has the property of being more flexible than either of the adhesives alone and may allow the apposed edges to move in conjunction with each other as a unit for a longer period of time and over a greater range of stresses than adhesives alone. This permits more rapid healing and establishment of integrity since the microgaps between the dermis edges are significantly reduced. By the time the scaffolds are sloughed from the wound site, there is greater strength and healing than that produced by adhesive alone or

  8. Lymphocyte binding to vascular endothelium in inflamed skin revisited: a central role for vascular adhesion protein-1 (VAP-1).

    PubMed

    Arvilommi, A M; Salmi, M; Kalimo, K; Jalkanen, S

    1996-04-01

    The binding of leukocytes to vascular endothelium and their migration into tissues is mediated by adhesion molecules on the endothelial cells and leukocytes. Vascular adhesion protein-1 (VAP-1) is a 170-180/90-kDa endothelial molecule expressed most prominently in high endothelial venules in peripheral lymph node (PLN) type lymphatic tissues. VAP-1 mediates lymphocyte binding to PLN, tonsil and synovium. The expression of VAP-1 is induced in inflammatory diseases such as arthritis and gut inflammation. We examined the expression, structure and function of VAP-1 in normal and inflamed skin and compared it to those of other adhesion molecules implicated in skin homing. In psoriasis lichen ruber planus, pemphigoid and allergic lesions, VAP-1 was markedly upregulated. The expression of VAP-1 was also increased in biopsies of healthy skin of the patients. The VAP-1 molecule induced in skin is decorated with abundant sialic acids. VAP-1 inflamed skin is functional, since inhibition with anti-VAP-1 monoclonal antibodies caused a 60% reduction in lymphocytes adhesion to vascular endothelium. Antibodies against E-selectin, which has been regarded as the major vascular addressin directing cutaneous lymphocyte traffic, and, surprisingly, against peripheral lymph node addressin (PNAd), caused inhibitions of 30% and 60%, respectively, in the frozen section adhesion assay. These findings suggest important roles also for VAP-1 and PNAd in lymphocyte homing into inflamed skin. PMID:8625974

  9. Different forms of human vascular adhesion protein-1 (VAP-1) in blood vessels in vivo and in cultured endothelial cells: implications for lymphocyte-endothelial cell adhesion models.

    PubMed

    Salmi, M; Jalkanen, S

    1995-10-01

    Vascular endothelium plays a pivotal role in controlling leukocyte extravasation from the blood into the tissues. Vascular adhesion protein-1 (VAP-1) is a novel endothelial cell molecule which mediates lymphocyte binding to the vascular lining (Salmi, M., and Jalkanen, S., Science 1992. 257:1407). In this study, we analyzed endothelial cell type-specific differences of VAP-1. In vivo, VAP-1 is a 90/170-kDa molecule which is mainly expressed on the lumenal surface and in cytoplasmic granules of peripheral lymph node-type postcapillary venules (high endothelial venules, HEV). In tonsil HEV, VAP-1 is modified with abundant sialic acids. VAP-1 is also detectable in the cytoplasm of human umbilical vein endothelial cells (HUVEC) and in an endothelial cell hybrid EaHy-926, although both cell types lack detectable surface VAP-1. Cultured endothelial cells do not express MECA-79-defined peripheral lymph node addressins either. VAP-1 was not translocated onto the endothelial cell surface after stimulation with multiple cytokines, mitogens or secretagogues which induced expression of other known endothelial adhesion molecules. Biochemical analyses revealed that VAP-1 is a approximately 180-kDa protein in these endothelial cell types. Digestions with neuraminidase, O-glycanase and N-glycanase, as well as treatment of cells with tunicamycin and benzyl-N-acetylgalactosaminide, did not alter the molecular mass of VAP-1 in EaHy-926. Pulse-chase experiments showed that VAP-1 is directly synthesized as a 180-kDa molecule without any detectable precursors. Thus, in cultured endothelial cells, VAP-1 is a 180-kDa protein which is devoid of post-translational modifications, and in particular, lacks the sialic acids crucial for the function of VAP-1 in tonsil vessels. Notably, the endothelial cell types commonly used as a model in studying lymphocyte-endothelial cell interactions lack surface expression of VAP-1 and peripheral node addressins, and hence are inherently of limited use in

  10. Nanoscale visualization of functional adhesion/excitability nodes at the intercalated disc

    PubMed Central

    Leo-Macias, Alejandra; Agullo-Pascual, Esperanza; Sanchez-Alonso, Jose L.; Keegan, Sarah; Lin, Xianming; Arcos, Tatiana; Feng-Xia-Liang; Korchev, Yuri E.; Gorelik, Julia; Fenyö, David; Rothenberg, Eli; Delmar, Mario

    2016-01-01

    Intercellular adhesion and electrical excitability are considered separate cellular properties. Studies of myelinated fibres, however, show that voltage-gated sodium channels (VGSCs) aggregate with cell adhesion molecules at discrete subcellular locations, such as the nodes of Ranvier. Demonstration of similar macromolecular organization in cardiac muscle is missing. Here we combine nanoscale-imaging (single-molecule localization microscopy; electron microscopy; and ‘angle view' scanning patch clamp) with mathematical simulations to demonstrate distinct hubs at the cardiac intercalated disc, populated by clusters of the adhesion molecule N-cadherin and the VGSC NaV1.5. We show that the N-cadherin-NaV1.5 association is not random, that NaV1.5 molecules in these clusters are major contributors to cardiac sodium current, and that loss of NaV1.5 expression reduces intercellular adhesion strength. We speculate that adhesion/excitability nodes are key sites for crosstalk of the contractile and electrical molecular apparatus and may represent the structural substrate of cardiomyopathies in patients with mutations in molecules of the VGSC complex. PMID:26787348

  11. Focal adhesions are foci for tyrosine-based signal transduction via GIV/Girdin and G proteins

    PubMed Central

    Lopez-Sanchez, Inmaculada; Kalogriopoulos, Nicholas; Lo, I-Chung; Kabir, Firooz; Midde, Krishna K.; Wang, Honghui; Ghosh, Pradipta

    2015-01-01

    GIV/Girdin is a multimodular signal transducer and a bona fide metastasis-related protein. As a guanidine exchange factor (GEF), GIV modulates signals initiated by growth factors (chemical signals) by activating the G protein Gαi. Here we report that mechanical signals triggered by the extracellular matrix (ECM) also converge on GIV-GEF via β1 integrins and that focal adhesions (FAs) serve as the major hubs for mechanochemical signaling via GIV. GIV interacts with focal adhesion kinase (FAK) and ligand-activated β1 integrins. Phosphorylation of GIV by FAK enhances PI3K-Akt signaling, the integrity of FAs, increases cell–ECM adhesion, and triggers ECM-induced cell motility. Activation of Gαi by GIV-GEF further potentiates FAK-GIV-PI3K-Akt signaling at the FAs. Spatially restricted signaling via tyrosine phosphorylated GIV at the FAs is enhanced during cancer metastasis. Thus GIV-GEF serves as a unifying platform for integration and amplification of adhesion (mechanical) and growth factor (chemical) signals during cancer progression. PMID:26446841

  12. Myelin Basic Protein Cleaves Cell Adhesion Molecule L1 and Improves Regeneration After Injury.

    PubMed

    Lutz, David; Kataria, Hardeep; Kleene, Ralf; Loers, Gabriele; Chaudhary, Harshita; Guseva, Daria; Wu, Bin; Jakovcevski, Igor; Schachner, Melitta

    2016-07-01

    Myelin basic protein (MBP) is a serine protease that cleaves neural cell adhesion molecule L1 and generates a transmembrane L1 fragment which facilitates L1-dependent functions in vitro, such as neurite outgrowth, neuronal cell migration and survival, myelination by Schwann cells as well as Schwann cell proliferation, migration, and process formation. Ablation and blocking of MBP or disruption of its proteolytic activity by mutation of a proteolytically active serine residue abolish L1-dependent cellular responses. In utero injection of adeno-associated virus encoding proteolytically active MBP into MBP-deficient shiverer mice normalizes differentiation, myelination, and synaptogenesis in the developing postnatal spinal cord, in contrast to proteolytically inactive MBP. Application of active MBP to the injured wild-type spinal cord and femoral nerve augments levels of a transmembrane L1 fragment, promotes remyelination, and improves functional recovery after injury. Application of MBP antibody impairs recovery. Virus-mediated expression of active MBP in the lesion site after spinal cord injury results in improved functional recovery, whereas injection of virus encoding proteolytically inactive MBP fails to do so. The present study provides evidence for a novel L1-mediated function of MBP in the developing spinal cord and in the injured adult mammalian nervous system that leads to enhanced recovery after acute trauma. PMID:26081148

  13. Synergic interaction between amyloid precursor protein and neural cell adhesion molecule promotes neurite outgrowth

    PubMed Central

    Chen, Keping; Lu, Huixia; Gao, Tianli; Xue, Xiulei; Wang, Chunling; Miao, Fengqin

    2016-01-01

    Alzheimer's disease (AD) is one of the most common neurodegenerative diseases worldwide. The main features of AD are the pathological changes of density and distribution of intracellular neurofibrillary tangles (NFT) and extracellular amyloid plaques. The processing of amyloid beta precursor protein (APP) to β-amyloid peptide (Aβ) is one of the critical events in the pathogenesis of AD. In this study, we evaluated the role of the interaction of neural cell adhesion molecule (NCAM) and APP in neurite outgrowth using two different experimental systems: PC12E2 cells and hippocampal neurons that were isolated from wild type, APP knock-in and APP knock-out mice. PC12E2 cells or hippocampal neurons were co-cultured with NCAM-negative or NCAM-positive fibroblasts L929 cells. We found that APP promoted neurite outgrowth of PC12E2 cells and hippocampal neurons in either the presence or absence of NCAM. Secreted APP can rescue the neurite outgrowth in hippocampal neurons from APP knock-out mice. The interaction of APP and NCAM had synergic effect in promoting neurite outgrowth in both PC12E2 cells and hippocampal neurons. Our results suggested that the interaction of APP with NCAM played an important role in AD development and therefore could be a potential therapeutic target for AD treatment. PMID:26883101

  14. The Prion Protein Controls Polysialylation of Neural Cell Adhesion Molecule 1 during Cellular Morphogenesis

    PubMed Central

    Mehrabian, Mohadeseh; Brethour, Dylan; Wang, Hansen; Xi, Zhengrui; Rogaeva, Ekaterina; Schmitt-Ulms, Gerold

    2015-01-01

    Despite its multi-faceted role in neurodegenerative diseases, the physiological function of the prion protein (PrP) has remained elusive. On the basis of its evolutionary relationship to ZIP metal ion transporters, we considered that PrP may contribute to the morphogenetic reprogramming of cells underlying epithelial-to-mesenchymal transitions (EMT). Consistent with this hypothesis, PrP transcription increased more than tenfold during EMT, and stable PrP-deficient cells failed to complete EMT in a mammalian cell model. A global comparative proteomics analysis identified the neural cell adhesion molecule 1 (NCAM1) as a candidate mediator of this impairment, which led to the observation that PrP-deficient cells fail to undergo NCAM1 polysialylation during EMT. Surprisingly, this defect was caused by a perturbed transcription of the polysialyltransferase ST8SIA2 gene. Proteomics data pointed toward β-catenin as a transcriptional regulator affected in PrP-deficient cells. Indeed, pharmacological blockade or siRNA-based knockdown of β-catenin mimicked PrP-deficiency in regards to NCAM1 polysialylation. Our data established the existence of a PrP-ST8SIA2-NCAM signaling loop, merged two mature fields of investigation and offer a simple model for explaining phenotypes linked to PrP. PMID:26288071

  15. Maximizing Fibroblast Adhesion on Protein-Coated Surfaces Using Microfluidic Cell Printing

    PubMed Central

    Davidoff, S.N.; Au, D.; Gale, B.K.; Brooks, B.D.; Brooks, A.E.

    2015-01-01

    translation of in vitro cell based assays to in vivo cellular response is imprecise at best. The advent of three-dimensional cell cultures in addition to bioreactor type microfluidics has improved the situation. However, these technical advances cannot be easily combined due to practical limitations. Development of a vertical microfluidic cell printer overcomes this obstacle, providing the ability to more closely recapitulate complex cellular environments and responses. As a proof of concept, we investigated the adhesion of fibroblasts under flow on protein-coated surfaces using a novel vertical microfluidic print head to isolate and manipulate both mechanical and biological factors as a model of fibroblast behavior during the foreign body response following implant insertion. A low flow rate with larger microfluidic channels onto a serum-coated surface has been determined to allow the highest density of viable fibroblasts to attach to the surface. While these insights into fibroblast surface attachment may lead to better material designs, the methods developed herein will certainly be useful as a biomaterials testing platform. PMID:26989480

  16. The Prion Protein Controls Polysialylation of Neural Cell Adhesion Molecule 1 during Cellular Morphogenesis.

    PubMed

    Mehrabian, Mohadeseh; Brethour, Dylan; Wang, Hansen; Xi, Zhengrui; Rogaeva, Ekaterina; Schmitt-Ulms, Gerold

    2015-01-01

    Despite its multi-faceted role in neurodegenerative diseases, the physiological function of the prion protein (PrP) has remained elusive. On the basis of its evolutionary relationship to ZIP metal ion transporters, we considered that PrP may contribute to the morphogenetic reprogramming of cells underlying epithelial-to-mesenchymal transitions (EMT). Consistent with this hypothesis, PrP transcription increased more than tenfold during EMT, and stable PrP-deficient cells failed to complete EMT in a mammalian cell model. A global comparative proteomics analysis identified the neural cell adhesion molecule 1 (NCAM1) as a candidate mediator of this impairment, which led to the observation that PrP-deficient cells fail to undergo NCAM1 polysialylation during EMT. Surprisingly, this defect was caused by a perturbed transcription of the polysialyltransferase ST8SIA2 gene. Proteomics data pointed toward β-catenin as a transcriptional regulator affected in PrP-deficient cells. Indeed, pharmacological blockade or siRNA-based knockdown of β-catenin mimicked PrP-deficiency in regards to NCAM1 polysialylation. Our data established the existence of a PrP-ST8SIA2-NCAM signaling loop, merged two mature fields of investigation and offer a simple model for explaining phenotypes linked to PrP. PMID:26288071

  17. β1 Integrin is an Adhesion Protein for Sperm Binding to Eggs

    PubMed Central

    Baessler, Keith A.; Lee, Younjoo; Sampson, Nicole S.

    2009-01-01

    We investigated the role of β1 integrin in mammalian fertilization and the mode of inhibition of fertilinβ-derived polymers. We determined that polymers displaying the Glu-Cys-Asp peptide from the fertilinβ disintegrin domain mediate inhibition of mammalian fertilization through a β1 integrin receptor on the egg surface. Inhibition of fertilization is a consequence of competition with sperm binding to the cell surface, not activation of an egg-signaling pathway. The presence of the β1 integrin on the egg surface increases the rate of sperm attachment, but does not alter the total number of sperm that can attach or fuse to the egg. We conclude that the presence of β1 integrin enhances the initial adhesion of sperm to the egg plasma membrane and that subsequent attachment and fusion are mediated by additional egg and sperm proteins present in the β1 integrin complex. Therefore, the mechanisms by which sperm fertilize wild-type and β1 knockout eggs are different. PMID:19338281

  18. Inhibition of protein adsorption and cell adhesion on PNIPAAm-grafted polyurethane surface: effect of graft molecular weight.

    PubMed

    Zhao, Tieliang; Chen, Hong; Zheng, Jun; Yu, Qian; Wu, Zhaoqiang; Yuan, Lin

    2011-06-15

    In this work, the effect of molecular weight (MW) of surface grafted poly(N-isopropylacrylamide) (PNIPAAm) on protein adsorption and cell adhesion was investigated systematically. PNIPAAm-grafted polyurethane (PU) surfaces of varying graft MW were prepared via conventional radical polymerization. The MW was controlled by adjusting the monomer concentration. Fibrinogen (Fg) and human serum albumin (HSA) were selected as model proteins and their adsorption from phosphate-buffered saline (PBS, pH 7.4) and blood plasma at 37°C was measured using a radiolabeling method and immunoblot analysis respectively. It was found that in both media, as the MW increased, the adsorption of these two proteins decreased gradually reaching a plateau value at MW above 7.9×10(4). Compared to the unmodified PU, the surface grafted with PNIPAAm of MW 14.6×10(4) reduced the adsorption of Fg and HSA in PBS by 91% and 86%, respectively. Moreover, the surfaces with higher MW PNIPAAm showed minimal adhesion of L929 cells presumably due to the absence of cell-adhesive proteins on the surfaces. PMID:21093225

  19. Staphylococcus aureus Fibronectin-Binding Protein A Mediates Cell-Cell Adhesion through Low-Affinity Homophilic Bonds

    PubMed Central

    Herman-Bausier, Philippe; El-Kirat-Chatel, Sofiane; Foster, Timothy J.

    2015-01-01

    ABSTRACT Staphylococcus aureus is an important opportunistic pathogen which is a leading cause of biofilm-associated infections on indwelling medical devices. The cell surface-located fibronectin-binding protein A (FnBPA) plays an important role in the accumulation phase of biofilm formation by methicillin-resistant S. aureus (MRSA), but the underlying molecular interactions are not yet established. Here, we use single-cell and single-molecule atomic force microscopy to unravel the mechanism by which FnBPA mediates intercellular adhesion. We show that FnBPA is responsible for specific cell-cell interactions that involve the FnBPA A domain and cause microscale cell aggregation. We demonstrate that the strength of FnBPA-mediated adhesion originates from multiple low-affinity homophilic interactions between FnBPA A domains on neighboring cells. Low-affinity binding by means of FnBPA may be important for biofilm dynamics. These results provide a molecular basis for the ability of FnBPA to promote cell accumulation during S. aureus biofilm formation. We speculate that homophilic interactions may represent a generic strategy among staphylococcal cell surface proteins for guiding intercellular adhesion. As biofilm formation by MRSA strains depends on proteins rather than polysaccharides, our approach offers exciting prospects for the design of drugs or vaccines to inhibit protein-dependent intercellular interactions in MRSA biofilms. PMID:26015495

  20. MEASUREMENTS OF CONFORMATION CHANGES DURING ADHESION OF LIPID PROTEIN (POLYLYSINE AND S-LAYER) SURFACES

    EPA Science Inventory

    The adhesion forces between various surfaces were measured using the "surface forces apparatus" technique which allows for the thickness of surface layers and the adhesion force between them to be directly measured in controlled vapor or liquid environments. hree types of biologi...

  1. Regulation of T-lymphocyte motility, adhesion and de-adhesion by a cell surface mechanism directed by low density lipoprotein receptor-related protein 1 and endogenous thrombospondin-1

    PubMed Central

    Talme, Toomas; Bergdahl, Eva; Sundqvist, Karl-Gösta

    2014-01-01

    T lymphocytes are highly motile and constantly reposition themselves between a free-floating vascular state, transient adhesion and migration in tissues. The regulation behind this unique dynamic behaviour remains unclear. Here we show that T cells have a cell surface mechanism for integrated regulation of motility and adhesion and that integrin ligands and CXCL12/SDF-1 influence motility and adhesion through this mechanism. Targeting cell surface-expressed low-density lipoprotein receptor-related protein 1 (LRP1) with an antibody, or blocking transport of LRP1 to the cell surface, perturbed the cell surface distribution of endogenous thrombospondin-1 (TSP-1) while inhibiting motility and potentiating cytoplasmic spreading on intercellular adhesion molecule 1 (ICAM-1) and fibronectin. Integrin ligands and CXCL12 stimulated motility and enhanced cell surface expression of LRP1, intact TSP-1 and a 130 000 MW TSP-1 fragment while preventing formation of a de-adhesion-coupled 110 000 MW TSP-1 fragment. The appearance of the 130 000 MW TSP-1 fragment was inhibited by the antibody that targeted LRP1 expression, inhibited motility and enhanced spreading. The TSP-1 binding site in the LRP1-associated protein, calreticulin, stimulated adhesion to ICAM-1 through intact TSP-1 and CD47. Shear flow enhanced cell surface expression of intact TSP-1. Hence, chemokines and integrin ligands up-regulate a dominant motogenic pathway through LRP1 and TSP-1 cleavage and activate an associated adhesion pathway through the LRP1–calreticulin complex, intact TSP-1 and CD47. This regulation of T-cell motility and adhesion makes pro-adhesive stimuli favour motile responses, which may explain why T cells prioritize movement before permanent adhesion. PMID:24877199

  2. Organ-selective regulation of vascular adhesion protein-1 expression in man.

    PubMed

    Arvilommi, A M; Salmi, M; Jalkanen, S

    1997-07-01

    Vascular adhesion protein-1 (VAP-1) is an endothelial molecule which mediates lymphocyte binding to endothelium in peripheral lymph nodes and at certain sites of inflammation. The expression of VAP-1 in vivo is strongly up-regulated in inflamed tissues, such as gut and skin. The purpose of this work was to examine the factors responsible for this induction of VAP-1. Since the expression of VAP-1 could not be induced in cultured endothelial cells with a large panel of mediators, we used an organ culture technique for the investigation of the regulation of VAP-1 expression in a more physiological micromilieu. Indeed, we found that the expression of endothelial VAP-1 could be up-regulated in human tonsillar tissue with interleukin (IL)-1, IL-4, tumor necrosis factor (TNF-alpha), interferon (IFN)-gamma and lipopolysaccharide, whereas histamine, thrombin, dibutyryl cAMP, N-formyl-Met-Leu-Phe (fMLP) and phorbol 12-myristate 13-acetate (PMA) had no effect. The induced VAP-1 protein was similar in molecular weight to the non-induced VAP-1, suggesting that VAP-1 synthesized de novo carries appropriate carbohydrate moieties. In contrast to tonsil organ culture, similar inductions performed with human appendix showed no up-regulation of VAP-1 expression, indicating that the regulation of VAP-1 expression exhibits organ-selective characteristics. Furthermore, in these tissues the smooth muscle cells, which constitutively express VAP-1, could not be stimulated to alter their level of expression of this molecule. In conclusion, the expression of VAP-1 can be markedly up-regulated with several mediators in tonsil but not in appendix organ culture, whereas cultured endothelial cells cannot be induced to express VAP-1. These results indicate that the expression of VAP-1 is regulated in a tissue- and cell type-selective manner, and a correct micromilieu is required for the up-regulation to occur. PMID:9247594

  3. Thrombospondin-related adhesive protein (TRAP) of Plasmodium falciparum: expression during sporozoite ontogeny and binding to human hepatocytes.

    PubMed Central

    Robson, K J; Frevert, U; Reckmann, I; Cowan, G; Beier, J; Scragg, I G; Takehara, K; Bishop, D H; Pradel, G; Sinden, R

    1995-01-01

    Plasmodium sporozoites collected from oocysts, haemocoel and salivary glands of the mosquito show profound differences in their biological properties such as motility, ability to induce protective immune response and infectivity for vertebrate host cells. Sporozoites from salivary glands are much more infectious than those from oocysts and haemocoel. Differential expression of proteins, such as the circumsporozoite (CS) protein and the thrombospondin-related adhesive protein (TRAP), implicated in sporozoite recognition and entry into hepatocytes may account for the development of infectivity during ontogeny. We have carried out a series of experiments to: (i) analyse the expression and localization of TRAP in P.falciparum sporozoites during development in the mosquito; and (ii) elucidate the biochemical and adhesive properties of recombinant TRAP. Our data indicate that TRAP is not expressed in oocysts, whereas variable amounts of CS protein are found in this parasite developmental stage. Hemocoel sporozoites display the distinct phenotypes TRAP- CS protein+ and TRAP+ CS protein+ at a frequency of 98.5 and 1.5% respectively. Salivary gland sporozoites are all TRAP+ CS protein+. We also provide experimental evidence showing that recombinant TRAP binds to the basolateral cell membrane of hepatocytes in the Disse's space and that sulfated glycoconjugates function as TRAP ligands on human hepatocytes. Images PMID:7664729

  4. Negative staining and immunoelectron microscopy of adhesion-deficient mutants of Streptococcus salivarius reveal that the adhesive protein antigens are separate classes of cell surface fibril.

    PubMed Central

    Weerkamp, A H; Handley, P S; Baars, A; Slot, J W

    1986-01-01

    The subcellular distribution of the cell wall-associated protein antigens of Streptococcus salivarius HB, which are involved in specific adhesive properties of the cells, was studied. Mutants which had lost the adhesive properties and lacked the antigens at the cell surface were compared with the parent strain. Immunoelectron microscopy of cryosections of cells labeled with affinity-purified, specific antisera and colloidal gold-protein A complexes was used to locate the antigens. Antigen C (AgC), a glycoprotein involved in attachment to host surfaces, was mainly located in the fibrillar layer outside the cell wall. A smaller amount of label was also found throughout the cytoplasmic area in the form of small clusters of gold particles, which suggests a macromolecular association. Mutant HB-7, which lacks the wall-associated AgC, accumulated AgC reactivity intracellularly. Intracellular AgC was often found associated with isolated areas of increased electron density, but sometimes seemed to fill the entire interior of the cell. Antigen B (AgB), a protein responsible for interbacterial coaggregation, was also located in the fibrillar layer, although its distribution differed from that of the wall-associated AgC since AgB was found predominantly in the peripheral areas. A very small amount of label was also found in the cytoplasmic area as discrete gold particles. Mutant HB-V5, which lacks wall-associated AgB, was not labeled in the fibrillar coat, but showed the same weak intracellular label as the parent strain. Immunolabeling with serum against AgD, another wall-associated protein but of unknown function, demonstrated its presence in the fibrillar layer of strain HB. Negatively stained preparations of whole cells of wild-type S. salivarius and mutants that had lost wall-associated AgB or AgC revealed that two classes of short fibrils are carried on the cell surface at the same time. AgB and AgC are probably located on separate classes of short, protease

  5. Src Kinase Determines the Dynamic Exchange of the Docking Protein NEDD9 (Neural Precursor Cell Expressed Developmentally Down-regulated Gene 9) at Focal Adhesions*

    PubMed Central

    Bradbury, Peta; Bach, Cuc T.; Paul, Andre; O'Neill, Geraldine M.

    2014-01-01

    Dynamic exchange of molecules between the cytoplasm and integrin-based focal adhesions provides a rapid response system for modulating cell adhesion. Increased residency time of molecules that regulate adhesion turnover contributes to adhesion stability, ultimately determining migration speed across two-dimensional surfaces. In the present study we test the role of Src kinase in regulating dynamic exchange of the focal adhesion protein NEDD9/HEF1/Cas-L. Using either chemical inhibition or fibroblasts genetically null for Src together with fluorescence recovery after photobleaching (FRAP), we find that Src significantly reduces NEDD9 exchange at focal adhesions. Analysis of NEDD9 mutant constructs with the two major Src-interacting domains disabled revealed the greatest effects were due to the NEDD9 SH2 binding domain. This correlated with a significant change in two-dimensional migratory speed. Given the emerging role of NEDD9 as a regulator of focal adhesion stability, the time of NEDD9 association at the focal adhesions is key in modulating rates of migration and invasion. Our study suggests that Src kinase activity determines NEDD9 exchange at focal adhesions and may similarly modulate other focal adhesion-targeted Src substrates to regulate cell migration. PMID:25059660

  6. Pregnancy-associated plasma protein A up-regulated by progesterone promotes adhesion and proliferation of trophoblastic cells.

    PubMed

    Wang, Jiao; Liu, Shuai; Qin, Hua-Min; Zhao, Yue; Wang, Xiao-Qi; Yan, Qiu

    2014-01-01

    Embryo implantation and development is a complex biological process for the establishment of the successful pregnancy. Progesterone is a critical factor in the regulation of embryo adhesion to uterine endometrium and proliferation. Although it has been reported that pregnancy-associated plasma protein A (PAPPA) is increased in pregnant women, the relationship between progesterone and PAPPA, and the effects of PAPPA on embryo adhesion and proliferation are still not clear. The present results showed that the serum level of progesterone and PAPPA was closely correlated by ELISA assay (p<0.01). PAPPA was detected in the villi of early embryo by RT-PCR, Western blot, immunohistochemistry and immunofluorescent staining. Moreover, PAPPA was significantly up-regulated by progesterone in trophoblastic (JAR) cells by Real-time PCR and ELISA assay (p<0.01); while the expression was decreased by the progesterone receptor inhibitor RU486. The down-regulation of PAPPA by siRNA transfection or up-regulation of PAPPA by progesterone treatment significantly decreased or increased the adhesion rate of trophoblastic cells to human uterine epithelial cell lines (RL95-2 and HEC-1A), respectively (p<0.01), as well as the proliferation of trophoblastic cells. In conclusion, PAPPA is up-regulated by progesterone, which promotes the adhesion and proliferation potential of trophoblastic cells. PMID:24817938

  7. Suppression of complement regulatory protein C1 inhibitor in vascular endothelial activation by inhibiting vascular cell adhesion molecule-1 action

    SciTech Connect

    Zhang, Haimou; Qin, Gangjian; Liang, Gang; Li, Jinan; Chiu, Isaac; Barrington, Robert A.; Liu, Dongxu . E-mail: dxliu001@yahoo.com

    2007-07-13

    Increased expression of adhesion molecules by activated endothelium is a critical feature of vascular inflammation associated with the several diseases such as endotoxin shock and sepsis/septic shock. Our data demonstrated complement regulatory protein C1 inhibitor (C1INH) prevents endothelial cell injury. We hypothesized that C1INH has the ability of an anti-endothelial activation associated with suppression of expression of adhesion molecule(s). C1INH blocked leukocyte adhesion to endothelial cell monolayer in both static assay and flow conditions. In inflammatory condition, C1INH reduced vascular cell adhesion molecule (VCAM-1) expression associated with its cytoplasmic mRNA destabilization and nuclear transcription level. Studies exploring the underlying mechanism of C1INH-mediated suppression in VCAM-1 expression were related to reduction of NF-{kappa}B activation and nuclear translocation in an I{kappa}B{alpha}-dependent manner. The inhibitory effects were associated with reduction of inhibitor I{kappa}B kinase activity and stabilization of the NF-{kappa}B inhibitor I{kappa}B. These findings indicate a novel role for C1INH in inhibition of vascular endothelial activation. These observations could provide the basis for new therapeutic application of C1INH to target inflammatory processes in different pathologic situations.

  8. Cleavage of Type I Collagen by Fibroblast Activation Protein-α Enhances Class A Scavenger Receptor Mediated Macrophage Adhesion

    PubMed Central

    Mazur, Anna; Holthoff, Emily; Vadali, Shanthi; Kelly, Thomas; Post, Steven R.

    2016-01-01

    Pathophysiological conditions such as fibrosis, inflammation, and tumor progression are associated with modification of the extracellular matrix (ECM). These modifications create ligands that differentially interact with cells to promote responses that drive pathological processes. Within the tumor stroma, fibroblasts are activated and increase the expression of type I collagen. In addition, activated fibroblasts specifically express fibroblast activation protein-α (FAP), a post-prolyl peptidase. Although FAP reportedly cleaves type I collagen and contributes to tumor progression, the specific pathophysiologic role of FAP is not clear. In this study, the possibility that FAP-mediated cleavage of type I collagen modulates macrophage interaction with collagen was examined using macrophage adhesion assays. Our results demonstrate that FAP selectively cleaves type I collagen resulting in increased macrophage adhesion. Increased macrophage adhesion to FAP-cleaved collagen was not affected by inhibiting integrin-mediated interactions, but was abolished in macrophages lacking the class A scavenger receptor (SR-A/CD204). Further, SR-A expressing macrophages localize with activated fibroblasts in breast tumors of MMTV-PyMT mice. Together, these results demonstrate that FAP-cleaved collagen is a substrate for SR-A-dependent macrophage adhesion, and suggest that by modifying the ECM, FAP plays a novel role in mediating communication between activated fibroblasts and macrophages. PMID:26934296

  9. Expression of E-Cadherin and N-Cadherin in Perinatal Hamster Ovary: Possible Involvement in Primordial Follicle Formation and Regulation by Follicle-Stimulating Hormone

    PubMed Central

    Wang, Cheng; Roy, Shyamal K.

    2010-01-01

    We examined the expression and hormonal regulation of E-cadherin (CDH1) and N-cadherin (CDH2) with respect to primordial follicle formation. Hamster Cdh1 and Cdh2 cDNA and amino acid sequences were more than 90% similar to those of the mouse, rat, and human. Although CDH1 expression remained exclusively in the oocytes during neonatal ovary development, CDH2 expression shifted from the oocytes to granulosa cells of primordial follicles on postnatal day (P)8. Subsequently, strong CDH2 expression was restricted to granulosa cells of growing follicles. Cdh2 mRNA levels in the ovary decreased from embryonic d 13 through P10 with a transient increase on P7, which was the day before the appearance of primordial follicles. Cdh1 mRNA levels decreased from embryonic d 13 through P3 and then showed a transient increase on P8, coinciding with the formation of primordial follicles. CDH1 and CDH2 expression were consistent with that of mRNA. Neutralization of FSH in utero impaired primordial follicle formation with an associated decrease in Cdh2 mRNA and CDH2, but an increase in Cdh1 mRNA and CDH1 expression. The altered expression was reversed by equine chorionic gonadotropin treatment on P1. Whereas a CDH2 antibody significantly reduced the formation of primordial and primary follicles in vitro, a CDH1 antibody had the opposite effect. This is the first evidence to suggest that primordial follicle formation requires a differential spatiotemporal expression and action of CDH1 and CDH2. Further, FSH regulation of primordial follicle formation may involve the action of CDH1 and CDH2. PMID:20219978

  10. Focal adhesion proteins connect IgE receptors to the cytoskeleton as revealed by micropatterned ligand arrays

    PubMed Central

    Torres, Alexis J.; Vasudevan, Lavanya; Holowka, David; Baird, Barbara A.

    2008-01-01

    Patterned surfaces that present specific ligands in spatially defined arrays are used to examine structural linkages between clustered IgE receptors (IgE-FcεRI) and the cytoskeleton in rat basophilic leukemia (RBL) mast cells. We showed with fluorescence microscopy that cytoskeletal F-actin concentrates in the same regions as cell surface IgE-FcεRI that bind to the micrometer-size patterned ligands. However, the proteins mediating these cytoskeletal connections and their functional relevance were not known. We now show that whereas the adaptor proteins ezrin and moesin do not detectably concentrate with the array of clustered IgE-FcεRI, focal adhesion proteins vinculin, paxillin, and talin, which are known to link F-actin with integrins, accumulate in these regions on the same time scale as F-actin. Moreover, colocalization of these focal adhesion proteins with clustered IgE-FcεRI is enhanced after addition of fibronectin-RGD peptides. Significantly, the most prominent rat basophilic leukemia cell integrin (α5) avoids the patterned regions occupied by the ligands and associates preferentially with exposed regions of the silicon substrate. Thus, spatial separation provided by the patterned surface reveals that particular focal adhesion proteins, which connect to the actin cytoskeleton, associate with ligand-cross-linked IgE-FcεRI, independently of integrins. We investigated the functional role of one of these proteins, paxillin, in IgE-FcεRI-mediated signaling by using small interfering RNA. From these results, we determine that paxillin reduces stimulated phosphorylation of the FcεRI β subunit but enhances stimulated Ca2+ release from intracellular stores. The results suggest that paxillin associated with clustered IgE-FcεRI has a net positive effect on FcεRI signaling. PMID:19004813

  11. Molecular cloning, structural analysis and functional expression of the proline-rich focal adhesion and microfilament-associated protein VASP.

    PubMed Central

    Haffner, C; Jarchau, T; Reinhard, M; Hoppe, J; Lohmann, S M; Walter, U

    1995-01-01

    The vasodilator-stimulated phosphoprotein (VASP), a substrate for cAMP- and cGMP-dependent protein kinases in vitro and in intact cells, is associated with actin filaments, focal adhesions and dynamic membrane regions. VASP, cloned here from human HL-60 and canine MDCK cells, is organized into three distinct domains. A central proline-rich domain contains a GPPPPP motif as a single copy and as a 3-fold tandem repeat, as well as three conserved phosphorylation sites for cyclic nucleotide-dependent protein kinases. A C-terminal domain contains a repetitive mixed-charge cluster which is predicted to form an alpha-helix. The hydrodynamic properties of purified human VASP together with the calculated molecular mass of cloned VASP suggest that the native protein is a homotetramer with an elongated structure. VASP over-expressed in transiently transfected BHK21 cells was predominantly detected at stress fibres, at focal adhesions and in F-actin-containing cell surface protrusions, whereas truncated VASP lacking the C-terminal domain was no longer concentrated at focal adhesions. These data indicate that the C-terminal domain is required for anchoring VASP at focal adhesion sites, whereas the central domain is suggested to mediate VASP interaction with profilin. Our results provide evidence for the structural basis by which VASP, both a target of the cAMP and cGMP signal transduction pathways and a component of the actin-based cytoskeleton, including the cytoskeleton-membrane interface, may be able to exchange signals between these networks. Images PMID:7828592

  12. Divalent cations and the protein surface co-ordinate the intensity of human platelet adhesion and P-selectin surface expression.

    PubMed

    Whiss, P A; Andersson, R G G

    2002-07-01

    At sites of blood vessel injury, platelets adhere to exposed vessel components, such as collagen, or immobilized fibrinogen derived from plasma or activated platelets. The divalent cations Mg(2+) and Ca(2+) are essential for platelet adhesion and activation, but Mg(2+) can also inhibit platelet activation. The present study evaluates, by an enzymatic method, the effects of various divalent cations on the adhesion of isolated human platelets to collagen, fibrinogen, albumin or plastic in vitro. By enzyme-linked immunosorbent assay, platelet surface expression of P-selectin was measured to estimate the state of activation on adherence. Mg(2+) increased platelet adhesion exclusively to collagen and fibrinogen at physiologically relevant concentrations. At higher concentrations, the adhesion declined. Ca(2+) induced a weak adhesion only to fibrinogen at physiological doses and a peak of increased adhesion to all protein-coated surfaces at 10 mmol/l. Mn(2+) elicited dose-dependent adhesion only to collagen and fibrinogen. Zn(2+), Ni(2+) and Cu(2+) increased the adhesion of platelets independently of the surface. Ca(2+) dose-dependently inhibited adhesion elicited by Mg(2+) to collagen and fibrinogen. No other combination of divalent cations elicited such an effect. Mg(2+)-dependent platelet adhesion to collagen and Ca(2+)-dependent adhesion to fibrinogen increased P-selectin expression. Thus, the present study shows that the outcome of the platelet adhesion depends on the surface and the access of divalent cations, which co-ordinate the intensity of platelet adhesion and P-selectin surface expression. PMID:12138368

  13. Role of Lactobacillus reuteri cell and mucus-binding protein A (CmbA) in adhesion to intestinal epithelial cells and mucus in vitro.

    PubMed

    Jensen, Hanne; Roos, Stefan; Jonsson, Hans; Rud, Ida; Grimmer, Stine; van Pijkeren, Jan-Peter; Britton, Robert A; Axelsson, Lars

    2014-04-01

    Lactobacillus reuteri, a symbiotic inhabitant of the gastrointestinal tract in humans and animals, is marketed as a probiotic. The ability to adhere to intestinal epithelial cells and mucus is an interesting property with regard to probiotic features such as colonization of the gastrointestinal tract and interaction with the host. Here, we present a study performed to elucidate the role of sortase (SrtA), four putative sortase-dependent proteins (SDPs), and one C-terminal membrane-anchored cell surface protein of Lactobacillus reuteri ATCC PTA 6475 in adhesion to Caco-2 cells and mucus in vitro. This included mutagenesis of the genes encoding these proteins and complementation of mutants. A null mutation in hmpref0536_10255 encoding srtA resulted in significantly reduced adhesion to Caco-2 cells and mucus, indicating involvement of SDPs in adhesion. Evaluation of the bacterial adhesion revealed that of the five putative surface protein mutants tested, only a null mutation in the hmpref0536_10633 gene, encoding a putative SDP with an LPxTG motif, resulted in a significant loss of adhesion to both Caco-2 cells and mucus. Complementation with the functional gene on a plasmid restored adhesion to Caco-2 cells. However, complete restoration of adhesion to mucus was not achieved. Overexpression of hmpref0536_10633 in strain ATCC PTA 6475 resulted in an increased adhesion to Caco-2 cells and mucus compared with the WT strain. We conclude from these results that, among the putative surface proteins tested, the protein encoded by hmpref0536_10633 plays a critical role in binding of Lactobacillus reuteri ATCC PTA 6475 to Caco-2 cells and mucus. Based on this, we propose that this LPxTG motif containing protein should be referred to as cell and mucus binding protein A (CmbA). PMID:24473252

  14. TM9/Phg1 and SadA proteins control surface expression and stability of SibA adhesion molecules in Dictyostelium

    PubMed Central

    Froquet, Romain; le Coadic, Marion; Perrin, Jackie; Cherix, Nathalie; Cornillon, Sophie; Cosson, Pierre

    2012-01-01

    TM9 proteins form a family of conserved proteins with nine transmembrane domains essential for cellular adhesion in many biological systems, but their exact role in this process remains unknown. In this study, we found that genetic inactivation of the TM9 protein Phg1A dramatically decreases the surface levels of the SibA adhesion molecule in Dictyostelium amoebae. This is due to a decrease in sibA mRNA levels, in SibA protein stability, and in SibA targeting to the cell surface. A similar phenotype was observed in cells devoid of SadA, a protein that does not belong to the TM9 family but also exhibits nine transmembrane domains and is essential for cellular adhesion. A contact site A (csA)-SibA chimeric protein comprising only the transmembrane and cytosolic domains of SibA and the extracellular domain of the Dictyostelium surface protein csA also showed reduced stability and relocalization to endocytic compartments in phg1A knockout cells. These results indicate that TM9 proteins participate in cell adhesion by controlling the levels of adhesion proteins present at the cell surface. PMID:22219373

  15. Physiological Osmotic Induction of Leptospira interrogans Adhesion: LigA and LigB Bind Extracellular Matrix Proteins and Fibrinogen▿

    PubMed Central

    Choy, Henry A.; Kelley, Melissa M.; Chen, Tammy L.; Møller, Annette K.; Matsunaga, James; Haake, David A.

    2007-01-01

    Transmission of leptospirosis occurs through contact of mucous membranes and abraded skin with freshwater contaminated by pathogenic Leptospira spp. Exposure to physiological osmolarity induces leptospires to express high levels of the Lig surface proteins containing imperfect immunoglobulin-like repeats that are shared or differ between LigA and LigB. We report that osmotic induction of Lig is accompanied by 1.6- to 2.5-fold increases in leptospiral adhesion to immobilized extracellular matrix and plasma proteins, including collagens I and IV, laminin, and especially fibronectin and fibrinogen. Recombinant LigA-unique and LigB-unique repeat proteins bind to these same host ligands. We found that the avidity of LigB in binding fibronectin is comparable to that of the Staphylococcus aureus FnBPA D repeats. Both LigA- and LigB-unique repeats interact with the amino-terminal fibrin- and gelatin-binding domains of fibronectin, which are also recognized by fibronectin-binding proteins mediating the adhesion of other microbial pathogens. In contrast, repeats common to both LigA and LigB do not bind these host proteins, and nonrepeat sequences in the carboxy-terminal domain of LigB show only weak interaction with fibronectin and fibrinogen. A functional role for the binding activity of LigA and LigB is suggested by the ability of the recombinants to inhibit leptospiral adhesion to fibronectin by 28% and 21%, respectively. The binding of LigA and LigB to multiple ligands present in different tissues suggests that these adhesins may be involved in the initial colonization and dissemination stages of leptospirosis. The characterization of the Lig adhesin function should aid the design of Lig-based vaccines and serodiagnostic tests. PMID:17296754

  16. Supporting data for characterization of non-coding RNAs associated with the Neuronal growth regulator 1 (NEGR1) adhesion protein.

    PubMed

    Kaur, Prameet; Tan, Jun Rong; Karolina, Dwi Setyowati; Sepramaniam, Sugunavathi; Armugam, Arunmozhiarasi; Peter Wong, Tsun-Hon; Jeyaseelan, Kandiah

    2016-06-01

    Long non-coding RNAs and microRNAs control gene expression to determine central nervous system development and function. Neuronal growth regulator 1 (NEGR1) is a cell adhesion molecule that plays an important role in neurite outgrowth during neuronal development and its precise expression is crucial for correct brain development. The data described here is related to the research article titled "A long non-coding RNA, BC048612 and a microRNA, miR-203 coordinate the gene expression of Neuronal growth regulator 1 (NEGR1) adhesion protein" [1]. This data article contains detailed bioinformatics analysis of genetic signatures at the Negr1 gene locus retrieved from the UCSC genome browser. This approach could be adopted to identify putative regulatory non-coding RNAs in other tissues and diseases. PMID:26977442

  17. In vivo modification of tyrosine residues in recombinant mussel adhesive protein by tyrosinase co-expression in Escherichia coli

    PubMed Central

    2012-01-01

    Background In nature, mussel adhesive proteins (MAPs) show remarkable adhesive properties, biocompatibility, and biodegradability. Thus, they have been considered promising adhesive biomaterials for various biomedical and industrial applications. However, limited production of natural MAPs has hampered their practical applications. Recombinant production in bacterial cells could be one alternative to obtain useable amounts of MAPs, although additional post-translational modification of tyrosine residues into 3,4-dihydroxyphenyl-alanine (Dopa) and Dopaquinone is required. The superior properties of MAPs are mainly attributed to the introduction of quinone-derived intermolecular cross-links. To solve this problem, we utilized a co-expression strategy of recombinant MAP and tyrosinase in Escherichia coli to successfully modify tyrosine residues in vivo. Results A recombinant hybrid MAP, fp-151, was used as a target for in vivo modification, and a dual vector system of pET and pACYC-Duet provided co-expression of fp-151 and tyrosinase. As a result, fp-151 was over-expressed and mainly obtained from the soluble fraction in the co-expression system. Without tyrosinase co-expression, fp-151 was over-expressed in an insoluble form in inclusion bodies. The modification of tyrosine residues in the soluble-expressed fp-151 was clearly observed from nitroblue tetrazolium staining and liquid-chromatography-mass/mass spectrometry analyses. The purified, in vivo modified, fp-151 from the co-expression system showed approximately 4-fold higher bulk-scale adhesive strength compared to in vitro tyrosinase-treated fp-151. Conclusion Here, we reported a co-expression system to obtain in vivo modified MAP; additional in vitro tyrosinase modification was not needed to obtain adhesive properties and the in vivo modified MAP showed superior adhesive strength compared to in vitro modified protein. It is expected that this co-expression strategy will accelerate the use of functional MAPs in

  18. Expression and organization of basement membranes and focal adhesion proteins in pregnant myometrium is regulated by uterine stretch.

    PubMed

    Shynlova, Oksana; Chow, Michelle; Lye, Stephen J

    2009-10-01

    The mechanisms underlying the preparation of the uterus for labor are not fully understood. We have previously found a significant increase in the expression of messenger RNA (mRNAs) encoding extracellular basement membrane (BM) proteins of the smooth muscle cells (SMCs) in late pregnant rat myometrium. At term, the myometrium is stretched by growing fetuses and these mechanical signals are transmitted from extracellular matrix into SMCs through focal adhesions (FA). The aim of this study was to investigate the effect of gravidity on the expression and spatiotemporal distribution of major BM proteins, laminin-gamma2 and collagen IV, as well as typical FA constituents, vinculin and paxillin, in the myometrium during gestation and parturition, using a unilaterally pregnant rat model. We found that the expression of laminin-gamma2 and collagen IV proteins increased significantly with gestational age (P < .05) and was dependent on gravidity whereas vinculin and paxillin proteins were not affected. Near term, BM proteins from gravid horn myometrium demonstrated increased extracellular immunostaining and major rearrangement from sporadic protein distribution to organized, continuous, and regular structures surrounding the plasma membrane of each myocyte. Examination of FA proteins revealed that paxillin was translocated from the cytoplasm to the cell periphery, while vinculin was sequestered specifically to FAs. At labor, BM and FA proteins, organized in similar bead-like structures, were localized on opposing sides of SMC plasma membrane into 2 different compartments. We suggest that these stretch-induced changes facilitate formation of stable cell-matrix adhesions and provide the molecular basis for optimal force transduction during labor contractions. PMID:19602722

  19. Identification, characterization, and expression levels of putative adhesive proteins from the tube-dwelling polychaete Sabellaria alveolata.

    PubMed

    Becker, Pierre T; Lambert, Aurélie; Lejeune, Annabelle; Lanterbecq, Déborah; Flammang, Patrick

    2012-10-01

    The shelter of the tube-dwelling polychaete Sabellaria alveolata is composed of mineral particles assembled with spots of a proteinaceous cement. The adhesive proteins constituting the cement were identified on the basis of their sequence similarity with proteins of a phylogenetically related species, Phragmatopoma californica. Two positively charged proteins, Sa-1 and Sa-2, share common features: they both have a mass of 22 kDa; are rich in glycine, tyrosine and basic residues; and show repeated peptide motifs. The consensus repeat of Sa-1 is KGAYGAKGLGYGNKAGYGAYG (occurring 6-8 times), while Sa-2 displays the consensus heptapeptide VHKAAWG (5 times) and undecapeptide VHKAAGYGGYG (8 times). Two variants of a serine-rich protein, Sa-3A (22 kDa) and Sa-3B (21 kDa), were also identified. Their serine residues account for 75 mol% and are probably phosphorylated, meaning that Sa-3 is very acidic and negatively charged. Moreover, tyrosine residues of all adhesive proteins are presumably modified into DOPA. Although protein sequences are not well-conserved between S. alveolata and P. californica, their main characteristics (including amino acid composition, post-translational modifications, repeated patterns, isoelectric point, and mass) are shared by both species. This suggests that these features are more important for their function than the primary structure of the proteins. The mRNA abundance for each protein was estimated by quantitative real-time PCR, revealing relative expression levels of about 5, 11, 1.5, and 1 for Sa-1, -2, -3A, and -3B, respectively. These levels could be indicative of charge neutralization phenomena or could reflect their function (interface vs. bulk) in the cement. PMID:23111133

  20. Adhesions of extracellular surface-layer associated proteins in Lactobacillus M5-L and Q8-L.

    PubMed

    Zhang, Yingchun; Xiang, Xinling; Lu, Qianhui; Zhang, Lanwei; Ma, Fang; Wang, Linlin

    2016-02-01

    Surface-layer associated proteins (SLAP) that envelop Lactobacillus paracasei ssp. paracasei M5-L and Lactobacillus casei Q8-L cell surfaces are involved in the adherence of these strain to the human intestinal cell line HT-29. To further elucidate some of the properties of these proteins, we assessed the yields and expressions of SLAP under different incubation conditions. An efficient and selective extraction of SLAP was obtained when cells of Lactobacillus were treated with 5 M LiCl at 37°C in aerobic conditions. The SLAP of Lactobacillus M5-L and Q8-L in cell extracts were visualized by SDS-PAGE and identified by Western blotting with sulfo-N-hydroxysuccinimide-biotin-labeled HT-29 cells as adhesion proteins. Atomic force microscopy contact imaging revealed that Lactobacillus strains M5-L and Q8-L normally display a smooth, homogeneous surface, whereas the surfaces of M5-L and Q8-L treated with 5 M LiCl were rough and more heterogeneous. Analysis of adhesion forces revealed that the initial adhesion forces of 1.41 and 1.28 nN obtained for normal Lactobacillus M5-L and Q8-L strains, respectively, decreased to 0.70 and 0.48 nN, respectively, following 5 M LiCl treatment. Finally, the dominant 45-kDa protein bands of Lactobacillus Q8-L and Lactobacillus M5-L were identified as elongation factor Tu and surface antigen, respectively, by liquid chromatography-tandem mass spectrometry. PMID:26709174

  1. A human salivary protein which promotes adhesion of Streptococcus mutans serotype c strains to hydroxyapatite.

    PubMed Central

    Kishimoto, E; Hay, D I; Gibbons, R J

    1989-01-01

    The aim of this study was to investigate the nature of one of the factors in human submandibular-sublingual (SMSL) saliva which promotes the adhesion of Streptococcus mutans serotype c strains to hydroxyapatite (HA) surfaces. Gel filtration chromatography of SMSL saliva on Trisacryl GF2000 gave a void volume peak which contained the major fraction of adhesion-promoting activity for S. mutans JBP to HA. Maximum adhesion-promoting activity, however, eluted slightly later than the maximum 220-nm absorbance of the void volume peak. Gel filtration of the void volume material after treatment with sodium dodecyl sulfate (SDS) gave an early-eluting larger peak followed by a smaller peak with which the adhesion-promoting activity was associated. SDS-polyacrylamide gel electrophoresis (SDS-PAGE) showed the presence of relatively slowly migrating material associated with the larger inactive peak, presumably mucin, and a faster-migrating band(s) associated with the smaller active peak. SDS-PAGE indicated molecular weights in the range of 300,000 to 350,000 by extrapolation from size standards. Comparison of SMSL from five individuals showed the presence of single bands or double bands associated with adhesion-promoting activity, indicating genetic polymorphism. The active material did not resemble either secretory immunoglobulin A, based on SDS-PAGE and immunoassay, or fibronectin, based on SDS-PAGE, and also differed in molecular weight from salivary mucins and salivary constituents previously reported to promote aggregation of certain oral bacteria, but a relationship to these materials cannot be excluded. This adhesion-promoting material may play a significant role in the initial colonization of tooth surfaces by S. mutans strains. Images PMID:2807544

  2. Circulating renalase, catecholamines, and vascular adhesion protein 1 in hypertensive patients.

    PubMed

    Maciorkowska, Dominika; Zbroch, Edyta; Malyszko, Jolanta

    2015-11-01

    The aim of the study was to estimate and correlate circulating levels of renalase, vascular adhesion protein-1 (VAP-1), catecholamines in patients with primary hypertension. The renalase, VAP-1, and catecholamines concentration was estimated in 121 hypertensive patients. The correlation between renalase, VAP-1 levels and catecholamine concentration in blood, blood pressure control, pharmacological therapy, and medical history were taken in to consideration. The median office blood pressure was 145.5/86 mm Hg and was significantly higher than the median home blood pressure measurement value, which was 135/80 mm Hg, P < .05. Circulating renalase and VAP-1 (Me 9.57 μg/mL and Me = 326.7 ng/mL) levels were significantly higher in patients with hypertension comparing to healthy individuals (3.83 μg/mL and 248.37 ng/mL, P < .05). The correlation between renalase and noradrenalin concentration in blood was observed (r = 0.549; P < .05), also the correlation between VAP-1 and noradrenaline was noticed (r = 0.21, P = .029). Renalase level was higher in patients with coronary artery disease and correlated with decreased ejection fraction. VAP-1 concentration correlated also with left ventricular ejection fraction (r = -0.23, P = .013). Hypertensive patients with diabetes mellitus had almost statistically significant higher VAP-1 concentration compared with hypertensive patients without diabetes mellitus (Me = 403.22 ng/mL vs. Me = 326,68 ng/mL, P = .064). In multiple regression analysis, renalase was predicted by plasma dopamine and norepinephrine as also diastolic office blood pressure and left ventricle ejection fraction. Circulating renalase and VAP-1 levels are elevated in patients with poor blood pressure control. Its correlation with noradrenalin concentration need further studies to find out the role of renalase as also VAP-1 in pathogenesis and treatment of hypertension. PMID:26403854

  3. Comparative genome-based identification of a cell wall-anchored protein from Lactobacillus plantarum increases adhesion of Lactococcus lactis to human epithelial cells.

    PubMed

    Zhang, Bo; Zuo, Fanglei; Yu, Rui; Zeng, Zhu; Ma, Huiqin; Chen, Shangwu

    2015-01-01

    Adhesion to host cells is considered important for Lactobacillus plantarum as well as other lactic acid bacteria (LAB) to persist in human gut and thus exert probiotic effects. Here, we sequenced the genome of Lt. plantarum strain NL42 originating from a traditional Chinese dairy product, performed comparative genomic analysis and characterized a novel adhesion factor. The genome of NL42 was highly divergent from its closest neighbors, especially in six large genomic regions. NL42 harbors a total of 42 genes encoding adhesion-associated proteins; among them, cwaA encodes a protein containing multiple domains, including five cell wall surface anchor repeat domains and an LPxTG-like cell wall anchor motif. Expression of cwaA in Lactococcus lactis significantly increased its autoaggregation and hydrophobicity, and conferred the new ability to adhere to human colonic epithelial HT-29 cells by targeting cellular surface proteins, and not carbohydrate moieties, for CwaA adhesion. In addition, the recombinant Lc. lactis inhibited adhesion of Staphylococcus aureus and Escherichia coli to HT-29 cells, mainly by exclusion. We conclude that CwaA is a novel adhesion factor in Lt. plantarum and a potential candidate for improving the adhesion ability of probiotics or other bacteria of interest. PMID:26370773

  4. Comparative genome-based identification of a cell wall-anchored protein from Lactobacillus plantarum increases adhesion of Lactococcus lactis to human epithelial cells

    PubMed Central

    Zhang, Bo; Zuo, Fanglei; Yu, Rui; Zeng, Zhu; Ma, Huiqin; Chen, Shangwu

    2015-01-01

    Adhesion to host cells is considered important for Lactobacillus plantarum as well as other lactic acid bacteria (LAB) to persist in human gut and thus exert probiotic effects. Here, we sequenced the genome of Lt. plantarum strain NL42 originating from a traditional Chinese dairy product, performed comparative genomic analysis and characterized a novel adhesion factor. The genome of NL42 was highly divergent from its closest neighbors, especially in six large genomic regions. NL42 harbors a total of 42 genes encoding adhesion-associated proteins; among them, cwaA encodes a protein containing multiple domains, including five cell wall surface anchor repeat domains and an LPxTG-like cell wall anchor motif. Expression of cwaA in Lactococcus lactis significantly increased its autoaggregation and hydrophobicity, and conferred the new ability to adhere to human colonic epithelial HT-29 cells by targeting cellular surface proteins, and not carbohydrate moieties, for CwaA adhesion. In addition, the recombinant Lc. lactis inhibited adhesion of Staphylococcus aureus and Escherichia coli to HT-29 cells, mainly by exclusion. We conclude that CwaA is a novel adhesion factor in Lt. plantarum and a potential candidate for improving the adhesion ability of probiotics or other bacteria of interest. PMID:26370773

  5. Involvement of NF-κB-mediated expression of galectin-3-binding protein in TNF-α-induced breast cancer cell adhesion.

    PubMed

    Noma, Naruto; Simizu, Siro; Kambayashi, Yusuke; Kabe, Yasuaki; Suematsu, Makoto; Umezawa, Kazuo

    2012-06-01

    Galectin-3-binding protein (G3BP) is highly expressed in various types of cancer and is thought to be involved in cancer malignancy; however, the role of G3BP in breast cancer cells is not fully understood. In this study, we investigated the role of NF-κB in the adhesion of breast cancer cells to a substrate by using (-)-DHMEQ, a specific inhibitor of NF-κB. (-)-DHMEQ inhibited both TNF-α-induced G3BP expression and cell adhesion in human breast cancer cell lines. We also found that knockdown of G3BP suppressed the adhesion, while its overexpression increased the adhesion. These data reveal that (-)-DHMEQ suppresses breast cancer cell adhesion by inhibiting NF-κB-regulated G3BP expression. PMID:22447108

  6. In-situ coupling between kinase activities and protein dynamics within single focal adhesions

    PubMed Central

    Wu, Yiqian; Zhang, Kaiwen; Seong, Jihye; Fan, Jason; Chien, Shu; Wang, Yingxiao; Lu, Shaoying

    2016-01-01

    The dynamic activation of oncogenic kinases and regulation of focal adhesions (FAs) are crucial molecular events modulating cell adhesion in cancer metastasis. However, it remains unclear how these events are temporally coordinated at single FA sites. Therefore, we targeted fluorescence resonance energy transfer (FRET)-based biosensors toward subcellular FAs to report local molecular events during cancer cell adhesion. Employing single FA tracking and cross-correlation analysis, we quantified the dynamic coupling characteristics between biochemical kinase activities and structural FA within single FAs. We show that kinase activations and FA assembly are strongly and sequentially correlated, with the concurrent FA assembly and Src activation leading focal adhesion kinase (FAK) activation by 42.6 ± 12.6 sec. Strikingly, the temporal coupling between kinase activation and individual FA assembly reflects the fate of FAs at later stages. The FAs with a tight coupling tend to grow and mature, while the less coupled FAs likely disassemble. During FA disassembly, however, kinase activations lead the disassembly, with FAK being activated earlier than Src. Therefore, by integrating subcellularly targeted FRET biosensors and computational analysis, our study reveals intricate interplays between Src and FAK in regulating the dynamic life of single FAs in cancer cells. PMID:27383747

  7. In-situ coupling between kinase activities and protein dynamics within single focal adhesions.

    PubMed

    Wu, Yiqian; Zhang, Kaiwen; Seong, Jihye; Fan, Jason; Chien, Shu; Wang, Yingxiao; Lu, Shaoying

    2016-01-01

    The dynamic activation of oncogenic kinases and regulation of focal adhesions (FAs) are crucial molecular events modulating cell adhesion in cancer metastasis. However, it remains unclear how these events are temporally coordinated at single FA sites. Therefore, we targeted fluorescence resonance energy transfer (FRET)-based biosensors toward subcellular FAs to report local molecular events during cancer cell adhesion. Employing single FA tracking and cross-correlation analysis, we quantified the dynamic coupling characteristics between biochemical kinase activities and structural FA within single FAs. We show that kinase activations and FA assembly are strongly and sequentially correlated, with the concurrent FA assembly and Src activation leading focal adhesion kinase (FAK) activation by 42.6 ± 12.6 sec. Strikingly, the temporal coupling between kinase activation and individual FA assembly reflects the fate of FAs at later stages. The FAs with a tight coupling tend to grow and mature, while the less coupled FAs likely disassemble. During FA disassembly, however, kinase activations lead the disassembly, with FAK being activated earlier than Src. Therefore, by integrating subcellularly targeted FRET biosensors and computational analysis, our study reveals intricate interplays between Src and FAK in regulating the dynamic life of single FAs in cancer cells. PMID:27383747

  8. The adhesive protein of Choromytilus chorus (Molina, 1782) and Aulacomya ater (Molina, 1782): a proline-rich and a glycine-rich polyphenolic protein.

    PubMed

    Burzio, L A; Saéz, C; Pardo, J; Waite, J H; Burzio, L O

    2000-06-15

    The adhesive polyphenolic proteins from Aulacomya ater and Choromytilus chorus with apparent molecular masses of 135000 and 105000, respectively, were digested with trypsin and the peptides produced resolved by reversed phase liquid chromatography. About 5 and 12 major peptides were obtained from the protein of A. ater and C. chorus, respectively. The major peptides were purified by reverse-phase chromatography and the amino acid sequence indicates that both polyphenolic proteins consisted of repeated sequence motifs in their primary structure. The major peptides of A. ater contain seven amino acids corresponding to the consensus sequence AGYGGXK, whereas the tyrosine was always found as 3, 4-dihydroxyphenylalanine (Dopa), the X residue in position 6 was either valine, leucine or isoleucine, and the carboxy terminal was either lysine or hydroxylysine. On the other hand, the major peptides of C. chorus ranged in size from 6 to 21 amino acids and the majority correspond to the consensus sequence AKPSKYPTGYKPPVK. Both proteins differ markedly in the sequence of their tryptic peptides, but they share the common characteristics of other adhesive proteins in having a tandem sequence repeat in their primary structure. PMID:11004549

  9. A role for the retinoblastoma protein as a regulator of mouse osteoblast cell adhesion: implications for osteogenesis and osteosarcoma formation.

    PubMed

    Sosa-García, Bernadette; Gunduz, Volkan; Vázquez-Rivera, Viviana; Cress, W Douglas; Wright, Gabriela; Bian, Haikuo; Hinds, Philip W; Santiago-Cardona, Pedro G

    2010-01-01

    The retinoblastoma protein (pRb) is a cell cycle regulator inactivated in most human cancers. Loss of pRb function results from mutations in the gene coding for pRb or for any of its upstream regulators. Although pRb is predominantly known as a cell cycle repressor, our data point to additional pRb functions in cell adhesion. Our data show that pRb regulates the expression of a wide repertoire of cell adhesion genes and regulates the assembly of the adherens junctions required for cell adhesion. We conducted our studies in osteoblasts, which depend on both pRb and on cell-to-cell contacts for their differentiation and function. We generated knockout mice in which the RB gene was excised specifically in osteoblasts using the cre-lox P system and found that osteoblasts from pRb knockout mice did not assemble adherens junction at their membranes. pRb depletion in wild type osteoblasts using RNAi also disrupted adherens junctions. Microarrays comparing pRb-expressing and pRb-deficient osteoblasts showed that pRb controls the expression of a number of cell adhesion genes, including cadherins. Furthermore, pRb knockout mice showed bone abnormalities consistent with osteoblast adhesion defects. We also found that pRb controls the function of merlin, a well-known regulator of adherens junction assembly, by repressing Rac1 and its effector Pak1. Using qRT-PCR, immunoblots, co-immunoprecipitation assays, and immunofluorescent labeling, we observed that pRb loss resulted in Rac1 and Pak1 overexpression concomitant with merlin inactivation by Pak1, merlin detachment from the membrane, and adherens junction loss. Our data support a pRb function in cell adhesion while elucidating the mechanism for this function. Our work suggests that in some tumor types pRb inactivation results in both a loss of cell cycle control that promotes initial tumor growth as well as in a loss of cell-to-cell contacts, which contributes to later stages of metastasis. PMID:21085651

  10. A protein fragment of streptococcal cell surface antigen I/II which prevents adhesion of Streptococcus mutans.

    PubMed Central

    Munro, G H; Evans, P; Todryk, S; Buckett, P; Kelly, C G; Lehner, T

    1993-01-01

    Attachment of Streptococcus mutans to the tooth surface involves a cell surface protein with an M(r) of 185,000, termed streptococcal antigen (SA) I/II. Four overlapping fragments of the gene encoding SA I/II were amplified by polymerase chain reaction, cloned, and expressed in Escherichia coli. The recombinant polypeptides were assayed for adhesion-binding activity to salivary receptors and for recognition by a panel of monoclonal antibodies (MAbs) raised against SA I/II. Two of the MAbs which are known to prevent colonization of S. mutans in vivo bound the recombinant polypeptide comprising residues 816 to 1161. In vitro adhesion of S. mutans to saliva-coated hydroxyapatite beads was also inhibited specifically by a polypeptide (residues 816 to 1213) encompassing the same region. The evidence from the MAbs preventing colonization of S. mutans and the adherence inhibition assay suggests that an adhesion-binding activity resides within the portion of SA I/II comprising residues 816 to 1213, which is highly conserved among oral streptococcal species. Images PMID:7691754

  11. β Integrin-like protein-mediated adhesion and its disturbances during cell cultivation of the mussel Mytilus trossulus.

    PubMed

    Maiorova, Mariia A; Odintsova, Nelly A

    2015-08-01

    In this study, we focus on the specific contribution of β integrin-like protein to adhesion-mediated events in molluscan larval cells in culture that could not have been investigated within the whole animal. An analysis of disturbances to cell-substratum adhesion, caused by the integrin receptor inhibiting Arg-Gly-Asp-Ser (RGDS)-peptide, the Ca(2+)/Mg(2+)-chelators and the stress influence of freezing-thawing, reveals that all these factors resulted in the partial destruction of the integrin-extracellular matrix (ECM) interaction in culture and, in particular, changes in the distribution and relative abundance of β integrin-positive cells. The experiments, carried out on selected substrates, found that β integrin-positive cells demonstrate different affinities for the substrates. This finding further supports the assumption that epithelial differentiation in cultivated cells of larval Mytilus may be mediated by β integrin-like proteins via binding to laminin; direct binding to other components of the ECM could not be demonstrated. The mussel β integrin-positive cells are not involved in myogenic or neuronal differentiation on any of the substrates but part of them has tubulin-positive cilia, forming some epithelia-like structures. Our data indicate that β integrin-positive cells are able to proliferate in vitro which suggests that they could participate in renewing the digestive epithelium in larvae. The findings provide evidence that the distribution pattern of β integrin-like protein depends on the cell type and the factors influencing the adhesion. PMID:25673210

  12. Resistance to protein adsorption and adhesion of fibroblasts on nanocrystalline diamond films: the role of topography and boron doping.

    PubMed

    Alcaide, María; Papaioannou, Stavros; Taylor, Andrew; Fekete, Ladislav; Gurevich, Leonid; Zachar, Vladimir; Pennisi, Cristian Pablo

    2016-05-01

    Boron-doped nanocrystalline diamond (BNCD) films exhibit outstanding electrochemical properties that make them very attractive for the fabrication of electrodes for novel neural interfaces and prosthetics. In these devices, the physicochemical properties of the electrode materials are critical to ensure an efficient long-term performance. The aim of this study was to investigate the relative contribution of topography and doping to the biological performance of BNCD films. For this purpose, undoped and boron-doped NCD films were deposited on low roughness (LR) and high roughness (HR) substrates, which were studied in vitro by means of protein adsorption and fibroblast growth assays. Our results show that BNCD films significantly reduce the adsorption of serum proteins, mostly on the LR substrates. As compared to fibroblasts cultured on LR BNCD films, cells grown on the HR BNCD films showed significantly reduced adhesion and lower growth rates. The mean length of fibronectin fibrils deposited by the cells was significantly increased in the BNCD coated substrates, mainly in the LR surfaces. Overall, the largest influence on protein adsorption, cell adhesion, proliferation, and fibronectin deposition was due to the underlying sub-micron topography, with little or no influence of boron doping. In perspective, BNCD films displaying surface roughness in the submicron range may be used as a strategy to reduce the fibroblast growth on the surface of neural electrodes. PMID:26975747

  13. Inhibition of S-fimbria-mediated adhesion to human ileostomy glycoproteins by a protein isolated from bovine colostrum.

    PubMed Central

    Ouwehand, A C; Conway, P L; Salminen, S J

    1995-01-01

    The aim of this study was to isolate and purify the component in bovine colostrum which is responsible for the inhibition of S-fimbria-mediated adhesion of Escherichia coli. Whey from defatted colostrum was fractionated by ultrafiltration, and the < 100K, < 30K, and < 10K fractions and the colostral whey were tested for inhibition of in vitro adhesion of radiolabelled S-fimbria-bearing E. coli to human ileostomy glycoproteins, which provide a model for human intestinal mucus. The inhibiting compound was purified from a dialyzed < 30K fraction with an anion exchange column which was eluted with a NaCl gradient (0 to 1.0 M). The compound was found to be a heat-resistant but pepsin-sensitive protein with an Mr of approximately 18,000 and an isoelectric point of approximately 5.75. The protein appears to block receptor sites for S-fimbriae on ileostomy glycoproteins, with steric hindrance being the most likely mechanism. Analysis of the amino acid sequence of the amino terminus of the 18K protein showed similarity with the sequence of beta-lactoglobulin. PMID:7591156

  14. SpyAD, a Moonlighting Protein of Group A Streptococcus Contributing to Bacterial Division and Host Cell Adhesion

    PubMed Central

    Gallotta, Marilena; Gancitano, Giovanni; Pietrocola, Giampiero; Mora, Marirosa; Pezzicoli, Alfredo; Tuscano, Giovanna; Chiarot, Emiliano; Nardi-Dei, Vincenzo; Taddei, Anna Rita; Rindi, Simonetta; Speziale, Pietro; Soriani, Marco; Bensi, Giuliano

    2014-01-01

    Group A streptococcus (GAS) is a human pathogen causing a wide repertoire of mild and severe diseases for which no vaccine is yet available. We recently reported the identification of three protein antigens that in combination conferred wide protection against GAS infection in mice. Here we focused our attention on the characterization of one of these three antigens, Spy0269, a highly conserved, surface-exposed, and immunogenic protein of unknown function. Deletion of the spy0269 gene in a GAS M1 isolate resulted in very long bacterial chains, which is indicative of an impaired capacity of the knockout mutant to properly divide. Confocal microscopy and immunoprecipitation experiments demonstrated that the protein was mainly localized at the cell septum and could interact in vitro with the cell division protein FtsZ, leading us to hypothesize that Spy0269 is a member of the GAS divisome machinery. Predicted structural domains and sequence homologies with known streptococcal adhesins suggested that this antigen could also play a role in mediating GAS interaction with host cells. This hypothesis was confirmed by showing that recombinant Spy0269 could bind to mammalian epithelial cells in vitro and that Lactococcus lactis expressing Spy0269 on its cell surface could adhere to mammalian cells in vitro and to mice nasal mucosa in vivo. On the basis of these data, we believe that Spy0269 is involved both in bacterial cell division and in adhesion to host cells and we propose to rename this multifunctional moonlighting protein as SpyAD (Streptococcus pyogenes Adhesion and Division protein). PMID:24778116

  15. Human epididymis protein 4 (HE4) plays a key role in ovarian cancer cell adhesion and motility

    SciTech Connect

    Lu, Renquan; Sun, Xinghui; Xiao, Ran; Zhou, Lei; Gao, Xiang; Guo, Lin

    2012-03-09

    Highlights: Black-Right-Pointing-Pointer We generated stable transduced HE4 overexpression and knockdown cells. Black-Right-Pointing-Pointer HE4 was associated with EOC cell adhesion and motility. Black-Right-Pointing-Pointer HE4 might have some effects on activation of EGFR-MAPK signaling pathway. Black-Right-Pointing-Pointer HE4 play an important role in EOC tumorigenicity. -- Abstract: Human epididymis protein 4 (HE4) is a novel and specific biomarker for epithelial ovarian cancer (EOC). We previously demonstrated that serum HE4 levels were significantly elevated in the majority of EOC patients but not in subjects with benign disease or healthy controls. However, the precise mechanism of HE4 protein function is unknown. In this study, we generated HE4-overexpressing SKOV3 cells and found that stably transduced cells promoted cell adhesion and migration. Knockdown of HE4 expression was achieved by stable transfection of SKOV3 cells with a construct encoding a short hairpin DNA directed against the HE4 gene. Correspondingly, the proliferation and spreading ability of HE4-expressed cells were inhibited by HE4 suppression. Mechanistically, impaired EGFR and Erk1/2 phosphorylation were observed in cells with HE4 knockdown. The phosphorylation was restored when the knockdown cells were cultured in conditioned medium containing HE4. Moreover, in vivo tumorigenicity showed that HE4 suppression markedly inhibited the growth of tumors. This suggests that expression of HE4 is associated with cancer cell adhesion, migration and tumor growth, which can be related to its effects on the EGFR-MAPK signaling pathway. Our results provide evidence of the cellular and molecular mechanisms that may underlie the motility-promoting role of HE4 in EOC progression. The role of HE4 as a target for gene-based therapy might be considered in future studies.

  16. Restoring E-cadherin-mediated cell-cell adhesion increases PTEN protein level and stability in human breast carcinoma cells

    SciTech Connect

    Li Zengxia; Wang Liying; Zhang Wen; Fu Yi; Zhao Hongbo; Hu Yali; Prins, Bram Peter; Zha Xiliang

    2007-11-09

    The phosphatase and tensin homolog deleted on chromosome 10 (PTEN) is a well-characterized tumor suppressor that negatively regulates cell growth and survival. Despite the critical role of PTEN in cell signaling, the mechanisms of its regulation are still under investigation. We reported here that PTEN expression could be controlled by overexpression or knock-down of E-cadherin in several mammary carcinoma cell lines. Furthermore, we showed that the accumulation of PTEN protein in E-cadherin overexpressing cells was due to increased PTEN protein stability rather than the regulation of its transcription. The proteasome-dependent PTEN degradation pathway was impaired after restoring E-cadherin expression. Moreover, maintenance of E-cadherin mediated cell-cell adhesion was necessary for its regulating PTEN. Altogether, our results suggested that E-cadherin mediated cell-cell adhesion was essential for preventing the proteasome degradation of PTEN, which might explain how breast carcinoma cells which lost cell-cell contact proliferate rapidly and are prone to metastasis.

  17. Plekhh2, a novel podocyte protein downregulated in human focal segmental glomerulosclerosis, is involved in matrix adhesion and actin dynamics.

    PubMed

    Perisic, Ljubica; Lal, Mark; Hulkko, Jenny; Hultenby, Kjell; Önfelt, Björn; Sun, Ying; Dunér, Fredrik; Patrakka, Jaakko; Betsholtz, Christer; Uhlen, Mathias; Brismar, Hjalmar; Tryggvason, Karl; Wernerson, Annika; Pikkarainen, Timo

    2012-11-01

    Pleckstrin homology domain-containing, family H (with MyTH4 domain), member 2 (Plekhh2) is a 1491-residue intracellular protein highly enriched in renal glomerular podocytes for which no function has been ascribed. Analysis of renal biopsies from patients with focal segmental glomerulosclerosis revealed a significant reduction in total podocyte Plekhh2 expression compared to controls. Sequence analysis indicated a putative α-helical coiled-coil segment as the only recognizable domain within the N-terminal half of the polypeptide, while the C-terminal half contains two PH, a MyTH4, and a FERM domain. We identified a phosphatidylinositol-3-phosphate consensus-binding site in the PH1 domain required for Plekhh2 localization to peripheral regions of cell lamellipodia. The N-terminal half of Plekkh2 is not necessary for lamellipodial targeting but mediates self-association. Yeast two-hybrid screening showed that Plekhh2 directly interacts through its FERM domain with the focal adhesion protein Hic-5 and actin. Plekhh2 and Hic-5 coprecipitated and colocalized at the soles of podocyte foot processes in situ and Hic-5 partially relocated from focal adhesions to lamellipodia in Plekhh2-expressing podocytes. In addition, Plekhh2 stabilizes the cortical actin cytoskeleton by attenuating actin depolymerization. Our findings suggest a structural and functional role for Plekhh2 in the podocyte foot processes. PMID:22832517

  18. The Adhesion G Protein-Coupled Receptor GPR56/ADGRG1 Is an Inhibitory Receptor on Human NK Cells.

    PubMed

    Chang, Gin-Wen; Hsiao, Cheng-Chih; Peng, Yen-Ming; Vieira Braga, Felipe A; Kragten, Natasja A M; Remmerswaal, Ester B M; van de Garde, Martijn D B; Straussberg, Rachel; König, Gabriele M; Kostenis, Evi; Knäuper, Vera; Meyaard, Linde; van Lier, René A W; van Gisbergen, Klaas P J M; Lin, Hsi-Hsien; Hamann, Jörg

    2016-05-24

    Natural killer (NK) cells possess potent cytotoxic mechanisms that need to be tightly controlled. Here, we explored the regulation and function of GPR56/ADGRG1, an adhesion G protein-coupled receptor implicated in developmental processes and expressed distinctively in mature NK cells. Expression of GPR56 was triggered by Hobit (a homolog of Blimp-1 in T cells) and declined upon cell activation. Through studying NK cells from polymicrogyria patients with disease-causing mutations in ADGRG1, encoding GPR56, and NK-92 cells ectopically expressing the receptor, we found that GPR56 negatively regulates immediate effector functions, including production of inflammatory cytokines and cytolytic proteins, degranulation, and target cell killing. GPR56 pursues this activity by associating with the tetraspanin CD81. We conclude that GPR56 inhibits natural cytotoxicity of human NK cells. PMID:27184850

  19. Surface modification of polydimethylsiloxane with photo-grafted poly(ethylene glycol) for micropatterned protein adsorption and cell adhesion.

    PubMed

    Sugiura, Shinji; Edahiro, Jun-ichi; Sumaru, Kimio; Kanamori, Toshiyuki

    2008-06-01

    In this study, we applied photo-induced graft polymerization to micropatterned surface modification of polydimethylsiloxane (PDMS) with poly(ethylene glycol). Two types of monomers, polyethylene glycol monoacrylate (PEGMA) and polyethylene glycol diacrylate (PEGDA), were tested for surface modification of PDMS. Changes in the surface hydrophilicity and surface element composition were characterized by contact angle measurement and electron spectroscopy for chemical analysis. The PEGMA-grafted PDMS surfaces gradually lost their hydrophilicity within two weeks. In contrast, the PEGDA-grafted PDMS surface maintained stable hydrophilic characteristics for more than two months. Micropatterned protein adsorption and micropatterned cell adhesion were successfully demonstrated using PEGDA-micropatterned PDMS surfaces, which were prepared by photo-induced graft polymerization using photomasks. The PEGDA-grafted PDMS exhibited useful characteristics for microfluidic devices (e.g. hydrophilicity, low protein adsorption, and low cell attachment). The technique presented in this study will be useful for surface modification of various research tools and devices. PMID:18242961

  20. New Insights into the Roles of Xin Repeat-Containing Proteins in Cardiac Development, Function, and Disease

    PubMed Central

    Wang, Qinchuan; Lin, Jenny Li-Chun; Erives, Albert J.; Lin, Cheng-I; Lin, Jim Jung-Ching

    2016-01-01

    Since the discovery of Xin repeat-containing proteins in 1996, the importance of Xin proteins in muscle development, function, regeneration, and disease has been continuously implicated. Most Xin proteins are localized to myotendinous junctions of the skeletal muscle and also to intercalated discs (ICDs) of the heart. The Xin gene is only found in vertebrates, which are characterized by a true chambered heart. This suggests that the evolutionary origin of the Xin gene may have played a key role in vertebrate origins. Diverse vertebrates including mammals possess two paralogous genes, Xinα (or Xirp1) and Xinβ (or Xirp2), and this review focuses on the role of their encoded proteins in cardiac muscles. Complete loss of mouse Xinβ (mXinβ) results in the failure of forming ICD, severe growth retardation, and early postnatal lethality. Deletion of mouse Xinα (mXinα) leads to late-onset cardiomyopathy with conduction defects. Molecular studies have identified three classes of mXinα-interacting proteins: catenins, actin regulators/modulators, and ion-channel subunits. Thus, mXinα acts as a scaffolding protein modulating the N-cadherin-mediated adhesion and ion-channel surface expression. Xin expression is significantly upregulated in early stages of stressed hearts, whereas Xin expression is downregulated in failing hearts from various human cardiomyopathies. Thus, mutations in these Xin loci may lead to diverse cardiomyopathies and heart failure. PMID:24725425

  1. Differential Expression of Adhesion-Related Proteins and MAPK Pathways Lead to Suitable Osteoblast Differentiation of Human Mesenchymal Stem Cells Subpopulations.

    PubMed

    Leyva-Leyva, Margarita; López-Díaz, Annia; Barrera, Lourdes; Camacho-Morales, Alberto; Hernandez-Aguilar, Felipe; Carrillo-Casas, Erika M; Arriaga-Pizano, Lourdes; Calderón-Pérez, Jaime; García-Álvarez, Jorge; Orozco-Hoyuela, Gabriel; Piña-Barba, Cristina; Rojas-Martínez, Augusto; Romero-Díaz, Víktor; Lara-Arias, Jorge; Rivera-Bolaños, Nancy; López-Camarillo, César; Moncada-Saucedo, Nidia; Galván-De los Santos, Alejandra; Meza-Urzúa, Fátima; Villarreal-Gómez, Luis; Fuentes-Mera, Lizeth

    2015-11-01

    Cellular adhesion enables communication between cells and their environment. Adhesion can be achieved throughout focal adhesions and its components influence osteoblast differentiation of human mesenchymal stem cells (hMSCs). Because cell adhesion and osteoblast differentiation are closely related, this article aimed to analyze the expression profiles of adhesion-related proteins during osteoblastic differentiation of two hMSCs subpopulations (CD105(+) and CD105(-)) and propose a strategy for assembling bone grafts based on its adhesion ability. In vitro experiments of osteogenic differentiation in CD105(-) cells showed superior adhesion efficiency and 2-fold increase of α-actinin expression compared with CD105(+) cells at the maturation stage. Interestingly, levels of activated β1-integrin increased in CD105(-) cells during the process. Additionally, the CD105(-) subpopulation showed 3-fold increase of phosphorylated FAK(Y397) compared to CD105(+) cells. Results also indicate that ERK1/2 was activated during CD105(-) bone differentiation and participation of mitogen-activated protein kinase (MAPK)-p38 in CD105(+) differentiation through a focal adhesion kinase (FAK)-independent pathway. In vivo trial demonstrated that grafts containing CD105(-) showed osteocytes embedded in a mineralized matrix, promoted adequate graft integration, increased host vascular infiltration, and efficient intramembranous repairing. In contrast, grafts containing CD105(+) showed deficient endochondral ossification and fibrocartilaginous tissue. Based on the expression of α-actinin, FAKy,(397) and ERK1/2 activation, we define maturation stage as critical for bone graft assembling. By in vitro assays, CD105(-) subpopulation showed superior adhesion efficiency compared to CD105(+) cells. Considering in vitro and in vivo assays, this study suggests that integration of a scaffold with CD105(-) subpopulation at the maturation stage represents an attractive strategy for clinical use in

  2. Effects of antibodies against cell surface protein antigen PAc-glucosyltransferase fusion proteins on glucan synthesis and cell adhesion of Streptococcus mutans.

    PubMed Central

    Yu, H; Nakano, Y; Yamashita, Y; Oho, T; Koga, T

    1997-01-01

    Cell surface protein antigen (PAc) and glucosyltransferases (GTFs) produced by Streptococcus mutans are considered to be major colonization factors of the organism, and the inhibition of these two factors is predicted to provide protection against dental caries. In this study, we have constructed fusion protein PAcA-GB, a fusion of the saliva-binding alanine-rich region (PAcA) of PAc with the glucan binding (GB) domain of GTF-I, an enzyme catalyzing the synthesis of water-insoluble glucan from sucrose, and fusion protein PAcA-SB, a fusion of PAcA with the sucrose binding (SB) domain of GTF-I. The recombinant fusion proteins were purified from cell extracts of Escherichia coli harboring the fusion genes, and rabbit antibodies against these fusion proteins were prepared. Water-insoluble glucan synthesis by cell-associated and cell-free GTF preparations from S. mutans as well as total glucan synthesis by GTF-I was markedly inhibited by anti-PAcA-GB immunoglobulin G (IgG) antibodies but not by anti-PAcA-SB IgG antibodies. Significant inhibition of the sucrose-independent and sucrose-dependent adhesion of S. mutans to saliva-coated hydroxyapatite beads was observed when anti-PAcA-GB antibodies were added to the reaction mixture. Anti-PAcA-SB antibodies inhibited the adhesion of S. mutans to the beads in the absence of sucrose but not in the presence of sucrose. Immunization with the fusion protein PAcA-GB may be useful for controlling the colonization of teeth by S. mutans. PMID:9169766

  3. A role for the WH-30 protein in sperm-sperm adhesion during rouleaux formation in the guinea pig.

    PubMed

    Flaherty, S P; Swann, N J; Primakoff, P; Myles, D G

    1993-03-01

    Mammalian spermatozoa participate in specific cell adhesion phenomena during their development and functional lifespan; this includes interaction with Sertoli cells, the zona pellucida, and the oolemma. In some species such as the guinea pig, an additional sperm-sperm adhesion occurs during epididymal maturation which results in the formation of rouleaux in which the sperm heads are stacked one upon the other and the periacrosomal plasma membranes of adjacent sperm are linked by periodic cross-bridges. In this study, we have used a monoclonal antibody to investigate the role of the WH-30 protein on the sperm surface in the formation of the junctional zones between adjacent guinea pig sperm in rouleaux. WH-30 monoclonal antibodies caused a dose- and time-dependent dissociation of rouleaux and an increase in the percentage of single, acrosome-intact sperm; there were no effects on sperm motility (maintained at 80-90%) or ultrastructure during the 120-min incubations. The maximal effect of about 80% single sperm was obtained with a 1:4 dilution of the WH-30 hybridoma supernatant or 5-50 micrograms/ml of purified WH-30 IgG. In contrast, incubation of sperm in AH-20 IgG, myeloma cell supernatants, or purified, nonspecific mouse IgG1 had no effect on rouleaux. Treatment of sperm with a WH-30 Fab fragment resulted in almost complete dissociation of rouleaux without any observed effect on sperm motility or acrosomal status. Surface labeling of sperm followed by immunoprecipitation and SDS-PAGE revealed that the WH-30 antibody recognizes a single polypeptide of 43-45 kDa. Using immunofluorescence, the WH-30 protein was localized over the entire surface of the sperm head (whole-head pattern), and immunogold labeling showed that WH-30 is localized in the glycocalyx on both the dorsal and ventral surfaces of the periacrosomal and postacrosomal plasma membranes. These results indicate that the WH-30 protein on the sperm surface is a cell adhesion protein which is involved in

  4. Polarity protein Crumbs homolog-3 (CRB3) regulates ectoplasmic specialization dynamics through its action on F-actin organization in Sertoli cells

    PubMed Central

    Gao, Ying; Lui, Wing-yee; Lee, Will M.; Cheng, C. Yan

    2016-01-01

    Crumbs homolog 3 (or Crumbs3, CRB3) is a polarity protein expressed by Sertoli and germ cells at the basal compartment in the seminiferous epithelium. CRB3 also expressed at the blood-testis barrier (BTB), co-localized with F-actin, TJ proteins occludin/ZO-1 and basal ES (ectoplasmic specialization) proteins N-cadherin/β-catenin at stages IV-VII only. The binding partners of CRB3 in the testis were the branched actin polymerization protein Arp3, and the barbed end-capping and bundling protein Eps8, illustrating its possible role in actin organization. CRB3 knockdown (KD) by RNAi in Sertoli cells with an established tight junction (TJ)-permeability barrier perturbed the TJ-barrier via changes in the distribution of TJ- and basal ES-proteins at the cell-cell interface. These changes were the result of CRB3 KD-induced re-organization of actin microfilaments, in which actin microfilaments were truncated, and extensively branched, thereby destabilizing F-actin-based adhesion protein complexes at the BTB. Using Polyplus in vivo-jetPEI as a transfection medium with high efficiency for CRB3 KD in the testis, the CRB3 KD testes displayed defects in spermatid and phagosome transport, and also spermatid polarity due to a disruption of F-actin organization. In summary, CRB3 is an actin microfilament regulator, playing a pivotal role in organizing actin filament bundles at the ES. PMID:27358069

  5. Polarity protein Crumbs homolog-3 (CRB3) regulates ectoplasmic specialization dynamics through its action on F-actin organization in Sertoli cells.

    PubMed

    Gao, Ying; Lui, Wing-Yee; Lee, Will M; Cheng, C Yan

    2016-01-01

    Crumbs homolog 3 (or Crumbs3, CRB3) is a polarity protein expressed by Sertoli and germ cells at the basal compartment in the seminiferous epithelium. CRB3 also expressed at the blood-testis barrier (BTB), co-localized with F-actin, TJ proteins occludin/ZO-1 and basal ES (ectoplasmic specialization) proteins N-cadherin/β-catenin at stages IV-VII only. The binding partners of CRB3 in the testis were the branched actin polymerization protein Arp3, and the barbed end-capping and bundling protein Eps8, illustrating its possible role in actin organization. CRB3 knockdown (KD) by RNAi in Sertoli cells with an established tight junction (TJ)-permeability barrier perturbed the TJ-barrier via changes in the distribution of TJ- and basal ES-proteins at the cell-cell interface. These changes were the result of CRB3 KD-induced re-organization of actin microfilaments, in which actin microfilaments were truncated, and extensively branched, thereby destabilizing F-actin-based adhesion protein complexes at the BTB. Using Polyplus in vivo-jetPEI as a transfection medium with high efficiency for CRB3 KD in the testis, the CRB3 KD testes displayed defects in spermatid and phagosome transport, and also spermatid polarity due to a disruption of F-actin organization. In summary, CRB3 is an actin microfilament regulator, playing a pivotal role in organizing actin filament bundles at the ES. PMID:27358069

  6. Facilitation of cell adhesion by immobilized dengue viral nonstructural protein 1 (NS1): arginine-glycine-aspartic acid structural mimicry within the dengue viral NS1 antigen.

    PubMed

    Chang, Hsin-Hou; Shyu, Huey-Fen; Wang, Yo-Ming; Sun, Der-Shan; Shyu, Rong-Hwa; Tang, Shiao-Shek; Huang, Yao-Shine

    2002-09-15

    Dengue virus infection causes life-threatening hemorrhagic fever. Increasing evidence implies that dengue viral nonstructural protein 1 (NS1) exhibits a tendency to elicit potentially hazardous autoantibodies, which show a wide spectrum of specificity against extracellular matrix and platelet antigens. How NS1 elicits autoantibodies remains unclear. To address the hypothesis that NS1 and matrix proteins may have structural and functional similarity, cell-matrix and cell-NS1 interactions were evaluated using a cell-adhesion assay. The present study showed that dengue NS1 immobilized on coverslips resulted in more cell adhesion than did the control proteins. This cell adhesion was inhibited by peptides containing arginine-glycine-aspartic acid (RGD), a motif important for integrin-mediated cell adhesion. In addition, anti-NS1 antibodies blocked RGD-mediated cell adhesion. Although there is no RGD motif in the NS1 protein sequence, these data indicate that RGD structural mimicry exists within the NS1 antigen. PMID:12198607

  7. Barnacle settlement and the adhesion of protein and diatom microfouling to xerogel films with varying surface energy and water wettability.

    PubMed

    Finlay, John A; Bennett, Stephanie M; Brewer, Lenora H; Sokolova, Anastasiya; Clay, Gemma; Gunari, Nikhil; Meyer, Anne E; Walker, Gilbert C; Wendt, Dean E; Callow, Maureen E; Callow, James A; Detty, Michael R

    2010-08-01

    Previous work has shown that organosilica-based xerogels have the potential to control biofouling. In this study, modifications of chemistry were investigated with respect to their resistance to marine slimes and to settlement of barnacle cyprids. Adhesion force measurements of bovine serum albumin (BSA)-coated atomic force microscopy (AFM) tips to xerogel surfaces prepared from aminopropylsilyl-, fluorocarbonsilyl-, and hydrocarbonsilyl-containing precursors, indicated that adhesion was significantly less on the xerogel surfaces in comparison to a poly(dimethylsiloxane) elastomer (PDMSE) standard. The strength of adhesion of BSA on the xerogels was highest on surfaces with the highest and the lowest critical surface tensions, gamma(C) and surface energies, gamma(S), and duplicated the 'Baier curve'. The attachment to and removal of cells of the diatom Navicula perminuta from a similar series of xerogel surfaces were examined. Initial attachment of cells was comparable on all of the xerogel surfaces, but the percentage removal of attached cells by hydrodynamic shear stress increased with gamma(C) and increased wettability as measured by the static water contact angle, theta(Ws), of the xerogel surfaces. The percentage removal of cells of Navicula was linearly correlated with both properties (R(2) = 0.74 for percentage removal as a function of theta(Ws) and R(2) = 0.69 for percentage removal as a function of gamma(C)). Several of the aminopropylsilyl-containing xerogels showed significantly greater removal of Navicula compared to a PDMSE standard. Cypris larvae of the barnacle B. amphitrite showed preferred settlement on hydrophilic/higher energy surfaces. Settlement was linearly correlated with theta(Ws) (R(2) = 0.84) and gamma(C) (R(2) = 0.84). Hydrophilic xerogels should prove useful as coatings for boats in regions where fouling is dominated by microfouling (protein and diatom slimes). PMID:20645195

  8. Platelet endothelial cell adhesion molecule-1 modulates endothelial cell motility through the small G-protein Rho.

    PubMed

    Gratzinger, Dita; Canosa, Sandra; Engelhardt, Britta; Madri, Joseph A

    2003-08-01

    Platelet endothelial cell adhesion molecule-1 (PECAM-1), an immunoglobulin family vascular adhesion molecule, is involved in endothelial cell migration and angiogenesis (1, 2). We found that endothelial cells lacking PECAM-1 exhibit increased single cell motility and extension formation but poor wound healing migration, reminiscent of cells in which Rho activity has been suppressed by overexpressing a GTPase-activating protein (3). The ability of PECAM-1 to restore wound healing migration to PECAM-1-deficient cells was independent of its extracellular domain or signaling via its immunoreceptor tyrosine-based inhibitory motif. PECAM-1-deficient endothelial cells had a selective defect in RhoGTP loading, and inhibition of Rho activity mimicked the PECAM-1-deficient phenotype of increased chemokinetic single cell motility at the expense of coordinated wound healing migration. The wound healing advantage of PECAM-1-positive endothelial cells was not only Rho mediated but pertussis toxin inhibitable, characteristic of migration mediated by heterotrimeric G-protein-linked seven-transmembrane receptor signaling such as signaling in response to the serum sphingolipid sphingosine-1-phosphate (S1P) (4, 5). Indeed, we found that the wound healing defect of PECAM-1 null endothelial cells is minimized in sphingolipid-depleted media; moreover, PECAM-1 null endothelial cells fail to increase their migration in response to S1P. We have also found that PECAM-1 localizes to rafts and that in its absence heterotrimeric G-protein components are differentially recruited to rafts, providing a potential mechanism for PECAM-1-mediated coordination of S1P signaling. PECAM-1 may thus support the effective S1P/RhoGTP signaling required for wound healing endothelial migration by allowing for the spatially directed, coordinated activation of Galpha signaling pathways. PMID:12890700

  9. Characterization of Palladin, a Novel Protein Localized to Stress Fibers and Cell Adhesions

    PubMed Central

    Parast, Mana M.; Otey, Carol A.

    2000-01-01

    Here, we describe the identification of a novel phosphoprotein named palladin, which colocalizes with α-actinin in the stress fibers, focal adhesions, cell–cell junctions, and embryonic Z-lines. Palladin is expressed as a 90–92-kD doublet in fibroblasts and coimmunoprecipitates in a complex with α-actinin in fibroblast lysates. A cDNA encoding palladin was isolated by screening a mouse embryo library with mAbs. Palladin has a proline-rich region in the NH2-terminal half of the molecule and three tandem Ig C2 domains in the COOH-terminal half. In Northern and Western blots of chick and mouse tissues, multiple isoforms of palladin were detected. Palladin expression is ubiquitous in embryonic tissues, and is downregulated in certain adult tissues in the mouse. To probe the function of palladin in cultured cells, the Rcho-1 trophoblast model was used. Palladin expression was observed to increase in Rcho-1 cells when they began to assemble stress fibers. Antisense constructs were used to attenuate expression of palladin in Rcho-1 cells and fibroblasts, and disruption of the cytoskeleton was observed in both cell types. At longer times after antisense treatment, fibroblasts became fully rounded. These results suggest that palladin is required for the normal organization of the actin cytoskeleton and focal adhesions. PMID:10931874

  10. ELMO1 increases expression of extracellular matrix proteins and inhibits cell adhesion to ECMs.

    PubMed

    Shimazaki, A; Tanaka, Y; Shinosaki, T; Ikeda, M; Watada, H; Hirose, T; Kawamori, R; Maeda, S

    2006-11-01

    We have previously identified the engulfment and cell motility 1 (ELMO1) as a susceptibility gene for diabetic nephropathy. To elucidate the role of ELMO1 in the pathogenesis of chronic renal injury, we examined the expression of Elmo1 in the kidney of a rat model for chronic glomerulonephritis (uninephrectomy plus anti-Thy1.1 antibody [E30] injection). We found that the expression of the Elmo1 was significantly increased in the renal cortex and glomeruli of uninephrectomized rats injected with E30 compared to controls. By in situ hybridization, the expression of Elmo1 was shown to be elevated in the diseased kidney, especially in glomerular epithelial cells. In COS cells, the overexpression of ELMO1 resulted in a substantial increase in fibronectin expression, whereas the depletion of the ELMO1 by small interfering RNA (siRNA) targeting ELMO1 significantly suppressed the fibronectin expression in ELMO1 overexpressing and control cells. We also found that the expression of integrin-linked kinase (ILK) was significantly increased in ELMO1 overexpressing cells, and the ELMO1-induced increase in fibronectin was partially, but significantly, inhibited by siRNA targeting ILK. Furthermore, we identified that the cell adhesion to ECMs was considerably inhibited in cells overexpressing ELMO1. These results suggest that the ELMO1 contributes to the development and progression of chronic glomerular injury through the dysregulation of ECM metabolism and the reduction in cell adhesive properties to ECMs. PMID:17021600

  11. Proteins implicated in the increase of adhesivity induced by suberoylanilide hydroxamic acid in leukemic cells.

    PubMed

    Grebeňová, D; Röselová, P; Pluskalová, M; Halada, P; Rösel, D; Suttnar, J; Brodská, B; Otevřelová, P; Kuželová, K

    2012-12-21

    We have previously shown that suberoylanilide hydroxamic acid (SAHA) treatment increases the adhesivity of leukemic cells to fibronectin at clinically relevant concentrations. Now, we present the results of the proteomic analysis of SAHA effects on leukemic cell lines using 2-DE and ProteomLab PF2D system. Histone acetylation at all studied acetylation sites reached the maximal level after 5 to 10 h of SAHA treatment. No difference in histone acetylation between subtoxic and toxic SAHA doses was observed. SAHA treatment induced cofilin phosphorylation at Ser3, an increase in vimentin and paxillin expression and a decrease in stathmin expression as confirmed by western-blotting and immunofluorescence microscopy. The interaction of cofilin with 14-3-3 epsilon was documented using both Duolink system and coimmunoprecipitation. However, this interaction was independent of cofilin Ser3 phosphorylation and the amount of 14-3-3-ε-bound cofilin did not rise following SAHA treatment. SAHA-induced increase in the cell adhesivity was associated with an increase in PAK phosphorylation in CML-T1 cells and was abrogated by simultaneous treatment with IPA-3, a PAK inhibitor. The effects of SAHA on JURL-MK1 cells were similar to those of other histone deacetylase inhibitors, tubastatin A and sodium butyrate. The proteome analysis also revealed several potential non-histone targets of histone deacetylases. PMID:23022583

  12. Specific degradation of the mucus adhesion-promoting protein (MapA) of Lactobacillus reuteri to an antimicrobial peptide.

    PubMed

    Bøhle, Liv Anette; Brede, Dag Anders; Diep, Dzung B; Holo, Helge; Nes, Ingolf F

    2010-11-01

    The intestinal flora of mammals contains lactic acid bacteria (LAB) that may provide positive health effects for the host. Such bacteria are referred to as probiotic bacteria. From a pig, we have isolated a Lactobacillus reuteri strain that produces an antimicrobial peptide (AMP). The peptide was purified and characterized, and it was unequivocally shown that the AMP was a well-defined degradation product obtained from the mucus adhesion-promoting protein (MapA); it was therefore termed AP48-MapA. This finding demonstrates how large proteins might inherit unexpected pleiotropic functions by conferring antimicrobial capacities on the producer. The MapA/AP48-MapA system is the first example where a large protein of an intestinal LAB is shown to give rise to such an AMP. It is also of particular interest that the protein that provides this AMP is associated with the binding of the bacterium producing it to the surface/lining of the gut. This finding gives us new perspective on how some probiotic bacteria may successfully compete in this environment and thereby contribute to a healthy microbiota. PMID:20833791

  13. The GPS Motif Is a Molecular Switch for Bimodal Activities of Adhesion Class G Protein-Coupled Receptors

    PubMed Central

    Prömel, Simone; Frickenhaus, Marie; Hughes, Samantha; Mestek, Lamia; Staunton, David; Woollard, Alison; Vakonakis, Ioannis; Schöneberg, Torsten; Schnabel, Ralf; Russ, Andreas P.; Langenhan, Tobias

    2012-01-01

    Summary Adhesion class G protein-coupled receptors (aGPCR) form the second largest group of seven-transmembrane-spanning (7TM) receptors whose molecular layout and function differ from canonical 7TM receptors. Despite their essential roles in immunity, tumorigenesis, and development, the mechanisms of aGPCR activation and signal transduction have remained obscure to date. Here, we use a transgenic assay to define the protein domains required in vivo for the activity of the prototypical aGPCR LAT-1/Latrophilin in Caenorhabditis elegans. We show that the GPCR proteolytic site (GPS) motif, the molecular hallmark feature of the entire aGPCR class, is essential for LAT-1 signaling serving in two different activity modes of the receptor. Surprisingly, neither mode requires cleavage but presence of the GPS, which relays interactions with at least two different partners. Our work thus uncovers the versatile nature of aGPCR activity in molecular detail and places the GPS motif in a central position for diverse protein-protein interactions. PMID:22938866

  14. A Short-Term Borrelia burgdorferi Infection Model Identifies Tissue Tropisms and Bloodstream Survival Conferred by Adhesion Proteins

    PubMed Central

    Caine, Jennifer A.

    2015-01-01

    Borrelia burgdorferi, the causative agent of Lyme disease in the United States, is able to persist in the joint, heart, skin, and central nervous system for the lifetime of its mammalian host. Borrelia species achieve dissemination to distal sites in part by entry into and travel within the bloodstream. Much work has been performed in vitro describing the roles of many B. burgdorferi outer surface proteins in adhesion to host cell surface proteins and extracellular matrix components, although the biological relevance of these interactions is only beginning to be explored in vivo. A need exists in the field for an in vivo model to define the biological roles of B. burgdorferi adhesins in tissue-specific vascular interactions. We have developed an in vivo model of vascular interaction of B. burgdorferi in which the bacteria are injected intravenously and allowed to circulate for 1 h. This model has shown that the fibronectin binding protein BB0347 has a tropism for joint tissue. We also have shown an importance of the integrin binding protein, P66, in binding to vasculature of the ear and heart. This model also revealed unexpected roles for Borrelia adhesins BBK32 and OspC in bacterial burdens in the bloodstream. The intravenous inoculation model of short-term infection provides new insights into critical B. burgdorferi interactions with the host required for initial survival and tissue colonization. PMID:26015482

  15. Interleukin-18 binding protein reduces b16 melanoma hepatic metastasis by neutralizing adhesiveness and growth factors of sinusoidal endothelium.

    PubMed

    Carrascal, Maria Teresa; Mendoza, Lorea; Valcárcel, Maria; Salado, Clarisa; Egilegor, Eider; Tellería, Naiara; Vidal-Vanaclocha, Fernando; Dinarello, Charles A

    2003-01-15

    We studied the role of endogenous interleukin (IL)-18 in hepatic metastasis by blocking this cytokine using the naturally occurring IL-18 binding protein (IL-18BP). A single i.p. dose of IL-18BP given 30 min before intrasplenic injection of murine B16 melanoma (B16M) cells reduced the number of hepatic metastatic foci by 75% and metastatic volume by 80%. Same treatment reduced the intrahepatic retention of luciferase-transfected B16M by 50% and abolished VCAM-1 up-regulation in the hepatic microvasculature, as assessed by reverse transcription-PCR, Western blot, and immunohistochemistry. Twelve hours after IL-18BP, hepatic sinusoidal endothelium (HSE) cells were isolated, and adhesion of B16M cells to these cultured HSE cells was reduced to the level of vehicle-treated mice. IL-18BP treatment of mice with established micrometastases resulted in a 25% decrease in metastasis number and 40% decrease in metastasis volume, suggesting inhibition of endogenous growth factors. Indeed, the addition of IL-18BP to normal HSE abolished the release of melanoma cell growth factor(s) induced by B16M. IL-18 promoted the in vitro growth of B16M and human melanoma cells, which was IL-1 dependent. These data demonstrate a significant role of endogenous IL-18 on hepatic metastasis by up-regulating melanoma cell adhesion to HSE cells and tumor growth, implicating a possible antimetastatic benefit of neutralizing IL-18. PMID:12543807

  16. Enhanced CLIP uncovers IMP protein-RNA targets in human pluripotent stem cells important for cell adhesion and survival

    PubMed Central

    Conway, Anne E.; Van Nostrand, Eric L.; Pratt, Gabriel A.; Aigner, Stefan; Wilbert, Melissa L.; Sundararaman, Balaji; Freese, Peter; Lambert, Nicole J.; Sathe, Shashank; Liang, Tiffany Y.; Essex, Anthony; Landais, Severine; Burge, Christopher B.; Jones, D. Leanne; Yeo, Gene W.

    2016-01-01

    SUMMARY Human pluripotent stem cells (hPSCs) require precise control of post-transcriptional RNA networks to maintain proliferation and survival. Using enhanced UV crosslinking and immunoprecipitation (eCLIP), we identify RNA targets of the IMP/IGF2BP family of RNA-binding proteins in hPSCs. At the broad region- and binding site-level IMP1 and IMP2 show reproducible binding to a large and overlapping set of 3′UTR-enriched targets. RNA Bind-N-Seq applied to recombinant full-length IMP1 and IMP2 reveals CA-rich motifs that are enriched in eCLIP-defined binding sites. We observe that IMP1 loss in hPSCs recapitulates IMP1 phenotypes, including a reduction in cell adhesion and an increase in cell death. For cell adhesion, in hPSCs we find IMP1 maintains levels of integrin mRNA, specifically regulating RNA stability of ITGB5. Additionally, we show IMP1 can be linked to hPSC survival via direct target BCL2. Thus, transcriptome-wide binding profiles identify hPSC targets modulating well-characterized IMP1 roles. PMID:27068461

  17. Analysis of the role of M24 protein in group A streptococcal adhesion and colonization by use of omega-interposon mutagenesis.

    PubMed Central

    Courtney, H S; Bronze, M S; Dale, J B; Hasty, D L

    1994-01-01

    We recently concluded that M protein mediates adherence of group A streptococci to HEp-2 tissue culture cells, because the N-terminal half of M protein blocked adherence and M+ strains attached in greater numbers than M- streptococci. To further assess the role of M protein in adhesion, an M-, isogenic mutant of M type M-, isogenic mutant of M type 24 group A streptococci was constructed by insertional inactivation of the emm24 gene with the omega-interposon flanked by emm24 gene sequences. Southern blot analysis confirmed that the omega-element inserted only into emm24. The M- isogenic mutant M24-omega 3 did not react with antiserum to M24 protein, not did it survive in whole human blood. Electron micrographs of M24-omega 3 showed a diminution of surface fibrillae and reduced binding of plasma components compared with the parent strain. The adhesion of the M+ parent to HEp-2 cells and to mouse oral epithelial cells was dramatically greater than the adhesion of the M24-omega 3 mutant, although there was no difference between the two in adhesion to human buccal cells. In addition, the parent strain was dramatically more effective than the M24-omega 3 mutant in colonizing the oral cavity of mice. These results indicate that the M24 protein can serve as an adhesin in streptococcal attachment to human cells in tissue culture and is important in the colonization of mouse mucosal surfaces. Images PMID:7927767

  18. Environmental toxicants perturb human Sertoli cell adhesive function via changes in F-actin organization mediated by actin regulatory proteins

    PubMed Central

    Xiao, Xiang; Mruk, Dolores D.; Tang, Elizabeth I.; Wong, Chris K.C.; Lee, Will M.; John, Constance M.; Turek, Paul J.; Silvestrini, Bruno; Cheng, C. Yan

    2014-01-01

    STUDY QUESTION Can human Sertoli cells cultured in vitro and that have formed an epithelium be used as a model to monitor toxicant-induced junction disruption and to better understand the mechanism(s) by which toxicants disrupt cell adhesion at the Sertoli cell blood–testis barrier (BTB)? SUMMARY ANSWER Our findings illustrate that human Sertoli cells cultured in vitro serve as a reliable system to monitor the impact of environmental toxicants on the BTB function. WHAT IS KNOWN ALREADY Suspicions of a declining trend in semen quality and a concomitant increase in exposures to environmental toxicants over the past decades reveal the need of an in vitro system that efficiently and reliably monitors the impact of toxicants on male reproductive function. Furthermore, studies in rodents have confirmed that environmental toxicants impede Sertoli cell BTB function in vitro and in vivo. STUDY DESIGN, SIZE AND DURATION We examined the effects of two environmental toxicants: cadmium chloride (0.5–20 µM) and bisphenol A (0.4–200 µM) on human Sertoli cell function. Cultured Sertoli cells from three men were used in this study, which spanned an 18-month period. PARTICIPANTS/MATERIALS, SETTING, METHODS Human Sertoli cells from three subjects were cultured in F12/DMEM containing 5% fetal bovine serum. Changes in protein expression were monitored by immunoblotting using specific antibodies. Immunofluorescence analyses were used to assess changes in the distribution of adhesion proteins, F-actin and actin regulatory proteins following exposure to two toxicants: cadmium chloride and bisphenol A (BPA). MAIN RESULTS AND THE ROLE OF CHANCE Human Sertoli cells were sensitive to cadmium and BPA toxicity. Changes in the localization of cell adhesion proteins were mediated by an alteration of the actin-based cytoskeleton. This alteration of F-actin network in Sertoli cells as manifested by truncation and depolymerization of actin microfilaments at the Sertoli cell BTB was caused by

  19. The tight-adhesion proteins TadGEF of Bradyrhizobium diazoefficiens USDA 110 are involved in cell adhesion and infectivity on soybean roots.

    PubMed

    Mongiardini, Elías J; Parisi, Gustavo D; Quelas, Juan I; Lodeiro, Aníbal R

    2016-01-01

    Adhesion of symbiotic bacteria to host plants is an essential early step of the infection process that leads to the beneficial interaction. In the Bradyrhizobium diazoefficiens-soybean symbiosis few molecular determinants of adhesion are known. Here we identified the tight-adhesion gene products TadGEF in the open-reading frames blr3941-blr3943 of the B. diazoefficiens USDA 110 complete genomic sequence. Predicted structure of TadG indicates a transmembrane domain and two extracytosolic domains, from which the C-terminal has an integrin fold. TadE and TadF are also predicted as bearing transmembrane segments. Mutants in tadG or the small cluster tadGEF were impaired in adhesion to soybean roots, and the root infection was delayed. However, nodule histology was not compromised by the mutations, indicating that these effects were restricted to the earliest contact of the B. diazoefficiens and root surfaces. Knowledge of preinfection determinants is important for development of inoculants that are applied to soybean crops worldwide. PMID:26686616

  20. Protein adsorption and cell adhesion on nanoscale bioactive coatings formed from poly(ethylene glycol) and albumin microgels

    PubMed Central

    Scott, Evan A.; Nichols, Michael D.; Cordova, Lee H.; George, Brandon J.; Jun, Young-Shin; Elbert, Donald L.

    2008-01-01

    Late-term thrombosis on drug-eluting stents is an emerging problem that might be addressed using extremely thin, biologically-active hydrogel coatings. We report a dip-coating strategy to covalently link poly(ethylene glycol) (PEG) to substrates, producing coatings with <≈100 nm thickness. Gelation of PEG-octavinylsulfone with amines in either bovine serum albumin (BSA) or PEG-octaamine was monitored by dynamic light scattering (DLS), revealing the presence of microgels before macrogelation. NMR also revealed extremely high end group conversions prior to macrogelation, consistent with the formation of highly crosslinked microgels and deviation from Flory-Stockmayer theory. Before macrogelation, the reacting solutions were diluted and incubated with nucleophile-functionalized surfaces. Using optical waveguide lightmode spectroscopy (OWLS) and quartz crystal microbalance with dissipation (QCM-D), we identified a highly hydrated, protein-resistant layer with a thickness of approximately 75 nm. Atomic force microscopy in buffered water revealed the presence of coalesced spheres of various sizes but with diameters less than about 100 nm. Microgel-coated glass or poly(ethylene terephthalate) exhibited reduced protein adsorption and cell adhesion. Cellular interactions with the surface could be controlled by using different proteins to cap unreacted vinylsulfone groups within the coating. PMID:18771802

  1. The small G-protein MglA connects to the MreB actin cytoskeleton at bacterial focal adhesions.

    PubMed

    Treuner-Lange, Anke; Macia, Eric; Guzzo, Mathilde; Hot, Edina; Faure, Laura M; Jakobczak, Beata; Espinosa, Leon; Alcor, Damien; Ducret, Adrien; Keilberg, Daniela; Castaing, Jean Philippe; Lacas Gervais, Sandra; Franco, Michel; Søgaard-Andersen, Lotte; Mignot, Tâm

    2015-07-20

    In Myxococcus xanthus the gliding motility machinery is assembled at the leading cell pole to form focal adhesions, translocated rearward to propel the cell, and disassembled at the lagging pole. We show that MglA, a Ras-like small G-protein, is an integral part of this machinery. In this function, MglA stimulates the assembly of the motility complex by directly connecting it to the MreB actin cytoskeleton. Because the nucleotide state of MglA is regulated spatially and MglA only binds MreB in the guanosine triphosphate-bound form, the motility complexes are assembled at the leading pole and dispersed at the lagging pole where the guanosine triphosphatase activating protein MglB disrupts the MglA-MreB interaction. Thus, MglA acts as a nucleotide-dependent molecular switch to regulate the motility machinery spatially. The function of MreB in motility is independent of its function in peptidoglycan synthesis, representing a coopted function. Our findings highlight a new function for the MreB cytoskeleton and suggest that G-protein-cytoskeleton interactions are a universally conserved feature. PMID:26169353

  2. The adhesion protein IgSF9b is coupled to neuroligin 2 via S-SCAM to promote inhibitory synapse development.

    PubMed

    Woo, Jooyeon; Kwon, Seok-Kyu; Nam, Jungyong; Choi, Seungwon; Takahashi, Hideto; Krueger, Dilja; Park, Joohyun; Lee, Yeunkum; Bae, Jin Young; Lee, Dongmin; Ko, Jaewon; Kim, Hyun; Kim, Myoung-Hwan; Bae, Yong Chul; Chang, Sunghoe; Craig, Ann Marie; Kim, Eunjoon

    2013-06-10

    Synaptic adhesion molecules regulate diverse aspects of synapse formation and maintenance. Many known synaptic adhesion molecules localize at excitatory synapses, whereas relatively little is known about inhibitory synaptic adhesion molecules. Here we report that IgSF9b is a novel, brain-specific, homophilic adhesion molecule that is strongly expressed in GABAergic interneurons. IgSF9b was preferentially localized at inhibitory synapses in cultured rat hippocampal and cortical interneurons and was required for the development of inhibitory synapses onto interneurons. IgSF9b formed a subsynaptic domain distinct from the GABAA receptor- and gephyrin-containing domain, as indicated by super-resolution imaging. IgSF9b was linked to neuroligin 2, an inhibitory synaptic adhesion molecule coupled to gephyrin, via the multi-PDZ protein S-SCAM. IgSF9b and neuroligin 2 could reciprocally cluster each other. These results suggest a novel mode of inhibitory synaptic organization in which two subsynaptic domains, one containing IgSF9b for synaptic adhesion and the other containing gephyrin and GABAA receptors for synaptic transmission, are interconnected through S-SCAM and neuroligin 2. PMID:23751499

  3. Localization of high-molecular-weight adhesion proteins of nontypeable Haemophilus influenzae by immunoelectron microscopy.

    PubMed Central

    Bakaletz, L O; Barenkamp, S J

    1994-01-01

    A family of high-molecular-weight (HMW) surface-exposed proteins important in the attachment of nontypeable Haemophilus influenzae (NTHi) to human epithelial cells was previously identified (J. W. St. Geme III, S. Falkow, and S. J. Barenkamp, Proc. Natl. Acad. Sci. USA 90:2875-2879, 1993). In the present investigation, indirect immunogold labeling and electron microscopy were used to localize these proteins on three clinical isolates of NTHi, mutants deficient in expression of one or both HMW proteins, and embedded sections of human oropharyngeal cells after incubation with NTHi strain 12. The filamentous material comprising the proteins was labeled with monoclonal antibodies directed against two prototype HMW proteins (HMW1 and HMW2) of prototype NTHi strain 12. Gold labeling was observed as a cap or discrete aggregate off one pole or centrally along one long axis of the bacterial cell. Heavily labeled, non-bacterial-cell-associated, disk-like aggregates of the HMW proteins were frequently noted in both bacterial preparations as well as in association with the oropharyngeal cell surface and intracellularly. Mutants demonstrated diminished labeling or an absence thereof, respectively, which correlated well with their previously demonstrated reduced ability or inability to adhere to Chang conjunctival epithelial cells in vitro. The Haemophilus HMW proteins share antigenic determinants with and demonstrate amino acid sequence similarity to the filamentous hemagglutinin protein of Bordetella pertussis, a critical adhesin of that organism. The studies presented here demonstrate that the Haemophilus proteins and B. pertussis filamentous hemagglutinin show impressive morphologic and perhaps additional functional similarity. Images PMID:7927710

  4. Comparison of adhesive properties of water- and phosphate-buffer-washed cottonseed meals with cottonseed protein isolate on bonding maple and poplar veneers

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Water- and phosphate buffer (35 mM Na2HPO4/NaH2PO4, pH 7.5)-washed cottonseed meals (abbreviated as WCM and BCM, respectively) could be low-cost and environmentally friendly protein-based adhesives as their preparation does not involve corrosive alkali and acid solutions that are needed for cottonse...

  5. Expression of cell-cell and cell-matrix adhesion proteins by sinusoidal endothelial cells in the normal and cirrhotic human liver.

    PubMed Central

    Couvelard, A.; Scoazec, J. Y.; Feldmann, G.

    1993-01-01

    We compared the expression of cell-cell and cell-matrix adhesion proteins by sinusoidal endothelial cells in normal human liver, in which the endothelial lining of hepatic sinusoids is discontinuous and devoid of basement membrane, and in cirrhosis, during which sinusoids might undergo a process of capillarization and acquire a continuous lining and a typical basement membrane. In normal liver, sinusoidal endothelial cells displayed a very restricted repertory of cell-adhesion molecules: the intercellular adhesion molecules PECAM-1 and CD34 were undetectable and only two integrins, alpha 1 beta 1 and alpha 5 beta 1, were present, whereas the laminin receptors alpha 6 beta 1 and alpha 2 beta 1 were undetectable and the beta 3 integrins were faintly expressed. In capillarized sinusoids, sinusoidal endothelial cells displayed striking changes in their repertory of cell-adhesion molecules, including the expression of PECAM-1 protein and messenger RNAs and the induction of the laminin receptors alpha 6 beta 1 and alpha 2 beta 1. Such changes co-localized with subendothelial laminin deposits. In conclusion, normal sinusoidal endothelial cells express a distinctive set of cell-adhesion molecules, adapted to their structural and microenvironmental characteristics, and this repertory is dramatically modified during sinusoidal capillarization, possibly as a consequence of the concomitant matrix changes. Images Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 PMID:8362973

  6. Rapid invasion of host cells by Toxoplasma requires secretion of the MIC2–M2AP adhesive protein complex

    PubMed Central

    Huynh, My-Hang; Rabenau, Karen E.; Harper, Jill M.; Beatty, Wandy L.; Sibley, L.David; Carruthers, Vern B.

    2003-01-01

    Vertebrate cells are highly susceptible to infection by obligate intracellular parasites such as Toxoplasma gondii, yet the mechanism by which these microbes breach the confines of their target cell is poorly understood. While it is thought that Toxoplasma actively invades by secreting adhesive proteins from internal organelles called micronemes, no genetic evidence is available to support this contention. Here, we report successful disruption of M2AP, a microneme protein tightly associated with an adhesive protein called MIC2. M2AP knockout parasites were >80% impaired in host cell entry. This invasion defect was likely due to defective expression of MIC2, which partially accumulated in the parasite endoplasmic reticulum and Golgi. M2AP knockout parasites were also unable to rapidly secrete MIC2, an event that normally accompanies parasite attachment to a target cell. These findings indicate a critical role for the MIC2–M2AP protein complex in parasite invasion. PMID:12727875

  7. Expression, purification, crystallization and preliminary X-ray analysis of the olfactomedin domain from the sea urchin cell-adhesion protein amassin

    SciTech Connect

    Hillier, Brian J.; Sundaresan, Vidyasankar; Stout, C. David; Vacquier, Victor D.

    2006-01-01

    The olfactomedin (OLF) domain from the sea urchin cell-adhesion protein amassin has been crystallized. A native data set extending to 2.7 Å has been collected using an in-house X-ray source. A family of animal proteins is emerging which contain a conserved protein motif known as an olfactomedin (OLF) domain. Novel extracellular protein–protein interactions occur through this domain. The OLF-family member amassin, from the sea urchin Strongylocentrotus purpuratus, has previously been identified to mediate a rapid cell-adhesion event resulting in a large aggregation of coelomocytes, the circulating immune cells. In this work, heterologous expression and purification of the OLF domain from amassin was carried out and initial crystallization trials were performed. A native data set has been collected, extending to 2.7 Å under preliminary cryoconditions, using an in-house generator. This work leads the way to the determination of the first structure of an OLF domain.

  8. An adhesive bone marrow scaffold and bone morphogenetic-2 protein carrier for cartilage tissue engineering.

    PubMed

    Simson, Jacob A; Strehin, Iossif A; Lu, Qiaozhi; Uy, Manuel O; Elisseeff, Jennifer H

    2013-03-11

    A chondroitin sulfate-bone marrow (CS-BM) adhesive hydrogel was used to localize rhBMP-2 to enhance articular cartilage tissue formation. Chondrocyte pellet culture revealed that 0.1 and 1 μg/mL of rhBMP-2 enhanced sulfated-GAG content. rhBMP-2 localization within the hydrogels was investigated, and it was found that BM, CS-NHS, and rhBMP-2 levels and time affected rhBMP-2 retention. Retention was modulated from 82 to 99% over a 3-week period for the material formulations investigated. To evaluate carrier efficacy, rhBMP-2 and bovine articular chondrocytes were encapsulated within CS-BM, and biochemical evaluation revealed significant increases in total collagen production with rhBMP-2. Histological analysis revealed more robust tissue formation and greater type-II collagen production with encapsulated rhBMP-2. Subsequently, a subcutaneous culture of hydrogels revealed increased total collagen, type-II to type-I collagen ratio, and sulfated GAG in samples carrying rhBMP-2. These findings indicate the development of a multifunctional system capable of localizing rhBMP-2 to enhance repair tissue quality. PMID:23320412

  9. Skelemin, a cytoskeletal M-disc periphery protein, contains motifs of adhesion/recognition and intermediate filament proteins.

    PubMed

    Price, M G; Gomer, R H

    1993-10-15

    In striated muscle, myofibrils are anchored to an interconnecting cytoskeleton of desmin intermediate filaments. Skelemin (195 kDa) may be a link between myofibrils and the intermediate filament cytoskeleton. Skelemin partitions with desmin to the insoluble cytoskeleton, and increases the thickness of reconstituted intermediate filaments. Concentrated at the M-disc periphery, skelemin may also contact myosin filaments. We used immunoscreening to isolate a mouse muscle cDNA which encodes a protein with a calculated molecular mass of 185 kDa. Anti-skelemin antibodies bound to the protein products of each of three nonoverlapping regions of the open reading frame. Antibodies directed against the protein products of each one-third of the cDNA react with a 195-kDa muscle protein and stain the M-disc indistinguishably from the original anti-skelemin antibodies, suggesting that the cDNA encodes skelemin. A single skelemin mRNA is detected in muscle but not non-muscle tissues, consistent with immunostaining results. Skelemin is a member of a family of myosin-associated proteins containing fibronectin type III and immunoglobulin superfamily C2 motifs. Skelemin is unique in this family in having intermediate filament core-like motifs, one near each terminus. We hypothesize that skelemin could interact with myosin or myosin-associated proteins through its fibronectin and/or immunoglobulin motifs, and with intermediate filaments through intermediate filament-like motifs. PMID:8408035

  10. Integrin cytoplasmic domain-associated protein 1alpha (ICAP-1alpha ) interacts directly with the metastasis suppressor nm23-H2, and both proteins are targeted to newly formed cell adhesion sites upon integrin engagement.

    PubMed

    Fournier, Henri-Noël; Dupé-Manet, Sandra; Bouvard, Daniel; Lacombe, Marie-Lise; Marie, Christiane; Block, Marc R; Albiges-Rizo, Corinne

    2002-06-01

    Cell adhesion-dependent signaling implicates cytoplasmic proteins interacting with the intracellular tails of integrins. Among those, the integrin cytoplasmic domain-associated protein 1alpha (ICAP-1alpha) has been shown to interact specifically with the beta(1) integrin cytoplasmic domain. Although it is likely that this protein plays an important role in controlling cell adhesion and migration, little is known about its actual function. To search for potential ICAP-1alpha-binding proteins, we used a yeast two-hybrid screen and identified the human metastatic suppressor protein nm23-H2 as a new partner of ICAP-1alpha. This direct interaction was confirmed in vitro, using purified recombinant ICAP-1alpha and nm23-H2, and by co-immunoprecipitation from CHO cell lysates over-expressing ICAP-1alpha. The physiological relevance of this interaction is provided by confocal fluorescence microscopy, which shows that ICAP-1alpha and nm23-H2 are co-localized in lamellipodia during the early stages of cell spreading. These adhesion sites are enriched in occupied beta(1) integrins and precede the formation of focal adhesions devoid of ICAP-1alpha and nm23-H2, indicating the dynamic segregation of components of matrix adhesions. This peripheral staining of ICAP-1alpha and nm23-H2 is only observed in cells spreading on fibronectin and collagen and is absent in cells spreading on poly-l-lysine, vitronectin, or laminin. This is consistent with the fact that targeting of both ICAP-1alpha and nm23-H2 to the cell periphery is dependent on beta(1) integrin engagement rather than being a consequence of cell adhesion. This finding represents the first evidence that the tumor suppressor nm23-H2 could act on beta(1) integrin-mediated cell adhesion by interacting with one of the integrin partners, ICAP-1alpha. PMID:11919189

  11. AND-34, a novel p130Cas-binding thymic stromal cell protein regulated by adhesion and inflammatory cytokines.

    PubMed

    Cai, D; Clayton, L K; Smolyar, A; Lerner, A

    1999-08-15

    We have characterized a novel cDNA whose steady state mRNA levels rise in the thymus 2 to 6 h following the induction of CD4+CD8+ thymocyte apoptosis by in vivo cross-linking of CD3 epsilon. This cDNA, AND-34-1, contains an open reading frame (ORF) encoding a protein with an amino-terminal Src homology 2 (SH2) domain and a carboxyl-terminal domain homologous to GDP-exchange factors (GEFs). Northern analysis demonstrates widespread expression of the AND-34 gene. Anti-CD3 epsilon treatment induces up-regulation of the AND-34 mRNA levels in total thymic RNA but not in RNA from purified thymocytes, suggesting that this transcript is derived from a thymic stromal cell population. IL-1 and TNF increase AND-34 transcript levels in thymic cortical reticular, thymic nurse, and fibroblast cell lines. In the thymic cortical reticular cell line, IL-1 and TNF induce a protein of the predicted 93-kDa size reactive with anti-AND-34 peptide antisera. Fifteen minutes of serum stimulation of vanadate-pretreated AND-34-1-transfected NIH3T3 fibroblasts induces tyrosine phosphorylation of AND-34 as well as coprecipitating 95-, 125-, and 130-kDa proteins. One of these tyrosine phosphorylated proteins is identified as p130Cas (Crk-associated substrate), a signaling molecule previously known to bind to a GDP-exchange factor (C3G) and inducibly associate with the focal adhesion complex. Consistent with such an association, AND-34 tyrosine phosphorylation is induced following adherence of trypsinized fibroblasts to fibronectin or poly-L -lysine-coated surfaces. PMID:10438950

  12. Modulation of endothelial cell adhesion to synthetic vascular grafts using biotinylated fibronectin in a dual ligand protein system

    NASA Astrophysics Data System (ADS)

    Anamelechi, Charles Chibuzor

    Over half a million coronary artery bypass operations are performed annually in the US yielding an annual health care cost of over 16 billion dollars. Only five percent of bypasses are repeat operations in spite of the procedures prevalence. Patients facing repeat coronary artery bypass operations often lack transplantable autologous arteries or veins, necessitating the use of substitutes. Unfortunately, synthetic small diameter vascular grafts have unacceptable patency rates, primarily due to lumenal thrombus formation and intimal thickening. Endothelial cells (EC) mediate the anti-thrombotic activity in healthy blood vessels, and due to the scarcity of suitable autologous vascular replacement, EC-seeded small diameter synthetic vascular grafts represent a clear, immediate, and practical solution. The fundamental goal of this project was to optimize the dual ligand (DL) system on synthetic vascular graft (SVG) surrogates to show enhanced cell adhesion, retention, and native functionality compared to fibronectin alone. Initially, two SVG surrogates were identified through characterization by x-ray photoelectron spectroscopy (XPS), atomic force microscopy (AFM), and 125I radiolabeling. The first modification to the DL system involved direct biotinylation of fibronectin (bFN) as a replacement for co-adsorption of FN with biotinylated bovine serum albumin (bBSA). This was analyzed with a Langmuir model using surface plasmon resonance (SPR) spectroscopy to verify the binding affinity of bFN and ELISA to detect the availability of the RGD binding motif post biotinylation. The second major change in this project examined cell binding and formation of focal adhesion after shifting from direct incubation of HUVECs with RGD-SA to sequentially adsorbing bFN(9) and RGD-SA prior to introducing unmodified HUVECs. These experiments were conducted under static seeding conditions. Next, dynamic cell seeding onto the sequentially adsorbed protein surface was examined as a function

  13. Induction of Fibronectin-Binding Proteins and Increased Adhesion of Quinolone-Resistant Staphylococcus aureus by Subinhibitory Levels of Ciprofloxacin

    PubMed Central

    Bisognano, Carmelo; Vaudaux, Pierre; Rohner, Peter; Lew, Daniel P.; Hooper, David C.

    2000-01-01

    We recently reported that strain EN1252a, a fluoroquinolone-resistant derivative of Staphylococcus aureus NCTC8325 with mutations in grlA and gyrA, expressed increased levels of fibronectin-binding proteins (FnBPs) and showed a significantly higher attachment to fibronectin-coated polymer surfaces after growth in the presence of subinhibitory concentrations of ciprofloxacin. The present study evaluated the occurrence and frequency of fluoroquinolone-induced FnBP-mediated adhesion in clinical isolates of fluoroquinolone-resistant methicillin-resistant S. aureus (MRSA) and methicillin-susceptible S. aureus (MSSA). Eight of ten MRSA isolates and four of six MSSA isolates with grlA and gyrA mutations exhibited significant increases in attachment to fibronectin-coated surfaces after growth in the presence of one-quarter the MIC of ciprofloxacin. Fluoroquinolone-induced FnBP-mediated adhesion of one clinical MRSA strain and the double mutant strain EN1252a also occurred on coverslips removed from the subcutaneous space of guinea pigs. For strain EN1252a, the regulation of fnb transcription by sub-MICs of ciprofloxacin was studied on reporter plasmids carrying fnb-luxAB fusions. One-quarter of the MIC of ciprofloxacin significantly increased fnbB, but not fnbA, promoter activity of the fluoroquinolone-resistant mutant but not its fluoroquinolone-susceptible parent ISP794. This response was abolished by pretreatment with rifampin, indicating an effect at the level of transcription. Activation of the fnbB promoter was not due to an indirect effect of ciprofloxacin on growth rate and still occurred in an agr mutant of strain EN1252a. These data suggest that sub-MIC levels of ciprofloxacin activate the fnbB promoter of some laboratory and clinical isolates, thus contributing to increased production of FnBP(s) and leading to higher levels of bacterial attachment to fibronectin-coated or subcutaneously implanted coverslips. PMID:10817688

  14. Investigation of mussel adhesive protein adsorption on polystyrene and poly(octadecyl methacrylate) using angle dependent XPS, ATR-FTIR, and AFM

    SciTech Connect

    Baty, A.M.; Suci, P.A.; Tyler, B.J.; Geesey, G.G.

    1996-02-10

    Despite many years of research effort, the molecular interactions that are responsible for microbial adhesion and fouling of surfaces remain obscure. An understanding of these interactions would contribute to the development of surfaces that resist colonization of microorganisms. The irreversible adsorption of mussel adhesive proteins (MAP) from the marine mussel Mytilus edulis has been investigated on polystyrene (PS) and poly(octadecyl methacrylate) (POMA) surfaces using angle resolved X-ray photoelectron spectroscopy (XPS), attenuated total reflection Fourier transform infrared (ATR-FTIR) spectrometry, and atomic force microscopy (AFM). Angle resolved XPS was used to quantify the elemental composition with depth of the upper 90 {angstrom} of the surface, and AFM was used to obtain the surface topography. The adsorption pattern of MAP, revealed by AFM images, is distinctly different on the two polymer surfaces and suggests that the substratum influences protein adhesion. The depth profiles of MAP, obtained from angle resolved XPS, show differences in nitrogen composition with depth for MAP adsorbed to PS and POMA. Infrared spectra of hydrated adsorbed MAP revealed significant differences in the amide III region and in two bands which may originate from residues in the tandemly repeated sequences of MAP. This data demonstrates that the chemistry of the polymer film that is present at the protein-polymer interface can influence protein-protein and protein-surface interactions.

  15. Cyclic di-GMP contributes to adaption and virulence of Bacillus thuringiensis through a riboswitch-regulated collagen adhesion protein.

    PubMed

    Tang, Qing; Yin, Kang; Qian, Hongliang; Zhao, Youwen; Wang, Wen; Chou, Shan-Ho; Fu, Yang; He, Jin

    2016-01-01

    Cyclic di-GMP is a ubiquitous second messenger that regulates diverse cellular processes in bacteria by binding to various protein or riboswitch effectors. In Bacillus thuringiensis BMB171, a c-di-GMP riboswitch termed Bc2 RNA resides in the 5'-untranslated region (5'-UTR) of an mRNA that encodes a collagen adhesion protein (Cap). The expression of cap was strongly repressed in parent strain BMB171 because of the presence of Bc2 RNA but was significantly promoted in the Bc2 RNA markerless deletion mutant. Bc2 RNA acts as a genetic "on" switch, which forms an anti-terminator structure to promote cap read-through transcription upon c-di-GMP binding. As a result, cap transcription was de-repressed under high c-di-GMP levels. Therefore, Bc2 RNA regulates cap expression using a repression/de-repression model. Bc2 RNA-regulated Cap was also found to be tightly associated with motility, aggregation, exopolysaccharide secretion, biofilm formation, and virulence of B. thuringiensis BMB171 against its host insect Helicoverpa armigera. PMID:27381437

  16. Cyclic di-GMP contributes to adaption and virulence of Bacillus thuringiensis through a riboswitch-regulated collagen adhesion protein

    PubMed Central

    Tang, Qing; Yin, Kang; Qian, Hongliang; Zhao, Youwen; Wang, Wen; Chou, Shan-Ho; Fu, Yang; He, Jin

    2016-01-01

    Cyclic di-GMP is a ubiquitous second messenger that regulates diverse cellular processes in bacteria by binding to various protein or riboswitch effectors. In Bacillus thuringiensis BMB171, a c-di-GMP riboswitch termed Bc2 RNA resides in the 5′-untranslated region (5′-UTR) of an mRNA that encodes a collagen adhesion protein (Cap). The expression of cap was strongly repressed in parent strain BMB171 because of the presence of Bc2 RNA but was significantly promoted in the Bc2 RNA markerless deletion mutant. Bc2 RNA acts as a genetic “on” switch, which forms an anti-terminator structure to promote cap read-through transcription upon c-di-GMP binding. As a result, cap transcription was de-repressed under high c-di-GMP levels. Therefore, Bc2 RNA regulates cap expression using a repression/de-repression model. Bc2 RNA-regulated Cap was also found to be tightly associated with motility, aggregation, exopolysaccharide secretion, biofilm formation, and virulence of B. thuringiensis BMB171 against its host insect Helicoverpa armigera. PMID:27381437

  17. The small G-protein MglA connects to the MreB actin cytoskeleton at bacterial focal adhesions

    PubMed Central

    Treuner-Lange, Anke; Macia, Eric; Guzzo, Mathilde; Hot, Edina; Faure, Laura M.; Jakobczak, Beata; Espinosa, Leon; Alcor, Damien; Ducret, Adrien; Keilberg, Daniela; Castaing, Jean Philippe; Lacas Gervais, Sandra; Franco, Michel

    2015-01-01

    In Myxococcus xanthus the gliding motility machinery is assembled at the leading cell pole to form focal adhesions, translocated rearward to propel the cell, and disassembled at the lagging pole. We show that MglA, a Ras-like small G-protein, is an integral part of this machinery. In this function, MglA stimulates the assembly of the motility complex by directly connecting it to the MreB actin cytoskeleton. Because the nucleotide state of MglA is regulated spatially and MglA only binds MreB in the guanosine triphosphate–bound form, the motility complexes are assembled at the leading pole and dispersed at the lagging pole where the guanosine triphosphatase activating protein MglB disrupts the MglA–MreB interaction. Thus, MglA acts as a nucleotide-dependent molecular switch to regulate the motility machinery spatially. The function of MreB in motility is independent of its function in peptidoglycan synthesis, representing a coopted function. Our findings highlight a new function for the MreB cytoskeleton and suggest that G-protein–cytoskeleton interactions are a universally conserved feature. PMID:26169353

  18. A novel COX-independent mechanism of sulindac sulfide involves cleavage of epithelial cell adhesion molecule protein.

    PubMed

    Liggett, Jason L; Min, Kyung-Won; Smolensky, Dmitriy; Baek, Seung Joon

    2014-08-01

    Non-steroidal anti-inflammatory drugs (NSAIDs) are extensively used over the counter to treat headaches and inflammation as well as clinically to prevent cancer among high-risk groups. The inhibition of cyclooxygenase (COX) activity by NSAIDs plays a role in their anti-tumorigenic properties. NSAIDs also have COX-independent activity which is not fully understood. In this study, we report a novel COX-independent mechanism of sulindac sulfide (SS), which facilitates a previously uncharacterized cleavage of epithelial cell adhesion molecule (EpCAM) protein. EpCAM is a type I transmembrane glycoprotein that has been implemented as an over-expressed oncogene in many cancers including colon, breast, pancreas, and prostate. We found EpCAM to be down-regulated by SS in a manner that is independent of COX activity, transcription regulation, de novo protein synthesis, and proteasomal degradation pathway. Our findings clearly demonstrate that SS drives cleavage of the extracellular portion of EpCAM near the N-terminus. This SS driven cleavage is blocked by a deleting amino acids 55-81 as well as simply mutating arginine residues at positions 80 and 81 to alanine of EpCAM. Proteolysis of EpCAM by SS may provide a novel mechanism by which NSAIDs affect anti-tumorigenesis at the post-translational level. PMID:24859349

  19. Nanoscale dynamics and protein adhesivity of alkylamine self-assembled monolayers on graphene.

    PubMed

    O'Mahony, S; O'Dwyer, C; Nijhuis, C A; Greer, J C; Quinn, A J; Thompson, D

    2013-06-18

    Atomic-scale molecular dynamics computer simulations are used to probe the structure, dynamics, and energetics of alkylamine self-assembled monolayer (SAM) films on graphene and to model the formation of molecular bilayers and protein complexes on the films. Routes toward the development and exploitation of functionalized graphene structures are detailed here, and we show that the SAM architecture can be tailored for use in emerging applications (e.g., electrically stimulated nerve fiber growth via the targeted binding of specific cell surface peptide sequences on the functionalized graphene scaffold). The simulations quantify the changes in film physisorption on graphene and the alkyl chain packing efficiency as the film surface is made more polar by changing the terminal groups from methyl (-CH3) to amine (-NH2) to hydroxyl (-OH). The mode of molecule packing dictates the orientation and spacing between terminal groups on the surface of the SAM, which determines the way in which successive layers build up on the surface, whether via the formation of bilayers of the molecule or the immobilization of other (macro)molecules (e.g., proteins) on the SAM. The simulations show the formation of ordered, stable assemblies of monolayers and bilayers of decylamine-based molecules on graphene. These films can serve as protein adsorption platforms, with a hydrophobin protein showing strong and selective adsorption by binding via its hydrophobic patch to methyl-terminated films and binding to amine-terminated films using its more hydrophilic surface regions. Design rules obtained from modeling the atomic-scale structure of the films and interfaces may provide input into experiments for the rational design of assemblies in which the electronic, physicochemical, and mechanical properties of the substrate, film, and protein layer can be tuned to provide the desired functionality. PMID:23301836

  20. Cell adhesion force microscopy

    PubMed Central

    Sagvolden, G.; Giaever, I.; Pettersen, E. O.; Feder, J.

    1999-01-01

    The adhesion forces of cervical carcinoma cells in tissue culture were measured by using the manipulation force microscope, a novel atomic force microscope. The forces were studied as a function of time and temperature for cells cultured on hydrophilic and hydrophobic polystyrene substrates with preadsorbed proteins. The cells attached faster and stronger at 37°C than at 23°C and better on hydrophilic than on hydrophobic substrates, even though proteins adsorb much better to the hydrophobic substrates. Because cell adhesion serves to control several stages in the cell cycle, we anticipate that the manipulation force microscope can help clarify some cell-adhesion related issues. PMID:9892657

  1. Biologically engineered protein-graft-poly(ethylene glycol) hydrogels: A cell-adhesive and plasmin-degradable biosynthetic material for tissue repair

    NASA Astrophysics Data System (ADS)

    Halstenberg, Sven

    2002-01-01

    The goal of the research presented in this dissertation was to create a biomimetic artificial material that exhibits functions of extracellular matrix relevant for improved nerve regeneration. Neural adhesion peptides were photoimmobilized on highly crosslinked poly(ethylene glycol)-based substrates that were otherwise non-adhesive. Neurons adhered in two-dimensional patterns for eleven hours, but no neurites extended. To enable neurite extension and nerve regeneration in three dimensions, and to address the need for specifically cell adhesive and cell degradable materials for clinical applications in tissue repair in general, an artificial protein was recombinantly expressed and purified that consisted of a repeating amino acid sequence based on fibrinogen and anti-thrombin III. The recombinant protein contained integrin-binding RGD sites, plasmin degradation sites, heparin binding sites, and six thiol-containing cysteine residues as grafting sites for poly(ethylene glycol) diacrylate via Michael-type conjugate addition. The resulting protein-graft-poly(ethylene glycol)acrylates were crosslinked by photopolymerization to form hydrogels. Although three-dimensional, RGD mediated and serine protease-dependent ingrowth of human fibroblasts into protein-graft-poly(ethylene glycol) hydrogels occurred, only surface neurite outgrowth was observed from chick dorsal root ganglia. Axonal outgrowth depended on the concentration of matrix-bound heparin, suggesting that improved mechanical strength of the hydrogels and possible immobilization of neuroactive factors due to the presence of heparin promoted neurite outgrowth. Together, the above results show that specific biological functions can be harnessed by protein-graft-poly(ethylene glycol) hydrogels to serve as matrices for tissue repair and regeneration. In particular, the two design objectives, specific cell adhesion and degradability by cell-associated proteases, were fulfilled by the material. In the future, this and

  2. Rescue of perfluorooctanesulfonate (PFOS)-mediated Sertoli cell injury by overexpression of gap junction protein connexin 43

    PubMed Central

    Li, Nan; Mruk, Dolores D.; Chen, Haiqi; Wong, Chris K. C.; Lee, Will M.; Cheng, C. Yan

    2016-01-01

    Perfluorooctanesulfonate (PFOS) is an environmental toxicant used in developing countries, including China, as a stain repellent for clothing, carpets and draperies, but it has been banned in the U.S. and Canada since the late 2000s. PFOS perturbed the Sertoli cell tight junction (TJ)-permeability barrier, causing disruption of actin microfilaments in cell cytosol, perturbing the localization of cell junction proteins (e.g., occluden-ZO-1, N-cadherin-ß-catenin). These changes destabilized Sertoli cell blood-testis barrier (BTB) integrity. These findings suggest that human exposure to PFOS might induce BTB dysfunction and infertility. Interestingly, PFOS-induced Sertoli cell injury associated with a down-regulation of the gap junction (GJ) protein connexin43 (Cx43). We next investigated if overexpression of Cx43 in Sertoli cells could rescue the PFOS-induced cell injury. Indeed, overexpression of Cx43 in Sertoli cells with an established TJ-barrier blocked the disruption in PFOS-induced GJ-intercellular communication, resulting in the re-organization of actin microfilaments, which rendered them similar to those in control cells. Furthermore, cell adhesion proteins that utilized F-actin for attachment became properly distributed at the cell-cell interface, resealing the disrupted TJ-barrier. In summary, Cx43 is a good target that might be used to manage PFOS-induced reproductive dysfunction. PMID:27436542

  3. Rescue of perfluorooctanesulfonate (PFOS)-mediated Sertoli cell injury by overexpression of gap junction protein connexin 43.

    PubMed

    Li, Nan; Mruk, Dolores D; Chen, Haiqi; Wong, Chris K C; Lee, Will M; Cheng, C Yan

    2016-01-01

    Perfluorooctanesulfonate (PFOS) is an environmental toxicant used in developing countries, including China, as a stain repellent for clothing, carpets and draperies, but it has been banned in the U.S. and Canada since the late 2000s. PFOS perturbed the Sertoli cell tight junction (TJ)-permeability barrier, causing disruption of actin microfilaments in cell cytosol, perturbing the localization of cell junction proteins (e.g., occluden-ZO-1, N-cadherin-ß-catenin). These changes destabilized Sertoli cell blood-testis barrier (BTB) integrity. These findings suggest that human exposure to PFOS might induce BTB dysfunction and infertility. Interestingly, PFOS-induced Sertoli cell injury associated with a down-regulation of the gap junction (GJ) protein connexin43 (Cx43). We next investigated if overexpression of Cx43 in Sertoli cells could rescue the PFOS-induced cell injury. Indeed, overexpression of Cx43 in Sertoli cells with an established TJ-barrier blocked the disruption in PFOS-induced GJ-intercellular communication, resulting in the re-organization of actin microfilaments, which rendered them similar to those in control cells. Furthermore, cell adhesion proteins that utilized F-actin for attachment became properly distributed at the cell-cell interface, resealing the disrupted TJ-barrier. In summary, Cx43 is a good target that might be used to manage PFOS-induced reproductive dysfunction. PMID:27436542

  4. Rescue of perfluorooctanesulfonate (PFOS)-mediated Sertoli cell injury by overexpression of gap junction protein connexin 43

    NASA Astrophysics Data System (ADS)

    Li, Nan; Mruk, Dolores D.; Chen, Haiqi; Wong, Chris K. C.; Lee, Will M.; Cheng, C. Yan

    2016-07-01

    Perfluorooctanesulfonate (PFOS) is an environmental toxicant used in developing countries, including China, as a stain repellent for clothing, carpets and draperies, but it has been banned in the U.S. and Canada since the late 2000s. PFOS perturbed the Sertoli cell tight junction (TJ)-permeability barrier, causing disruption of actin microfilaments in cell cytosol, perturbing the localization of cell junction proteins (e.g., occluden-ZO-1, N-cadherin-ß-catenin). These changes destabilized Sertoli cell blood-testis barrier (BTB) integrity. These findings suggest that human exposure to PFOS might induce BTB dysfunction and infertility. Interestingly, PFOS-induced Sertoli cell injury associated with a down-regulation of the gap junction (GJ) protein connexin43 (Cx43). We next investigated if overexpression of Cx43 in Sertoli cells could rescue the PFOS-induced cell injury. Indeed, overexpression of Cx43 in Sertoli cells with an established TJ-barrier blocked the disruption in PFOS-induced GJ-intercellular communication, resulting in the re-organization of actin microfilaments, which rendered them similar to those in control cells. Furthermore, cell adhesion proteins that utilized F-actin for attachment became properly distributed at the cell-cell interface, resealing the disrupted TJ-barrier. In summary, Cx43 is a good target that might be used to manage PFOS-induced reproductive dysfunction.

  5. The role of heparan sulfate on adhesion of 47 and 51 kDa outer membrane proteins of Helicobacter pylori to gastric cancer cells.

    PubMed

    López-Bolaños, Claudia C; Guzmán-Murillo, Maria A; Ruiz-Bustos, Eduardo; Ascencio, Felipe

    2009-04-01

    Helicobacter pylori is a common gastrointestinal pathogenic bacterium in humans and the usual preference for the stomach's outer membrane proteins (OMPs) are antigens involved in the adhesion process. Through SDS-PAGE and blotting analyses, using horseradish peroxidase-labeled heparan sulfate (HRP-HS) as a probe, we identified H. pylori OMPs with affinity for heparan sulfate (OMP-HS). Biotin-streptavidin bacterial-adhesion assay was used to evaluate participation of OMP-HS in the adhesion of H. pylori to semi-confluent HeLa S3 and Kato III cell monolayers. The results provide evidence that induction of antibodies against 2 OMP-HSs (HSBP-47 and HSBP-51) could reduce binding of H. pylori to both cell lines and induce detachment of cell-bound bacteria from infected cultured cells. PMID:19396245

  6. Bone morphogenic protein 4 produced in endothelial cells by oscillatory shear stress induces monocyte adhesion by stimulating reactive oxygen species production from a nox1-based NADPH oxidase.

    PubMed

    Sorescu, George P; Song, Hannah; Tressel, Sarah L; Hwang, Jinah; Dikalov, Sergey; Smith, Debra A; Boyd, Nolan L; Platt, Manu O; Lassègue, Bernard; Griendling, Kathy K; Jo, Hanjoong

    2004-10-15

    Atherosclerosis is an inflammatory disease occurring preferentially in arterial regions exposed to disturbed flow conditions including oscillatory shear stress (OS). OS exposure induces endothelial expression of bone morphogenic protein 4 (BMP4), which in turn may activate intercellular adhesion molecule-1 (ICAM-1) expression and monocyte adhesion. OS is also known to induce monocyte adhesion by producing reactive oxygen species (ROS) from reduced nicotinamide adenine dinucleotide phosphate (NADPH) oxidases, raising the possibility that BMP4 may stimulate the inflammatory response by ROS-dependent mechanisms. Here we show that ROS scavengers blocked ICAM-1 expression and monocyte adhesion induced by BMP4 or OS in endothelial cells (ECs). Similar to OS, BMP4 stimulated H2O2 and O2- production in ECs. Next, we used ECs obtained from p47phox-/- mice (MAE-p47-/-), which do not produce ROS in response to OS, to determine the role of NADPH oxidases. Similar to OS, BMP4 failed to induce monocyte adhesion in MAE-p47-/-, but it was restored when the cells were transfected with p47phox plasmid. Moreover, OS-induced O2- production was blocked by noggin (a BMP antagonist), suggesting a role for BMP. Furthermore, OS increased gp91phox (nox2) and nox1 mRNA levels while decreasing nox4. In contrast, BMP4 induced nox1 mRNA expression, whereas nox2 and nox4 were decreased or not affected, respectively. Also, OS-induced monocyte adhesion was blocked by knocking down nox1 with the small interfering RNA (siRNA). Finally, BMP4 siRNA inhibited OS-induced ROS production and monocyte adhesion. Together, these results suggest that BMP4 produced in ECs by OS stimulates ROS release from the nox1-dependent NADPH oxidase leading to inflammation, a critical early atherogenic step. PMID:15388638

  7. Characterization of a Cell Surface Protein of Clostridium difficile with Adhesive Properties

    PubMed Central

    Waligora, Anne-Judith; Hennequin, Claire; Mullany, Peter; Bourlioux, Pierre; Collignon, Anne; Karjalainen, Tuomo

    2001-01-01

    Our laboratory has previously shown that Clostridium difficile adherence to cultured cells is enhanced after heat shock at 60°C and that it is mediated by a proteinaceous surface component. The present study was undertaken to identify the surface molecules of this bacterium that could play a role in its adherence to the intestine. The cwp66 gene, encoding a cell surface-associated protein of C. difficile 79-685, was isolated by immunoscreening of a C. difficile gene library with polyclonal antibodies against C. difficile heated at 60°C. The Cwp66 protein (66 kDa) contains two domains, each carrying three imperfect repeats and one presenting homologies to the autolysin CwlB of Bacillus subtilis. A survey of 36 strains of C. difficile representing 11 serogroups showed that the 3′ portion of the cwp66 gene is variable; this was confirmed by sequencing of cwp66 from another strain, C-253. Two recombinant protein fragments corresponding to the two domains of Cwp66 were expressed in fusion with glutathione S-transferase in Escherichia coli and purified by affinity chromatography using gluthatione-Sepharose 4B. Antibodies raised against the two domains recognized Cwp66 in bacterial surface extracts. By immunoelectron microscopy, the C-terminal domain was found to be cell surface exposed. When used as inhibitors in cell binding studies, the antibodies and protein fragments partially inhibited adherence of C. difficile to cultured cells, confirming that Cwp66 is an adhesin, the first to be identified in clostridia. PMID:11254569

  8. The Ras suppressor Rsu-1 binds to the LIM 5 domain of the adaptor protein PINCH1 and participates in adhesion-related functions

    SciTech Connect

    Dougherty, Gerard W.; Chopp, Treasa; Qi Shengmei; Cutler, Mary Lou . E-mail: mcutler@usuhs.mil

    2005-05-15

    Rsu-1 is a highly conserved leucine rich repeat (LRR) protein that is expressed ubiquitously in mammalian cells. Rsu-1 was identified based on its ability to inhibit transformation by Ras, and previous studies demonstrated that ectopic expression of Rsu-1 inhibited anchorage-independent growth of Ras-transformed cells and human tumor cell lines. Using GAL4-based yeast two-hybrid screening, the LIM domain protein, PINCH1, was identified as the binding partner of Rsu-1. PINCH1 is an adaptor protein that localizes to focal adhesions and it has been implicated in the regulation of adhesion functions. Subdomain mapping in yeast revealed that Rsu-1 binds to the LIM 5 domain of PINCH1, a region not previously identified as a specific binding domain for any other protein. Additional testing demonstrated that PINCH2, which is highly homologous to PINCH1, except in the LIM 5 domain, does not interact with Rsu-1. Glutathione transferase fusion protein binding studies determined that the LRR region of Rsu-1 interacts with PINCH1. Transient expression studies using epitope-tagged Rsu-1 and PINCH1 revealed that Rsu-1 co-immunoprecipitated with PINCH1 and colocalized with vinculin at sites of focal adhesions in mammalian cells. In addition, endogenous P33 Rsu-1 from 293T cells co-immunoprecipitated with transiently expressed myc-tagged PINCH1. Furthermore, RNAi-induced reduction in Rsu-1 RNA and protein inhibited cell attachment, and while previous studies demonstrated that ectopic expression of Rsu-1 inhibited Jun kinase activation, the depletion of Rsu-1 resulted in activation of Jun and p38 stress kinases. These studies demonstrate that Rsu-1 interacts with PINCH1 in mammalian cells and functions, in part, by altering cell adhesion.

  9. Unbinding and unfolding of adhesion protein complexes through stretching: interplay between shear and tensile mechanical clamps.

    PubMed

    Różycki, Bartosz; Mioduszewski, Łukasz; Cieplak, Marek

    2014-11-01

    Using coarse-grained molecular dynamics simulations, we analyze mechanically induced dissociation and unfolding of the protein complex CD48-2B4. This heterodimer is an indispensable component of the immunological system: 2B4 is a receptor on natural killer cells whereas CD48 is expressed on surfaces of various immune cells. So far, its mechanostability has not been assessed either experimentally or theoretically. We find that the dissociation processes strongly depend on the direction of pulling and may take place in several pathways. Interestingly, the CD48-2B4 interface can be divided into three distinct patches that act as units when resisting the pulling forces. At experimentally accessible pulling speeds, the characteristic mechanostability forces are in the range between 100 and 200 pN, depending on the pulling direction. These characteristic forces need not be associated with tensile forces involved in the act of separation of the complex because prior shear-involving unraveling within individual proteins may give rise to a higher force peak. PMID:25142868

  10. Changes in cell adhesivity and cytoskeleton-related proteins during imatinib-induced apoptosis of leukemic JURL-MK1 cells.

    PubMed

    Kuželová, K; Pluskalová, M; Grebeňová, D; Pavlásková, K; Halada, P; Hrkal, Z

    2010-12-15

    The fusion protein Bcr-Abl, which is the molecular cause of chronic myelogenous leukemia (CML) interacts in multiple points with signaling pathways regulating the cellular adhesivity and cytoskeleton architecture and dynamics. We explored the effects of imatinib mesylate, an inhibitor of Bcr-Abl protein used in front-line CML therapy, on the adhesivity of JURL-MK1 cells to fibronectin and searched for underlying changes in the cell proteome. As imatinib induces apoptosis of JURL-MK1 cells, we used three different caspase inhibitors to discriminate between direct consequences of Bcr-Abl inhibition and secondary changes related to the apoptosis. Imatinib treatment caused a transient increase in JURL-MK1 cell adhesivity to fibronectin, possibly due to the switch off of Bcr-Abl activity. Subsequently, we observed a number of changes including a decrease in cell adhesivity, F-actin decomposition, reduction of integrin β1, CD44, and paxillin expression levels and a marked increase in cofilin phophorylation at Ser3. These events were generally related to the proceeding apoptosis but they differed in their sensitivity to the individual caspase inhibitors. PMID:20830748

  11. Implication of sortase-dependent proteins of Streptococcus thermophilus in adhesion to human intestinal epithelial cell lines and bile salt tolerance.

    PubMed

    Kebouchi, Mounira; Galia, Wessam; Genay, Magali; Soligot, Claire; Lecomte, Xavier; Awussi, Ahoefa Ablavi; Perrin, Clarisse; Roux, Emeline; Dary-Mourot, Annie; Le Roux, Yves

    2016-04-01

    Streptococcus thermophilus (ST) is a lactic acid bacterium widely used in dairy industry and displays several properties which could be beneficial for host. The objective of this study was to investigate, in vitro, the implication of sortase A (SrtA) and sortase-dependent proteins (SDPs) in the adhesion of ST LMD-9 strain to intestinal epithelial cells (IECs) and resistance to bile salt mixture (BSM; taurocholoate, deoxycholate, and cholate). The effect of mutations in prtS (protease), mucBP (MUCin-Binding Protein), and srtA genes in ST LMD-9 in these mechanisms were examined. The HT29-MTX, HT29-CL.16E, and Caco-2 TC7 cell lines were used. HT29-MTX and HT29-CL.16E cells express different mucins found in the gastro intestinal tract; whereas, Caco-2 TC7 express cell surface proteins found in the small intestine. All mutants showed different adhesion profiles depending on cell lines. The mutation in genes srtA and mucBP leads to a significant decrease in LMD-9 adhesion capacity to Caco-2 TC7 cells. A mutation in mucBP gene has also shown a significant decrease in LMD-9 adhesion capacity to HT29-CL.16E cells. However, no difference was observed using HT29-MTX cells. Furthermore, ST LMD-9 and srtA mutant were resistant to BSM up to 3 mM. Contrariwise, no viable bacteria were detected for prtS and mucBP mutants at this concentration. Two conclusions could be drawn. First, SDPs could be involved in the LMD-9 adhesion depending on the cell lines indicating the importance of eukaryotic-cell surface components in adherence. Second, SDPs could contribute to resistance to bile salts probably by maintaining the cell membrane integrity. PMID:26820650

  12. Folliculin, the product of the Birt-Hogg-Dube tumor suppressor gene, interacts with the adherens junction protein p0071 to regulate cell-cell adhesion.

    PubMed

    Medvetz, Doug A; Khabibullin, Damir; Hariharan, Venkatesh; Ongusaha, Pat P; Goncharova, Elena A; Schlechter, Tanja; Darling, Thomas N; Hofmann, Ilse; Krymskaya, Vera P; Liao, James K; Huang, Hayden; Henske, Elizabeth P

    2012-01-01

    Birt-Hogg-Dube (BHD) is a tumor suppressor gene syndrome associated with fibrofolliculomas, cystic lung disease, and chromophobe renal cell carcinoma. In seeking to elucidate the pathogenesis of BHD, we discovered a physical interaction between folliculin (FLCN), the protein product of the BHD gene, and p0071, an armadillo repeat containing protein that localizes to the cytoplasm and to adherens junctions. Adherens junctions are one of the three cell-cell junctions that are essential to the establishment and maintenance of the cellular architecture of all epithelial tissues. Surprisingly, we found that downregulation of FLCN leads to increased cell-cell adhesion in functional cell-based assays and disruption of cell polarity in a three-dimensional lumen-forming assay, both of which are phenocopied by downregulation of p0071. These data indicate that the FLCN-p0071 protein complex is a negative regulator of cell-cell adhesion. We also found that FLCN positively regulates RhoA activity and Rho-associated kinase activity, consistent with the only known function of p0071. Finally, to examine the role of Flcn loss on cell-cell adhesion in vivo, we utilized keratin-14 cre-recombinase (K14-cre) to inactivate Flcn in the mouse epidermis. The K14-Cre-Bhd(flox/flox) mice have striking delays in eyelid opening, wavy fur, hair loss, and epidermal hyperplasia with increased levels of mammalian target of rapamycin complex 1 (mTORC1) activity. These data support a model in which dysregulation of the FLCN-p0071 interaction leads to alterations in cell adhesion, cell polarity, and RhoA signaling, with broad implications for the role of cell-cell adhesion molecules in the pathogenesis of human disease, including emphysema and renal cell carcinoma. PMID:23139756

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

    SciTech Connect

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

    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.

  14. The adhesion G protein-coupled receptor GPR56 is a cell-autonomous regulator of oligodendrocyte development

    PubMed Central

    Giera, Stefanie; Deng, Yiyu; Luo, Rong; Ackerman, Sarah D.; Mogha, Amit; Monk, Kelly R.; Ying, Yanqin; Jeong, Sung-Jin; Makinodan, Manabu; Bialas, Allison R.; Chang, Bernard S.; Stevens, Beth; Corfas, Gabriel; Piao, Xianhua

    2015-01-01

    Mutations in GPR56, a member of the adhesion G protein-coupled receptor family, cause a human brain malformation called bilateral frontoparietal polymicrogyria (BFPP). Magnetic resonance imaging (MRI) of BFPP brains reveals myelination defects in addition to brain malformation. However, the cellular role of GPR56 in oligodendrocyte development remains unknown. Here, we demonstrate that loss of Gpr56 leads to hypomyelination of the central nervous system in mice. GPR56 levels are abundant throughout early stages of oligodendrocyte development, but are downregulated in myelinating oligodendrocytes. Gpr56-knockout mice manifest with decreased oligodendrocyte precursor cell (OPC) proliferation and diminished levels of active RhoA, leading to fewer mature oligodendrocytes and a reduced number of myelinated axons in the corpus callosum and optic nerves. Conditional ablation of Gpr56 in OPCs leads to a reduced number of mature oligodendrocytes as seen in constitutive knockout of Gpr56. Together, our data define GPR56 as a cell-autonomous regulator of oligodendrocyte development. PMID:25607655

  15. Expression, purification and crystallization of a BH domain from the GTPase regulatory protein associated with focal adhesion kinase.

    PubMed

    Sheffield, P J; Derewenda, U; Taylor, J; Parsons, T J; Derewenda, Z S

    1999-01-01

    Signaling by small GTPases is down-regulated by GTPase activating proteins (GAPs) which enhance the rate of GTP hydrolysis. The activity of GAPs specific for Rho GTPases resides in the BH domain, many homologues of which are found in any mammalian genome. One of them was identified in the GTPase regulator associated with focal-adhesion kinase (GRAF). It shares approximately 20% sequence identity with p50RhoGAP. This GAP activates RhoA and Cdc42Hs, but not Rac. In order to dissect the molecular basis of this specificity, a 231-residue-long fragment corresponding to the BH domain of GRAF has been expressed, purified and crystallized. Trigonal crystals, of space group P3(1)21 or P3(2)21, with unit-cell dimensions a = b = 63.5, c = 90.38 A were grown from solutions of PEG 6000. Data to 2.15 A were collected from a flash-frozen sample on an R-AXIS IV imaging-plate detector mounted on a rotating anode X-ray generator. PMID:10232922

  16. Controllable degradation of medical magnesium by electrodeposited composite films of mussel adhesive protein (Mefp-1) and chitosan.

    PubMed

    Jiang, Ping-Li; Hou, Rui-Qing; Chen, Cheng-Dong; Sun, Lan; Dong, Shi-Gang; Pan, Jin-Shan; Lin, Chang-Jian

    2016-09-15

    To control the degradation rate of medical magnesium in body fluid environment, biocompatible films composed of Mussel Adhesive Protein (Mefp-1) and chitosan were electrodeposited on magnesium surface in cathodic constant current mode. The compositions and structures of the films were characterized by atomic force microscope (AFM), scanning electron microscope (SEM) and infrared reflection absorption spectroscopy (IRAS). And the corrosion protection performance was investigated using electrochemical measurements and immersion tests in simulated body fluid (Hanks' solution). The results revealed that Mefp-1 and chitosan successfully adhered on the magnesium surface and formed a protective film. Compared with either single Mefp-1 or single chitosan film, the composite film of chitosan/Mefp-1/chitosan (CPC (chitosan/Mefp-1/chitosan)) exhibited lower corrosion current density, higher polarization resistance and more homogenous corrosion morphology and thus was able to effectively control the degradation rate of magnesium in simulated body environment. In addition, the active attachment and spreading of MC3T3-E1 cells on the CPC film coated magnesium indicated that the CPC film was significantly able to improve the biocompatibility of the medical magnesium. PMID:27309944

  17. Extracellular matrix protein ITGBL1 promotes ovarian cancer cell migration and adhesion through Wnt/PCP signaling and FAK/SRC pathway.

    PubMed

    Sun, Li; Wang, Defeng; Li, Xiaotian; Zhang, Lingling; Zhang, Hui; Zhang, Yingjie

    2016-07-01

    Despite the advances in cancer treatment and the progresses in tumor biological, ovarian cancer remains a bad situation. In current study, we found a novel extracellular matrix protein, ITGBL1, which is highly expressed in ovarian cancer tissues by immunohistochemistry examination. The expression pattern of ITGBL1 in malignant tissues inspired us to investigate its role in ovarian cancer progression. Both loss- and gain-function assays revealed that ITGBL1 could promote ovarian cancer cell migration and adhesion. As it's a secreted protein, we further used recombinant ITGBL1 protein treated cancer cells and found that ITGBL1 promotes cell migration and adhesion in a concentration dependent manner. Furthermore, we found that ITGBL1 not only influences the activity of Wnt/PCP signaling but also affects FAK/src pathway in vitro. Taken together, our results suggest that highly expressed ITGBL1 could promotes cancer cell migration and adhesion in ovarian cancer and as a secreted protein, ITGBL1 might be a novel biomarker for ovarian cancer diagnosis. PMID:27261588

  18. Protein adsorption and cell adhesion on three-dimensional polycaprolactone scaffolds with respect to plasma modification by etching and deposition techniques

    NASA Astrophysics Data System (ADS)

    Myung, Sung Woon; Ko, Yeong Mu; Kim, Byung Hoon

    2014-11-01

    In this work, protein adsorption and cell adhesion on three-dimensional (3D) polycaprolactone (PCL) scaffolds treated by plasma etching and deposition were performed. The 3D PCL scaffold used as a substrate of a bone tissue was fabricated by recent rapid prototype techniques. To increase surface properties, such as hydrophilicity, roughness, and surface chemistry, through good protein adhesion on scaffolds, oxygen (O2) plasma etching and acrylic acid or allyamine plasma deposition were performed on the 3D PCL scaffolds. The O2 plasma etching induced the formation of random nanoporous structures on the roughened surfaces of the 3D PCL scaffolds. The plasma deposition with acrylic acid and allyamine induced the chemical modification for introducing a functional group. The protein adsorption increased on the O2 plasma-etched surface compared with an untreated 3D PCL scaffold. MC3T3-E1 cells adhered bioactively on the etched and deposited surface compared with the untreated surface. The present plasma modification might be sought as an effective technique for enhancing protein adsorption and cell adhesion.

  19. Hybrid chitosan/β-1,3-glucan matrix of bone scaffold enhances osteoblast adhesion, spreading and proliferation via promotion of serum protein adsorption.

    PubMed

    Przekora, Agata; Benko, Aleksandra; Blazewicz, Marta; Ginalska, Grazyna

    2016-01-01

    Initial protein adsorption to the material surface is crucial for osteoblast adhesion, survival, and rapid proliferation resulting in intensive new bone formation. The aim of this study was to demonstrate that modification of a chitosan matrix of chitosan/hydroxyapatite (chit/HA) biomaterial for bone tissue engineering applications with linear β-1,3-glucan (curdlan) leads to promotion of serum protein adsorption to the resultant scaffold (chit/glu/HA) and thus in enhancement of osteoblast adhesion, spreading and proliferation. Fabricated biomaterials were pre-adsorbed with different protein solutions and then protein adsorption and osteoblast behavior on the scaffolds were compared. Moreover, surface chemical composition, wettability and surface energy of biomaterials were compared. Modification of the chitosan matrix with β-1,3-glucan introduces a greater polarpart in the resultant chitosan/β-1,3-glucan matrix presumably resulting from more OH groups within the curdlan structure. Moreover, FTIR-ATR results suggest that there might be some sort of chemical interaction between the NH group of chitosan and the OH group of β-1,3-glucan. As a consequence, the chit/glu/HA scaffold adsorbs significantly more adhesion proteins that are crucial for osteoblasts compared to the chit/HA material, providing a higher density culture of well-spread osteoblasts on its surface. Obtained results revealed that not only is chit/glu/HA biomaterial a promising scaffold for bone tissue engineering applications, but the specific polysaccharide chit/glu matrix itself is promising for use in the biomedical material field to modify various biomaterials in order to enhance osteoblast adhesion and proliferation on their surfaces. PMID:27388048

  20. Library Screen Identifies Enterococcus faecalis CcpA, the Catabolite Control Protein A, as an Effector of Ace, a Collagen Adhesion Protein Linked to Virulence

    PubMed Central

    Gao, Peng; Pinkston, Kenneth L.; Bourgogne, Agathe; Cruz, Melissa R.; Garsin, Danielle A.; Murray, Barbara E.

    2013-01-01

    The Enterococcus faecalis cell wall-anchored protein Ace is an important virulence factor involved in cell adhesion and infection. Expression of Ace on the cell surface is affected by many factors, including stage of growth, culture temperature, and environmental components, such as serum, urine, and collagen. However, the mechanisms that regulate or modulate Ace display are not well understood. With interest in identifying genes associated with Ace expression, we utilized a whole-cell enzyme-linked immunosorbent assay (ELISA)-based screening method to identify mutants from a transposon insertion mutant library which exhibited distinct Ace surface expression profiles. We identified a ccpA insertion mutant which showed significantly decreased levels of Ace surface expression at early growth phase versus those of wild-type OG1RF. Confirmation of the observation was achieved through flow cytometry and complementation analysis. Compared to the wild type, the E. faecalis ccpA mutant had an impaired ability to adhere to collagen when grown to early exponential phase, consistent with the lack of Ace expression in the early growth phase. As a key component of carbon catabolite regulation, CcpA has been previously reported to play a critical role in regulating expression of proteins involved in E. faecalis carbohydrate uptake and utilization. Our discovery is the first to associate CcpA with the production of a major E. faecalis virulence factor, providing new insights into the regulation of E. faecalis pathogenesis. PMID:23974022

  1. Dynamic and Static Interactions between p120 Catenin and E-Cadherin Regulate the Stability of Cell-Cell Adhesion

    SciTech Connect

    Ishiyama, Noboru; Lee, Seung-Hye; Liu, Shuang; Li, Guang-Yao; Smith, Matthew J.; Reichardt, Louis F.; Ikura, Mitsuhiko

    2010-04-26

    The association of p120 catenin (p120) with the juxtamembrane domain (JMD) of the cadherin cytoplasmic tail is critical for the surface stability of cadherin-catenin cell-cell adhesion complexes. Here, we present the crystal structure of p120 isoform 4A in complex with the JMD core region (JMD{sub core}) of E-cadherin. The p120 armadillo repeat domain contains modular binding pockets that are complementary to electrostatic and hydrophobic properties of the JMD{sub core}. Single-residue mutations within the JMD{sub core}-binding site of p120 abolished its interaction with E- and N-cadherins in vitro and in cultured cells. These mutations of p120 enabled us to clearly differentiate between N-cadherin-dependent and -independent steps of neuronal dendritic spine morphogenesis crucial for synapse development. NMR studies revealed that p120 regulates the stability of cadherin-mediated cell-cell adhesion by associating with the majority of the JMD, including residues implicated in clathrin-mediated endocytosis and Hakai-dependent ubiquitination of E-cadherin, through its discrete dynamic and static binding sites.

  2. Hybrid GPCR/cadherin (Celsr) proteins in rat testis are expressed with cell type specificity and exhibit differential Sertoli cell-germ cell adhesion activity.

    PubMed

    Beall, Stephanie A; Boekelheide, Kim; Johnson, Kamin J

    2005-01-01

    Spermatogenesis requires Sertoli cell-germ cell adhesion for germ cell survival and maturation. Cadherins are a diverse superfamily of adhesion proteins; structurally unique members of this superfamily (celsr cadherins) are hybrid molecules containing extracellular cadherin repeats connected to a G protein-coupled receptor transmembrane motif. Here we demonstrate postnatal testicular mRNA expression of the 3 celsr paralogs (celsr1, celsr2, and celsr3), protein localization of celsr2 and celsr3, and functional analysis of celsr2 adhesion activity in primary Sertoli cell-germ cell co-cultures. Evaluation of celsr mRNA levels during a postnatal time course indicated that celsr1 and celsr2 were Sertoli cell and/or early-stage germ cell products, whereas celsr3 was expressed in later-stage germ cells. Cell type-specific expression was verified using the Sertoli cell line 93RS2, where celsr1 and celsr2 mRNA, but not celsr3, were detected. Immunostaining of testicular cryosections resulted in celsr2 protein localization to a spokelike pattern in the basal seminiferous epithelium and punctate figures in the apical epithelium, consistent with both Sertoli cell and germ cell expression. Celsr3 localized to punctate structures in the adluminal epithelium from postnatal day 40, consistent with elongate spermatid expression. The subcellular localization of celsr2 was examined further to define its localization in Sertoli cells and germ cells. Celsr2 localized to the Golgi complex in Sertoli cells and germ cells. In addition, germ cell celsr2 localized to a rab7-positive structure, which may be an endocytic compartment. Neither celsr2 nor celsr3 immunostaining was present at classic cadherin-based adhesion junctions. Nonetheless, the addition of a recombinant celsr2 protein fragment consisting of extracellular cadherin domains 4 through 8 to Sertoli cell-germ cell co-cultures resulted in germ cell detachment from Sertoli cells. Collectively, these data indicate that celsr

  3. Composites containing albumin protein or cyanoacrylate adhesives and biodegradable scaffolds: I. Acute wound closure study in a rat model

    NASA Astrophysics Data System (ADS)

    Hoffman, Grant T.; Soller, Eric C.; Heintzelman, Douglas L.; Duffy, Mark T.; Bloom, Jeffrey N.; Gilmour, Travis M.; Gonnerman, Krista N.; McNally-Heintzelman, Karen M.

    2004-07-01

    Composite adhesives composed of biodegradable scaffolds impregnated with a biological or synthetic adhesive were investigated for use in wound closure as an alternative to using either one of the adhesives alone. Two different scaffold materials were investigated: (i) a synthetic biodegradable material fabricated from poly(L-lactic-co-glycolic acid); and (ii) a biological material, small intestinal sub mucosa, manufactured by Cook BioTech. The biological adhesive was composed of 50%(w/v) bovine serum albumin solder and 0.5mg/ml indocyanine green dye mixed in deionized water, and activated with an 808-nm diode laser. The synthetic adhesive was Ethicon's Dermabond, a 2-octyl-cyanoacrylate. The tensile strength of skin incisions repaired ex vivo in a rat model, by adhesive alone or in combination with a scaffold, as well as the time-to-failure, were measured and compared. The tensile strength of repairs formed using the scaffold-enhanced biological adhesives were on average, 80% stronger than their non-enhanced counterparts, with an accompanying increase in the time-to-failure of the repairs. These results support the theory that a scaffold material with an irregular surface that bridges the wound provides a stronger, more durable and consistent adhesion, due to the distribution of the tensile stress forces over the many micro-adhesions provided by the irregular surface, rather than the one large continuous adhesive contact. This theory is also supported by several previous ex vivo experiments demonstrating enhanced tensile strength of irregular versus smooth scaffold surfaces in identical tissue repairs performed on bovine thoracic aorta, liver, spleen, small intestine and lung tissue.

  4. Strain-specific diversity of mucus-binding proteins in the adhesion and aggregation properties of Lactobacillus reuteri.

    PubMed

    Mackenzie, Donald A; Jeffers, Faye; Parker, Mary L; Vibert-Vallet, Amandine; Bongaerts, Roy J; Roos, Stefan; Walter, Jens; Juge, Nathalie

    2010-11-01

    Mucus-binding proteins (MUBs) have been revealed as one of the effector molecules involved in mechanisms of the adherence of lactobacilli to the host; mub, or mub-like, genes are found in all of the six genomes of Lactobacillus reuteri that are available. We recently reported the crystal structure of a Mub repeat from L. reuteri ATCC 53608 (also designated strain 1063), revealing an unexpected recognition of immunoglobulins. In the current study, we explored the diversity of the ATCC 53608 mub gene, and MUB expression levels in a large collection of L. reuteri strains isolated from a range of vertebrate hosts. This analysis revealed that the MUB was only detectable on the cell surface of two highly related isolates when using antibodies that were raised against the protein. There was considerable variation in quantitative mucus adhesion in vitro among L. reuteri strains, and mucus binding showed excellent correlation with the presence of cell-surface ATCC 53608 MUB. ATCC 53608 MUB presence was further highly associated with the autoaggregation of L. reuteri strains in washed cell suspensions, suggesting a novel role of this surface protein in cell aggregation. We also characterized MUB expression in representative L. reuteri strains. This analysis revealed that one derivative of strain 1063 was a spontaneous mutant that expressed a C-terminally truncated version of MUB. This frameshift mutation was caused by the insertion of a duplicated 13 nt sequence at position 4867 nt in the mub gene, producing a truncated MUB also lacking the C-terminal LPxTG region, and thus unable to anchor to the cell wall. This mutant, designated 1063N (mub-4867(i)), displayed low mucus-binding and aggregation capacities, further providing evidence for the contribution of cell-wall-anchored MUB to such phenotypes. In conclusion, this study provided novel information on the functional attributes of MUB in L. reuteri, and further demonstrated that MUB and MUB-like proteins

  5. Crystal structure of linoleate 13R-manganese lipoxygenase in complex with an adhesion protein.

    PubMed

    Chen, Yang; Wennman, Anneli; Karkehabadi, Saeid; Engström, Åke; Oliw, Ernst H

    2016-08-01

    The crystal structure of 13R-manganese lipoxygenase (MnLOX) of Gaeumannomyces graminis (Gg) in complex with zonadhesin of Pichia pastoris was solved by molecular replacement. Zonadhesin contains β-strands in two subdomains. A comparison of Gg-MnLOX with the 9S-MnLOX of Magnaporthe oryzae (Mo) shows that the protein fold and the geometry of the metal ligands are conserved. The U-shaped active sites differ mainly due to hydrophobic residues of the substrate channel. The volumes and two hydrophobic side pockets near the catalytic base may sanction oxygenation at C-13 and C-9, respectively. Gly-332 of Gg-MnLOX is positioned in the substrate channel between the entrance and the metal center. Replacements with larger residues could restrict oxygen and substrate to reach the active site. C18 fatty acids are likely positioned with C-11 between Mn(2+)OH2 and Leu-336 for hydrogen abstraction and with one side of the 12Z double bond shielded by Phe-337 to prevent antarafacial oxygenation at C-13 and C-11. Phe-347 is positioned at the end of the substrate channel and replacement with smaller residues can position C18 fatty acids for oxygenation at C-9. Gg-MnLOX does not catalyze the sequential lipoxygenation of n-3 fatty acids in contrast to Mo-MnLOX, which illustrates the different configurations of their substrate channels. PMID:27313058

  6. Reversible Conformational Change in the Plasmodium falciparum Circumsporozoite Protein Masks Its Adhesion Domains

    PubMed Central

    Herrera, Raul; Anderson, Charles; Kumar, Krishan; Molina-Cruz, Alvaro; Nguyen, Vu; Burkhardt, Martin; Reiter, Karine; Shimp, Richard; Howard, Randall F.; Srinivasan, Prakash; Nold, Michael J.; Ragheb, Daniel; Shi, Lirong; DeCotiis, Mark; Aebig, Joan; Lambert, Lynn; Rausch, Kelly M.; Muratova, Olga; Jin, Albert; Reed, Steven G.; Sinnis, Photini; Barillas-Mury, Carolina; Duffy, Patrick E.; MacDonald, Nicholas J.

    2015-01-01

    The extended rod-like Plasmodium falciparum circumsporozoite protein (CSP) is comprised of three primary domains: a charged N terminus that binds heparan sulfate proteoglycans, a central NANP repeat domain, and a C terminus containing a thrombospondin-like type I repeat (TSR) domain. Only the last two domains are incorporated in RTS,S, the leading malaria vaccine in phase 3 trials that, to date, protects about 50% of vaccinated children against clinical disease. A seroepidemiological study indicated that the N-terminal domain might improve the efficacy of a new CSP vaccine. Using a panel of CSP-specific monoclonal antibodies, well-characterized recombinant CSPs, label-free quantitative proteomics, and in vitro inhibition of sporozoite invasion, we show that native CSP is N-terminally processed in the mosquito host and undergoes a reversible conformational change to mask some epitopes in the N- and C-terminal domains until the sporozoite interacts with the liver hepatocyte. Our findings show the importance of understanding processing and the biophysical change in conformation, possibly due to a mechanical or molecular signal, and may aid in the development of a new CSP vaccine. PMID:26169272

  7. Reversible Conformational Change in the Plasmodium falciparum Circumsporozoite Protein Masks Its Adhesion Domains.

    PubMed

    Herrera, Raul; Anderson, Charles; Kumar, Krishan; Molina-Cruz, Alvaro; Nguyen, Vu; Burkhardt, Martin; Reiter, Karine; Shimp, Richard; Howard, Randall F; Srinivasan, Prakash; Nold, Michael J; Ragheb, Daniel; Shi, Lirong; DeCotiis, Mark; Aebig, Joan; Lambert, Lynn; Rausch, Kelly M; Muratova, Olga; Jin, Albert; Reed, Steven G; Sinnis, Photini; Barillas-Mury, Carolina; Duffy, Patrick E; MacDonald, Nicholas J; Narum, David L

    2015-10-01

    The extended rod-like Plasmodium falciparum circumsporozoite protein (CSP) is comprised of three primary domains: a charged N terminus that binds heparan sulfate proteoglycans, a central NANP repeat domain, and a C terminus containing a thrombospondin-like type I repeat (TSR) domain. Only the last two domains are incorporated in RTS,S, the leading malaria vaccine in phase 3 trials that, to date, protects about 50% of vaccinated children against clinical disease. A seroepidemiological study indicated that the N-terminal domain might improve the efficacy of a new CSP vaccine. Using a panel of CSP-specific monoclonal antibodies, well-characterized recombinant CSPs, label-free quantitative proteomics, and in vitro inhibition of sporozoite invasion, we show that native CSP is N-terminally processed in the mosquito host and undergoes a reversible conformational change to mask some epitopes in the N- and C-terminal domains until the sporozoite interacts with the liver hepatocyte. Our findings show the importance of understanding processing and the biophysical change in conformation, possibly due to a mechanical or molecular signal, and may aid in the development of a new CSP vaccine. PMID:26169272

  8. Haemophilus influenzae P4 Interacts With Extracellular Matrix Proteins Promoting Adhesion and Serum Resistance.

    PubMed

    Su, Yu-Ching; Mukherjee, Oindrilla; Singh, Birendra; Hallgren, Oskar; Westergren-Thorsson, Gunilla; Hood, Derek; Riesbeck, Kristian

    2016-01-15

    Interaction with the extracellular matrix (ECM) is one of the successful colonization strategies employed by nontypeable Haemophilus influenzae (NTHi). Here we identified Haemophilus lipoprotein e (P4) as a receptor for ECM proteins. Purified recombinant P4 displayed a high binding affinity for laminin (Kd = 9.26 nM) and fibronectin (Kd = 10.19 nM), but slightly less to vitronectin (Kd = 16.51 nM). A P4-deficient NTHi mutant showed a significantly decreased binding to these ECM components. Vitronectin acquisition conferred serum resistance to both P4-expressing NTHi and Escherichia coli transformants. P4-mediated bacterial adherence to pharynx, type II alveolar, and bronchial epithelial cells was mainly attributed to fibronectin. Importantly, a significantly reduced bacterial infection was observed in the middle ear of the Junbo mouse model when NTHi was devoid of P4. In conclusion, our data provide new insight into the role of P4 as an important factor for Haemophilus colonization and subsequent respiratory tract infection. PMID:26153407

  9. Mechanism of a cytosolic O-glycosyltransferase essential for the synthesis of a bacterial adhesion protein

    PubMed Central

    Chen, Yu; Seepersaud, Ravin; Bensing, Barbara A.; Sullam, Paul M.; Rapoport, Tom A.

    2016-01-01

    O-glycosylation of Ser and Thr residues is an important process in all organisms, which is only poorly understood. Such modification is required for the export and function of adhesin proteins that mediate the attachment of pathogenic Gram-positive bacteria to host cells. Here, we have analyzed the mechanism by which the cytosolic O-glycosyltransferase GtfA/B of Streptococcus gordonii modifies the Ser/Thr-rich repeats of adhesin. The enzyme is a tetramer containing two molecules each of GtfA and GtfB. The two subunits have the same fold, but only GtfA contains an active site, whereas GtfB provides the primary binding site for adhesin. During a first phase of glycosylation, the conformation of GtfB is restrained by GtfA to bind substrate with unmodified Ser/Thr residues. In a slow second phase, GtfB recognizes residues that are already modified with N-acetylglucosamine, likely by converting into a relaxed conformation in which one interface with GtfA is broken. These results explain how the glycosyltransferase modifies a progressively changing substrate molecule. PMID:26884191

  10. Abdominal Adhesions

    MedlinePlus

    ... Abdominal Adhesions 1 Ward BC, Panitch A. Abdominal adhesions: current and novel therapies. Journal of Surgical Research. 2011;165(1):91– ... are abdominal adhesions and intestinal obstructions ... generally do not require treatment. Surgery is the only way to treat abdominal ...

  11. A Novel Domain Cassette Identifies Plasmodium falciparum PfEMP1 Proteins Binding ICAM-1 and Is a Target of Cross-Reactive, Adhesion-Inhibitory Antibodies

    PubMed Central

    Bengtsson, Anja; Joergensen, Louise; Rask, Thomas S.; Olsen, Rebecca W.; Andersen, Marianne A.; Turner, Louise; Theander, Thor G.; Higgins, Matthew K.; Craig, Alister; Brown, Alan

    2013-01-01

    Cerebral Plasmodium falciparum malaria is characterized by adhesion of infected erythrocytes (IEs) to the cerebral microvasculature. This has been linked to parasites expressing the structurally related group A subset of the P. falciparum erythrocyte membrane protein 1 (PfEMP1) family of IE adhesion ligands and to IEs with affinity for ICAM-1. However, recent evidence has cast doubt on both these associations, tempering hopes of the feasibility of developing a vaccine based on ICAM-1–binding PfEMP1. In this study, we report the identification of a domain cassette (DC) present in group A var genes from six genetically distinct P. falciparum parasites. The three domains in the cassette, which we call DC4, had a high level of sequence identity and cluster together phylogenetically. Erythrocytes infected by these parasites and selected in vitro for expression of DC4 adhered specifically to ICAM-1. The ICAM-1–binding capacity of DC4 was mapped to the C-terminal third of its Duffy-binding–like β3 domain. DC4 was the target of broadly cross-reactive and adhesion-inhibitory IgG Abs, and levels of DC4-specific and adhesion-inhibitory IgG increased with age among P. falciparum–exposed children. Our study challenges earlier conclusions that group A PfEMP1 proteins are not central to ICAM-1–specific IE adhesion and support the feasibility of developing a vaccine preventing cerebral malaria by inhibiting cerebral IE sequestration. PMID:23209327

  12. Antisense RNA inactivation of gene expression of a cell-cell adhesion protein (gp64) in the cellular slime mold Polysphondylium pallidum.

    PubMed

    Funamoto, S; Ochiai, H

    1996-05-01

    The gp64 protein of Polysphondylium pallidum has been shown to mediate EDTA-stable cell-cell adhesion. To explore the functional role of gp64, we made an antisense RNA expression construct designed to prevent the gene expression of gp64; the construct was introduced into P. pallidum cells and the transformants were characterised. The antisense RNA-expressing clone L3mc2 which had just been harvested at the growth phase tended to re-form in aggregates smaller in size than did the parental cells in either the presence or absence of 10 mM EDTA. In contrast, 6.5-hour starved L3mc2 cells remained considerably dissociated from each other after 5 minutes gyrating, although aggregation gradually increased by 50% during a further 55 minutes gyrating in the presence of 10 mM EDTA. Correspondingly, L3mc2 lacked specifically the cell-cell adhesion protein, gp64. We therefore conclude that the gp64 protein is involved in forming the EDTA-resistant cell-cell contact. In spite of the absence of gp64, L3mc2 exhibited normal developmental processes, a fact which demonstrates that another cell-cell adhesion system exists in the development of Polysphondylium. This is the first report in which an antisense RNA technique was successfully applied to Polysphondylium. PMID:8743948

  13. Seafood delicacy makes great adhesive

    ScienceCinema

    Idaho National Laboratory - Frank Roberto, Heather Silverman

    2010-01-08

    Technology from Mother Nature is often hard to beat, so Idaho National Laboratory scientistsgenetically analyzed the adhesive proteins produced by blue mussels, a seafood delicacy. Afterobtaining full-length DNA sequences encoding these proteins, reprod

  14. Seafood delicacy makes great adhesive

    SciTech Connect

    Idaho National Laboratory - Frank Roberto, Heather Silverman

    2008-03-26

    Technology from Mother Nature is often hard to beat, so Idaho National Laboratory scientistsgenetically analyzed the adhesive proteins produced by blue mussels, a seafood delicacy. Afterobtaining full-length DNA sequences encoding these proteins, reprod

  15. Y-box-binding protein-1 (YB-1) promotes cell proliferation, adhesion and drug resistance in diffuse large B-cell lymphoma.

    PubMed

    Miao, Xiaobing; Wu, Yaxun; Wang, Yuchan; Zhu, Xinghua; Yin, Haibing; He, Yunhua; Li, Chunsun; Liu, Yushan; Lu, Xiaoyun; Chen, Yali; Shen, Rong; Xu, Xiaohong; He, Song

    2016-08-15

    YB-1 is a multifunctional protein, which has been shown to correlate with resistance to treatment of various tumor types. This study investigated the expression and biologic function of YB-1 in diffuse large B-cell lymphoma (DLBCL). Immunohistochemical analysis showed that the expression statuses of YB-1 and pYB-1(S102) were reversely correlated with the clinical outcomes of DLBCL patients. In addition, we found that YB-1 could promote the proliferation of DLBCL cells by accelerating the G1/S transition. Ectopic expression of YB-1 could markedly increase the expression of cell cycle regulators cyclin D1 and cyclin E. Furthermore, we found that adhesion of DLBCL cells to fibronectin (FN) could increase YB-1 phosphorylation at Ser102 and pYB-1(S102) nuclear translocation. In addition, overexpression of YB-1 could increase the adhesion of DLBCL cells to FN. Intriguingly, we found that YB-1 overexpression could confer drug resistance through cell-adhesion dependent and independent mechanisms in DLBCL. Silencing of YB-1 could sensitize DLBCL cells to mitoxantrone and overcome cell adhesion-mediated drug resistance (CAM-DR) phenotype in an AKT-dependent manner. PMID:27397581

  16. Enhanced Adhesion and OspC Protein Synthesis of the Lyme Disease Spirochete Borrelia Burgdorferi Cultivated in a Host-Derived Tissue Co-Culture System

    PubMed Central

    Şen, Ece; Sigal, Leonard H.

    2013-01-01

    Background: The adhesion process of Borrelia burgdorferi to susceptible host cell has not yet been completely understood regarding the function of OspA, OspB and OspC proteins and a conflict exists in the infection process. Aims: The adhesion rates of pathogenic (low BSK medium passaged or susceptible rat joint tissue co-cultivated) or non-pathogenic Borrelia burgdorferi (high BSK medium passaged) isolate (FNJ) to human umbilical vein endothelial cells (HUVEC) cultured on coverslips and the synthesis of OspA and OspC proteins were investigated to analyze the infection process of this bacterium. Study Design: In-vitro study. Methods: Spirochetes were cultured in BSK medium or in a LEW/N rat tibiotarsal joint tissue feeder layer supported co-culture system using ESG co-culture medium and labelled with 3H-adenine for 48 hours. SDS-PAGE, Western Blotting, Immunogold A labeling as well as radiolabeling experiments were used to compare pathogenic or non pathogenic spirochetes during the adhesion process. Results: Tissue co-cultured B. burgdorferi adhered about ten times faster than BSK-grown spirochetes. Trypsin inhibited attachment to HUVEC and co-culture of trypsinized spirochetes with tissues reversed the inhibition. Also, the synthesis of OspC protein by spirochetes was increased in abundance after tissue co-cultures, as determined by SDS-PAGE and by electron microscopy analysis of protein A-immunogold staining by anti-OspC antibodies. OspA protein was synthesized in similar quantities in all Borrelia cultures analyzed by the same techniques. Conclusion: Low BSK passaged or tissue co-cultured pathogenic Lyme disease spirochetes adhere to HUVEC faster than non-pathogenic high BSK passaged forms of this bacterium. Spirochetes synthesized OspC protein during host tissue-associated growth. However, we did not observe a reduction of OspA synthesis during host tissue co-cultivation in vitro. PMID:25207103

  17. Biophysically inspired model for functionalized nanocarrier adhesion to cell surface: roles of protein expression and mechanical factors

    PubMed Central

    Ramakrishnan, N.; Tourdot, Richard W.; Eckmann, David M.; Ayyaswamy, Portonovo S.; Muzykantov, Vladimir R.; Radhakrishnan, Ravi

    2016-01-01

    In order to achieve selective targeting of affinity–ligand coated nanoparticles to the target tissue, it is essential to understand the key mechanisms that govern their capture by the target cell. Next-generation pharmacokinetic (PK) models that systematically account for proteomic and mechanical factors can accelerate the design, validation and translation of targeted nanocarriers (NCs) in the clinic. Towards this objective, we have developed a computational model to delineate the roles played by target protein expression and mechanical factors of the target cell membrane in determining the avidity of functionalized NCs to live cells. Model results show quantitative agreement with in vivo experiments when specific and non-specific contributions to NC binding are taken into account. The specific contributions are accounted for through extensive simulations of multivalent receptor–ligand interactions, membrane mechanics and entropic factors such as membrane undulations and receptor translation. The computed NC avidity is strongly dependent on ligand density, receptor expression, bending mechanics of the target cell membrane, as well as entropic factors associated with the membrane and the receptor motion. Our computational model can predict the in vivo targeting levels of the intracellular adhesion molecule-1 (ICAM1)-coated NCs targeted to the lung, heart, kidney, liver and spleen of mouse, when the contributions due to endothelial capture are accounted for. The effect of other cells (such as monocytes, etc.) do not improve the model predictions at steady state. We demonstrate the predictive utility of our model by predicting partitioning coefficients of functionalized NCs in mice and human tissues and report the statistical accuracy of our model predictions under different scenarios. PMID:27429783

  18. Contributions of adhesive proteins to the cellular and bacterial response to surfaces treated with bioactive polymers: case of poly(sodium styrene sulfonate) grafted titanium surfaces.

    PubMed

    Felgueiras, Helena P; Aissa, Ines Ben; Evans, Margaret D M; Migonney, Véronique

    2015-11-01

    The research developed on functionalized model or prosthetic surfaces with bioactive polymers has raised the possibility to modulate and/or control the biological in vitro and in vivo responses to synthetic biomaterials. The mechanisms underlying the bioactivity exhibited by sulfonated groups on surfaces involves both selective adsorption and conformational changes of adsorbed proteins. Indeed, surfaces functionalized by grafting poly(sodium styrene sulfonate) [poly(NaSS)] modulate the cellular and bacterial response by inducing specific interactions with fibronectin (Fn). Once implanted, a biomaterial surface is exposed to a milieu of many proteins that compete for the surface which dictates the subsequent biological response. Once understood, this can be controlled by dictating exposure of active binding sites. In this in vitro study, we report the influence of binary mixtures of proteins [albumin (BSA), Fn and collagen type I (Col I)] adsorbed on poly(NaSS) grafted Ti6Al4V on the adhesion and differentiation of MC3T3-E1 osteoblast-like cells and the adhesion and proliferation of Staphylococcus aureus (S. aureus). Outcomes showed that poly(NaSS) stimulated cell spreading, attachment strength, differentiation and mineralization, whatever the nature of protein provided at the interface compared with ungrafted Ti6Al4V (control). While in competition, Fn and Col I were capable of prevailing over BSA. Fn played an important role in the early interactions of the cells with the surface, while Col I was responsible for increased alkaline phosphatase, calcium and phosphate productions associated with differentiation. Poly(NaSS) grafted surfaces decreased the adhesion of S. aureus and the presence of Fn on these chemically altered surfaces increased bacterial resistance ≈70% compared to the ungrafted Ti6Al4V. Overall, our study showed that poly(NaSS) grafted Ti6Al4V selectively adsorbed proteins (particularly Fn) promoting the adhesion and differentiation of osteoblast

  19. Upregulation of adhesion complex proteins and fibronectin by human keratinocytes treated with an aqueous extract from the leaves of Chromolaena odorata (Eupolin).

    PubMed

    Phan, T T; Allen, J; Hughes, M A; Cherry, G; Wojnarowska, F

    2000-01-01

    The fresh leaves and extract of the plant Chromolaena odorata are a traditional herbal treatment in developing countries for burns, soft tissue wounds and skin infections. We have previously shown that the extract had an effect on the growth and proliferation of keratinocytes and fibroblasts in culture. This study has demonstrated that Eupolin extract increased expression of several components of the adhesion complex and fibronectin by human keratinocytes. Using indirect immunofluorescence we found increased expression (dose-dependent) of laminin 5, laminin 1, collagen IV, and fibronectin. The expression of the b1 and b4 integrins was upregulated by the extract at low concentrations (0.1 and 1 microg/ml), but the expression was decreased at higher doses of Eupolin (10 microg-150 microg/ml). A number of clinical studies carried out by Vietnamese and international medical investigators have demonstrated the efficacy of this extract on the wound healing process. In this study we have shown that Eupolin stimulated the expression of many proteins of the adhesion complex and fibronectin by human keratinocytes. The adhesion complex proteins are essential to stabilise epithelium and this effect could contribute to the clinical efficacy of Eupolin in healing. PMID:11056422

  20. DDB2 (damaged-DNA binding 2) protein: a new modulator of nanomechanical properties and cell adhesion of breast cancer cells

    NASA Astrophysics Data System (ADS)

    Barbieux, Claire; Bacharouche, Jalal; Soussen, Charles; Hupont, Sébastien; Razafitianamaharavo, Angélina; Klotz, Rémi; Pannequin, Rémi; Brie, David; Bécuwe, Philippe; Francius, Grégory; Grandemange, Stéphanie

    2016-02-01

    DDB2, known for its role in DNA repair, was recently shown to reduce mammary tumor invasiveness by inducing the transcription of IκBα, an inhibitor of NF-κB activity. Since cellular adhesion is a key event during the epithelial to mesenchymal transition (EMT) leading to the invasive capacities of breast tumor cells, the aim of this study was to investigate the role of DDB2 in this process. Thus, using low and high DDB2-expressing MDA-MB231 and MCF7 cells, respectively, in which DDB2 expression was modulated experimentally, we showed that DDB2 overexpression was associated with a decrease of adhesion abilities on glass and plastic areas of breast cancer cells. Then, we investigated cell nanomechanical properties by atomic force microscopy (AFM). Our results revealed significant changes in the Young's Modulus value and the adhesion force in MDA-MB231 and MCF7 cells, whether DDB2 was expressed or not. The cell stiffness decrease observed in MDA-MB231 and MCF7 expressing DDB2 was correlated with a loss of the cortical actin-cytoskeleton staining. To understand how DDB2 regulates these processes, an adhesion-related gene PCR-Array was performed. Several adhesion-related genes were differentially expressed according to DDB2 expression, indicating that important changes are occurring at the molecular level. Thus, this work demonstrates that AFM technology is an important tool to follow cellular changes during tumorigenesis. Moreover, our data revealed that DDB2 is involved in early events occurring during metastatic progression of breast cancer cells and will contribute to define this protein as a new marker of metastatic progression in this type of cancer.

  1. Dynamic Regulation of a Cell Adhesion Protein Complex Including CADM1 by Combinatorial Analysis of FRAP with Exponential Curve-Fitting

    PubMed Central

    Sakurai-Yageta, Mika; Maruyama, Tomoko; Suzuki, Takashi; Ichikawa, Kazuhisa; Murakami, Yoshinori

    2015-01-01

    Protein components of cell adhesion machinery show continuous renewal even in the static state of epithelial cells and participate in the formation and maintenance of normal epithelial architecture and tumor suppression. CADM1 is a tumor suppressor belonging to the immunoglobulin superfamily of cell adhesion molecule and forms a cell adhesion complex with an actin-binding protein, 4.1B, and a scaffold protein, MPP3, in the cytoplasm. Here, we investigate dynamic regulation of the CADM1-4.1B-MPP3 complex in mature cell adhesion by fluorescence recovery after photobleaching (FRAP) analysis. Traditional FRAP analysis were performed for relatively short period of around 10min. Here, thanks to recent advances in the sensitive laser detector systems, we examine FRAP of CADM1 complex for longer period of 60 min and analyze the recovery with exponential curve-fitting to distinguish the fractions with different diffusion constants. This approach reveals that the fluorescence recovery of CADM1 is fitted to a single exponential function with a time constant (τ) of approximately 16 min, whereas 4.1B and MPP3 are fitted to a double exponential function with two τs of approximately 40-60 sec and 16 min. The longer τ is similar to that of CADM1, suggesting that 4.1B and MPP3 have two distinct fractions, one forming a complex with CADM1 and the other present as a free pool. Fluorescence loss in photobleaching analysis supports the presence of a free pool of these proteins near the plasma membrane. Furthermore, double exponential fitting makes it possible to estimate the ratio of 4.1B and MPP3 present as a free pool and as a complex with CADM1 as approximately 3:2 and 3:1, respectively. Our analyses reveal a central role of CADM1 in stabilizing the complex with 4.1B and MPP3 and provide insight in the dynamics of adhesion complex formation. PMID:25780926

  2. Regulatory and junctional proteins of the blood-testis barrier in human Sertoli cells are modified by monobutyl phthalate (MBP) and bisphenol A (BPA) exposure.

    PubMed

    de Freitas, André Teves Aquino Gonçalves; Ribeiro, Mariana Antunes; Pinho, Cristiane Figueiredo; Peixoto, André Rebelo; Domeniconi, Raquel Fantin; Scarano, Wellerson R

    2016-08-01

    The blood-testis barrier (BTB) is responsible for providing a protected environment and coordinating the spermatogenesis. Endocrine disruptors (EDs) might lead to infertility, interfering in the BTB structure and modulation. This study aimed to correlate the actions of two EDs, monobutyl phthalate (MBP) and bisphenol A (BPA) in different periods of exposure, in a low toxicity dose to the human Sertoli cells (HSeC) and its effects on the proteins of the BTB and regulatory proteins involved in its modulation. HSeC cells were exposed to MBP (10μM) and BPA (20μM) for 6 and 48h. Western Blot assay indicated that MBP was able to reduce the expression of occludin, ZO-1, N-cadherin and Androgen Receptor (AR), while BPA leads to a reduction of occludin, ZO-1, β-catenin and AR. TGF-β2 and F-actin were not modified. Phalloidin and Hematoxylin and Eosin assay revealed phenotically disruption in Sertoli cells adhesion, without changes in F-actin expression or localization. Our data suggested both EDs present potential for disrupting the structure and maintenance of the human BTB by AR dependent pathway. PMID:26922907

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

    SciTech Connect

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

    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 increased

  4. Investigating the BSA protein adsorption and bacterial adhesion of Al-alloy surfaces after creating a hierarchical (micro/nano) superhydrophobic structure.

    PubMed

    Moazzam, Parisa; Razmjou, Amir; Golabi, Mohsen; Shokri, Dariush; Landarani-Isfahani, Amir

    2016-09-01

    Bacterial adhesion and subsequent biofilm formation on metals such as aluminum (Al) alloys lead to serious issues in biomedical and industrial fields from both an economical and health perspective. Here, we showed that a careful manipulation of Al surface characteristics via a facile two-steps superhydrophobic modification can provide not only biocompatibility and an ability to control protein adsorption and bacterial adhesion, but also address the issue of apparent long-term toxicity of Al-alloys. To find out the roles of surface characteristics, surface modification and protein adsorption on microbial adhesion and biofilm formation, the surfaces were systematically characterized by SEM, EDX, XPS, AFM, FTIR, water contact angle (WCA) goniometry, surface free energy (SFE) measurement, MTT, Bradford, Lowry and microtiter plate assays and also flow-cytometry and potentiostat analyses. Results showed that WCA and SFE changed from 70° to 163° and 36.3 to 0.13 mN m(-1) , respectively. The stable and durable modification led to a substantial reduction in static/dynamic BSA adsorption. The effect of such a treatment on the biofilm formation was analyzed by using three different bacteria of Pseudomonas aeruginosa, Staphylococcus epidermidis, and Staphylococcus aureus. The microtiter plate assay and flow cytometry analysis showed that the modification not only could substantially reduce the bacterial adhesion but this biofouling resistance is independent of bacterium type. An excellent cell viability after exposure of HeLa cells to waters incubated with the modified samples was observed. Finally, the corrosion rate reduced sharply from 856.6 to 0.119 MPY after superhydrophobic modifications, which is an excellent stable corrosion inhibition property. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 104A: 2220-2233, 2016. PMID:27104583

  5. Protein 4.1R regulates cell adhesion, spreading, migration and motility of mouse keratinocytes by modulating surface expression of β1 integrin

    PubMed Central

    Chen, Lixiang; Hughes, Richard A.; Baines, Anthony J.; Conboy, John; Mohandas, Narla; An, Xiuli

    2011-01-01

    Protein 4.1R is a membrane-cytoskeleton adaptor protein that has diverse roles in controlling the cell surface expression and/or function of transmembrane proteins, and in organizing F-actin. 4.1R is expressed in keratinocytes, but its role in these cells has not been explored. Here, we have investigated the role of 4.1R in skin using 4.1R−/− mice. Cell adhesion, spreading, migration and motility were significantly impaired in 4.1R−/− keratinocytes, and 4.1R−/− mice exhibited defective epidermal wound healing. Cultured 4.1R−/− keratinocytes on fibronectin failed to form actin stress fibres and focal adhesions. Furthermore, in the absence of 4.1R, the surface expression, and consequently the activity of β1 integrin were reduced. These data enabled the identification of a functional role for protein 4.1R in keratinocytes by modulating the surface expression of β1 integrin, possibly through a direct association between 4.1R and β1 integrin. PMID:21693581

  6. Protein 4.1R regulates cell adhesion, spreading, migration and motility of mouse keratinocytes by modulating surface expression of beta1 integrin.

    PubMed

    Chen, Lixiang; Hughes, Richard A; Baines, Anthony J; Conboy, John; Mohandas, Narla; An, Xiuli

    2011-07-15

    Protein 4.1R is a membrane-cytoskeleton adaptor protein that has diverse roles in controlling the cell surface expression and/or function of transmembrane proteins, and in organizing F-actin. 4.1R is expressed in keratinocytes, but its role in these cells has not been explored. Here, we have investigated the role of 4.1R in skin using 4.1R(-/-) mice. Cell adhesion, spreading, migration and motility were significantly impaired in 4.1R(-/-) keratinocytes, and 4.1R(-/-) mice exhibited defective epidermal wound healing. Cultured 4.1R(-/-) keratinocytes on fibronectin failed to form actin stress fibres and focal adhesions. Furthermore, in the absence of 4.1R, the surface expression, and consequently the activity of β1 integrin were reduced. These data enabled the identification of a functional role for protein 4.1R in keratinocytes by modulating the surface expression of β1 integrin, possibly through a direct association between 4.1R and β1 integrin. PMID:21693581

  7. Getting from A to B-exploring the activation motifs of the class B adhesion G protein-coupled receptor subfamily G member 4/GPR112.

    PubMed

    Peeters, Miriam C; Mos, Iris; Lenselink, Eelke B; Lucchesi, Martina; IJzerman, Adriaan P; Schwartz, Thue W

    2016-05-01

    The adhesion G protein-coupled receptors [ADGRs/class B2 G protein-coupled receptors (GPCRs)] constitute an ancient family of GPCRs that have recently been demonstrated to play important roles in cellular and developmental processes. Here, we describe a first insight into the structure-function relationship of ADGRs using the family member ADGR subfamily G member 4 (ADGRG4)/GPR112 as a model receptor. In a bioinformatics approach, we compared conserved, functional elements of the well-characterized class A and class B1 secretin-like GPCRs with the ADGRs. We identified several potential equivalent motifs and subjected those to mutational analysis. The importance of the mutated residues was evaluated by examining their effect on the high constitutive activity of the N-terminally truncated ADGRG4/GPR112 in a 1-receptor-1-G protein Saccharomyces cerevisiae screening system and was further confirmed in a transfected mammalian human embryonic kidney 293 cell line. We evaluated the results in light of the crystal structures of the class A adenosine A2A receptor and the class B1 corticotropin-releasing factor receptor 1. ADGRG4 proved to have functionally important motifs resembling class A, class B, and combined elements, but also a unique highly conserved ADGR motif (H3.33). Given the high conservation of these motifs and residues across the adhesion GPCR family, it can be assumed that these are general elements of ADGR function.-Peeters, M. C., Mos, I., Lenselink, E. B., Lucchesi, M., IJzerman, A. P., Schwartz, T. W. Getting from A to B-exploring the activation motifs of the class B adhesion G protein-coupled receptor subfamily G member 4/GPR112. PMID:26823453

  8. RhoGAP68F controls transport of adhesion proteins in Rab4 endosomes to modulate epithelial morphogenesis of Drosophila leg discs

    PubMed Central

    de Madrid, Beatriz Hernandez; Greenberg, Lina; Hatini, Victor

    2015-01-01

    SUMMARY Elongation and invagination of epithelial tissues are fundamental developmental processes that contribute to the morphogenesis of embryonic and adult structures and are dependent on coordinated remodeling of cell-cell contacts. The morphogenesis of Drosophila leg imaginal discs depends on extensive remodeling of cell contacts and thus provides a useful system with which to investigate the underlying mechanisms. The small Rho GTPase regulator RhoGAP68F has been previously implicated in leg morphogenesis. It consists of an N-terminal Sec14 domain and a C-terminal GAP domain. Here we examined the molecular function and role of RhoGAP68F in epithelial remodeling. We find that depletion of RhoGAP68F impairs epithelial remodeling from a pseudostratified to simple, while overexpression of RhoGAP68F causes tears of lateral cell-cell contacts and thus impairs epithelial integrity. We show that the RhoGAP68F protein localizes to Rab4 recycling endosomes and forms a complex with the Rab4 protein. The Sec14 domain is sufficient for localizing to Rab4 endosomes, while the activity of the GAP domain is dispensable. RhoGAP68F, in turn, inhibits the scission and movement of Rab4 endosomes involved in transport the adhesion proteins Fasciclin3 and E-cadherin back to cell-cell contacts. Expression of RhoGAP68F is upregulated during prepupal development suggesting that RhoGAP68F decreases the transport of key adhesion proteins to the cell surface during this developmental stage to decrease the strength of adhesive cell-cell contacts and thereby facilitate epithelial remodeling and leg morphogenesis. PMID:25617722

  9. The GafD protein of the G (F17) fimbrial complex confers adhesiveness of Escherichia coli to laminin.

    PubMed Central

    Saarela, S; Westerlund-Wikström, B; Rhen, M; Korhonen, T K

    1996-01-01

    Escherichia coli IHE11088(pRR-5) expressing the G (F17) fimbria adhered to immobilized laminin as well as to reconstituted basement membranes. No adhesion was seen with the plasmidless strain IHE11088 or with the deletion derivative IHE11088(pHUB110), which expresses the G-fimbrial filament with a defective GafD lectin and lacks N-acetyl-D-glucosamine-specific binding. Adhesion of IHE11088(pRR-5) to laminin and to reconstituted basement membranes was specifically inhibited by N-acetyl-D-glucosamine, and adhesion was abolished after N-glycosidase F treatment of laminin. The results show that the GafD lectin binds to laminin carbohydrate and suggest a novel function for the F17 fimbria in binding to mammalian basement membranes. PMID:8698525

  10. Serum Vascular Adhesion Protein-1 Predicts End-Stage Renal Disease in Patients with Type 2 Diabetes

    PubMed Central

    Nien, Feng-Jung; Wu, Vin-Cent; Jiang, Yi-Der; Chang, Tien-Jyun; Kao, Hsien-Li; Lin, Mao-Shin; Wei, Jung-Nan; Lin, Cheng-Hsin; Shih, Shyang-Rong; Hung, Chi-Sheng; Chuang, Lee-Ming

    2016-01-01

    Background Diabetes is the leading cause of end-stage renal disease (ESRD) worldwide. Vascular adhesion protein-1 (VAP-1) participates in inflammation and catalyzes the deamination of primary amines into aldehydes, hydrogen peroxide, and ammonia, both of which are involved in the pathogenesis of diabetic complications. We have shown that serum VAP-1 is higher in patients with diabetes and in patients with chronic kidney disease (CKD), and can predict cardiovascular mortality in subjects with diabetes. In this study, we investigated if serum VAP-1 can predict ESRD in diabetic subjects. Methods In this prospective cohort study, a total of 604 type 2 diabetic subjects were enrolled between 1996 to 2003 at National Taiwan University Hospital, Taiwan, and were followed for a median of 12.36 years. The development of ESRD was ascertained by linking our database with the nationally comprehensive Taiwan Society Nephrology registry. Serum VAP-1 concentrations at enrollment were measured by time-resolved immunofluorometric assay. Results Subjects with serum VAP-1 in the highest tertile had the highest incidence of ESRD (p<0.001). Every 1-SD increase in serum VAP-1 was associated with a hazard ratio of 1.55 (95%CI 1.12–2.14, p<0.01) for the risk of ESRD, adjusted for smoking, history of cardiovascular disease, body mass index, hypertension, HbA1c, duration of diabetes, total cholesterol, use of statins, ankle-brachial index, estimated GFR, and proteinuria. We developed a risk score comprising serum VAP-1, HbA1c, estimated GFR, and proteinuria, which could predict ESRD with good performance (area under the ROC curve = 0.9406, 95%CI 0.8871–0.9941, sensitivity = 77.3%, and specificity = 92.8%). We also developed an algorithm based on the stage of CKD and a risk score including serum VAP-1, which can stratify these subjects into 3 categories with an ESRD risk of 0.101%/year, 0.131%/year, and 2.427%/year, respectively. Conclusions In conclusion, serum VAP-1 can predict ESRD

  11. Isolation of surface (S) layer protein carrying Lactobacillus species from porcine intestine and faeces and characterization of their adhesion properties to different host tissues.

    PubMed

    Jakava-Viljanen, Miia; Palva, Airi

    2007-10-01

    Surface-layer proteins (Slps) of lactobacilli have been shown to confer tissue adherence. This study aimed to isolate and identify Slps carrying Lactobacillus species from the porcine intestine and faeces and to characterize these S-layer-expressing strains for their ability to adhere to the pig and human intestinal cells and to extracellular matrix (ECM) proteins. In total 99 strains, putatively belonging to the genus Lactobacillus, were isolated as pure cultures. SDS-PAGE and a gene probe specific for the Lactobacillus brevis ATCC 8287 S-layer protein gene (slpA) were used to screen the presence of strains possessing putative Slps. Eight of the 99 pure cultures exhibited Slps according to the SDS-PAGE analyses. In these strains the presence of genes encoding Slps was confirmed by PCR and partial sequencing. Only one isolate of the 99 strains gave a positive hybridisation signal with the L. brevis slpA probe but did not appear to produce S-layer protein. Their taxonomic identification, based on phenotyping and the 16S rRNA sequences, revealed that the eight S-layer protein-producing strains were closely related to Lactobacillus amylovorus, Lactobacillus sobrius and Lactobacillus crispatus. The strain with the slpA positive hybridisation result was identified as Lactobacillus mucosae. The SDS-extractable protein profile, the size of the putative S-layer protein and binding capability of the strains varied greatly, even among the isolates belonging to the same Lactobacillus cluster. Removal of the intact Slps from the bacterial surface by extraction with guanidine hydrochloride reduced the adhesion of some strains to fibronectin and laminin, whereas, the adhesiveness to laminin increased with some strains. PMID:17544232

  12. Proteomic dataset of the sea urchin Paracentrotus lividus adhesive organs and secreted adhesive.

    PubMed

    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. PMID:27182547

  13. Proteomic dataset of the sea urchin Paracentrotus lividus adhesive organs and secreted adhesive

    PubMed Central

    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

    2016-01-01

    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. PMID:27182547

  14. Abdominal Adhesions

    MedlinePlus

    ... Adhesions 1 Ward BC, Panitch A. Abdominal adhesions: current and novel therapies. Journal of Surgical Research. 2011;165(1):91–111. Seek Help for ... and how to participate, visit the NIH Clinical Research Trials and You website ... Foundation for Functional Gastrointestinal Disorders 700 West Virginia ...

  15. Leucocyte cellular adhesion molecules.

    PubMed

    Yong, K; Khwaja, A

    1990-12-01

    Leucocytes express adhesion promoting receptors which mediate cell-cell and cell-matrix interactions. These adhesive interactions are crucial to the regulation of haemopoiesis and thymocyte maturation, the direction and control of leucocyte traffic and migration through tissues, and in the development of immune and non-immune inflammatory responses. Several families of adhesion receptors have been identified (Table). The leucocyte integrin family comprises 3 alpha beta heterodimeric membrane glycoproteins which share a common beta subunit, designated CD18. The alpha subunits of each of the 3 members, lymphocyte function associated antigen-1 (LFA-1), macrophage antigen-1 (Mac-1) and p150,95 are designated CD11a, b and c respectively. These adhesion molecules play a critical part in the immune and inflammatory responses of leucocytes. The leucocyte integrin family is, in turn, part of the integrin superfamily, members of which are evolutionally, structurally and functionally related. Another Integrin subfamily found on leucocytes is the VLA group, so-called because the 'very late activation antigens' VLA-1 and VLA-2 were originally found to appear late in T-cell activation. Members of this family function mainly as extracellular matrix adhesion receptors and are found both on haemopoietic and non-haemopoietic cells. They play a part in diverse cellular functions including tissue organisation, lymphocyte recirculation and T-cell immune responses. A third integrin subfamily, the cytoadhesins, are receptors on platelets and endothelial cells which bind extracellular matrix proteins. A second family of adhesion receptors is the immunoglobulin superfamily, members of which include CD2, LFA-3 and ICAM-1, which participate in T-cell adhesive interactions, and the antigen-specific receptors of T and B cells, CD4, CD8 and the MHC Class I and II molecules. A recently recognised family of adhesion receptors is the selectins, characterised by a common lectin domain. Leucocyte

  16. Streptococcus suis Type 2 SSU0587 Protein is a Beta-Galactosidase That Contributes to Bacterial Adhesion but Not to Virulence in Mice

    PubMed Central

    TANG, Yulong; ZHANG, Xiaoyan; YIN, Yulong; HARDWIDGE, Philip R.; FANG, Weihuan

    2014-01-01

    ABSTRACT Bacterial surface proteins play key roles in virulence and often contribute to bacterial adhesion and invasion. We discovered that the Streptococcus suis type 2 (SS2) gene SSU0587 encodes a protein of 1,491 amino acids that possesses β-galactosidase activity. The surface association of the protein was dependent upon sortase activity. Deleting SSU0587 from clinical SS2 isolate JX081101 caused a loss of both β-galactosidase activity and adherence to microvascular endothelial cells. Deleting SSU0587 had no measurable impact on either invasion of microvascular endothelial cells or on virulence in a murine infection model, although the concentration of JX081101ΔSSU0587 was reduced in the brains of infected mice, as compared with the pathogen loads of the wild-type strain. PMID:24670993

  17. Streptococcus suis type 2 SSU0587 protein is a beta-galactosidase that contributes to bacterial adhesion but not to virulence in mice.

    PubMed

    Tang, Yulong; Zhang, Xiaoyan; Yin, Yulong; Hardwidge, Philip R; Fang, Weihuan

    2014-07-01

    Bacterial surface proteins play key roles in virulence and often contribute to bacterial adhesion and invasion. We discovered that the Streptococcus suis type 2 (SS2) gene SSU0587 encodes a protein of 1,491 amino acids that possesses β-galactosidase activity. The surface association of the protein was dependent upon sortase activity. Deleting SSU0587 from clinical SS2 isolate JX081101 caused a loss of both β-galactosidase activity and adherence to microvascular endothelial cells. Deleting SSU0587 had no measurable impact on either invasion of microvascular endothelial cells or on virulence in a murine infection model, although the concentration of JX081101ΔSSU0587 was reduced in the brains of infected mice, as compared with the pathogen loads of the wild-type strain. PMID:24670993

  18. In vivo adhesion of malignant B cells to bone marrow microvasculature is regulated by α4β1 cytoplasmic-binding proteins.

    PubMed

    Martínez-Moreno, M; Leiva, M; Aguilera-Montilla, N; Sevilla-Movilla, S; Isern de Val, S; Arellano-Sánchez, N; Gutiérrez, N C; Maldonado, R; Martínez-López, J; Buño, I; García-Marco, J A; Sánchez-Mateos, P; Hidalgo, A; García-Pardo, A; Teixidó, J

    2016-04-01

    Multiple myeloma (MM) and chronic lymphocytic leukemia (CLL) cells must attach to the bone marrow (BM) microvasculature before lodging in the BM microenvironment. Using intravital microscopy (IVM) of the BM calvariae we demonstrate that the α4β1 integrin is required for MM and CLL cell firm arrest onto the BM microvasculature, while endothelial P-selectin and E-selectin mediate cell rolling. Talin, kindlin-3 and ICAP-1 are β1-integrin-binding partners that regulate β1-mediated cell adhesion. We show that talin and kindlin-3 cooperatively stimulate high affinity and strength of α4β1-dependent MM and CLL cell attachment, whereas ICAP-1 negatively regulates this adhesion. A functional connection between talin/kindlin-3 and Rac1 was found to be required for MM cell attachment mediated by α4β1. Importantly, IVM analyses with talin- and kindlin-3-silenced MM cells indicate that these proteins are needed for cell arrest on the BM microvasculature. Instead, MM cell arrest is repressed by ICAP-1. Moreover, MM cells silenced for talin and kindlin-3, and cultured on α4β1 ligands showed higher susceptibility to bortezomib-mediated cell apoptosis. Our results highlight the requirement of α4β1 and selectins for the in vivo attachment of MM and CLL cells to the BM microvasculature, and indicate that talin, kindlin-3 and ICAP-1 differentially control physiological adhesion by regulating α4β1 activity. PMID:26658839

  19. UV-induced graft copolymerization of monoacrylate-poly(ethylene glycol) onto poly(3-hydroxyoctanoate) to reduce protein adsorption and platelet adhesion.

    PubMed

    Kim, Hyung Woo; Chung, Chung Wook; Rhee, Young Ha

    2005-03-01

    Homogeneous solutions of poly(3-hydroxyoctanoate) (PHO) and the monoacrylate-poly(ethylene glycol) (PEGMA) monomer in chloroform were irradiated with UV light to obtain PEGMA-grafted PHO (PEGMA-g-PHO) copolymers. Variables affecting the degree of grafting (DG), such as the time of UV irradiation and the concentrations of the PEGMA monomer and initiator, were investigated. The PEGMA-g-PHO copolymers were characterized by measuring the water contact angle, molecular weight, thermal transition temperatures and mechanical properties, as well as by nuclear magnetic resonance spectroscopy. The results from all of these measurements indicate that PEGMA groups were present on the PHO polymer. The protein adsorption and platelet adhesion on the PEGMA-g-PHO surfaces were examined using poly(L-lactide) (PLLA) surfaces as the control. The proteins and platelets had a significantly lower tendency to adhere to the PEGMA-g-PHO copolymers than to PLLA. The graft copolymer with a high DG of PEGMA was very effective in reducing the protein adsorption and platelet adhesion and did not activate the platelets. The results obtained in this study suggest that PEGMA-g-PHO copolymers have the potential to be used as blood-contacting devices in a broad range of biomedical applications. PMID:15769515

  20. Mapping the laminin-binding and adhesive domain of the cell surface-associated Hlp/LBP protein from Mycobacterium leprae.

    PubMed

    Soares de Lima, Cristiana; Zulianello, Laurence; Marques, Maria Angela de Melo; Kim, Heejin; Portugal, Michelle Iespa; Antunes, Sérgio Luiz; Menozzi, Franco Dante; Ottenhoff, Tom Henricus Maria; Brennan, Patrick Joseph; Pessolani, Maria Cristina Vidal

    2005-07-01

    Binding of Mycobacterium leprae to and invasion of Schwann cells (SC) represent a crucial step that initiates nerve damage in leprosy. We and others have described that M. leprae colonization of the peripheral nerve system may be mediated in part by a surface-exposed histone-like protein (Hlp), characterized as a laminin-binding protein (LBP). Hlp/LBP has also been shown to play a role in the binding of mycobacteria to alveolar epithelial cells and macrophages. In the present study we report that M. leprae expresses Hlp/LBP protein during the course of human infection. Additionally, we analyzed the interaction of Hlp/LBP with the extracellular matrix and host cell surface. We show that Hlp/LBP, besides laminin, also binds heparin and heparan sulfate. Testing truncated recombinant Hlp molecules corresponding to the N-terminal (rHlp-N) and the C-terminal (rHlp-C) domains of the protein, we established that interaction of Hlp/LBP with laminin-2 and heparin is mainly mediated by the C-terminal domain of the protein. Moreover, the same domain was found to be involved in Hlp/LBP-mediating bacterial binding to human SC. Finally, evidence is shown suggesting that M. leprae produces a post-translationally modified Hlp/LBP containing methyllysine residues. Methylation of the lysine residues, however, seems not to affect the adhesive properties of Hlp/LBP. Taken together, our observations reinforce the involvement of Hlp/LBP as an adhesin in mycobacterial infections and define its highly positive C-terminal region as the major adhesive domain of this protein. PMID:15919224

  1. Effects of the knockdown of death-associated protein 3 expression on cell adhesion, growth and migration in breast cancer cells.

    PubMed

    Wazir, Umar; Sanders, Andrew J; Wazir, Ahmad M A; Ye, Lin; Jiang, Wen G; Ster, Irina C; Sharma, Anup K; Mokbel, Kefah

    2015-05-01

    The death-associated protein 3 (DAP3) is a highly conserved phosphoprotein involved in the regulation of autophagy. A previous clinical study by our group suggested an association between low DAP3 expression and clinicopathological parameters of human breast cancer. In the present study, we intended to determine the role of DAP3 in cancer cell behaviour in the context of human breast cancer. We developed knockdown sub-lines of MCF7 and MDA-MB-231, and performed growth, adhesion, invasion assays and electric cell-substrate impedance sensing (ECIS) studies of post-wound migration of the cells. In addition, we studied the mRNA expression of caspase 8 and 9, death ligand signal enhancer (DELE), IFN-β promoter stimulator 1 (IPS1), cyclin D1 and p21 in the control and knockdown sub-lines. The knockdown sub-lines of MCF7 and MDA-MB-231 had significantly increased adhesion and decreased growth when compared to the controls. Furthermore, invasion and migration were significantly increased in the MDA-MB-231DAP3kd cells vs. the controls. The expression of caspase 9 and IPS1, known components of the apoptosis pathway, were significantly reduced in the MCF7DAP3kd cells (p=0.05 and p=0.003, respectively). We conclude that DAP3 silencing contributes to breast carcinogenesis by increasing cell adhesion, migration and invasion. It is possible that this may be due to the activity of focal adhesion kinase further downstream of the anoikis pathway. Further research in this direction would be beneficial in increasing our understanding of the mechanisms underlying human breast cancer. PMID:25738636

  2. Planar Cell Polarity (PCP) Protein Vangl2 Regulates Ectoplasmic Specialization Dynamics via Its Effects on Actin Microfilaments in the Testes of Male Rats.

    PubMed

    Chen, Haiqi; Mruk, Dolores D; Lee, Will M; Cheng, C Yan

    2016-05-01

    Planar cell polarity (PCP) proteins confer polarization of a field of cells (eg, elongating/elongated spermatids) within the plane of an epithelium such as the seminiferous epithelium of the tubule during spermatogenesis. In adult rat testes, Sertoli and germ cells were found to express PCP core proteins (eg, Van Gogh-like 2 [Vangl2]), effectors, ligands, and signaling proteins. Vangl2 expressed predominantly by Sertoli cells was localized at the testis-specific, actin-rich ectoplasmic specialization (ES) at the Sertoli-spermatid interface in the adluminal compartment and also Sertoli-Sertoli interface at the blood-testis barrier (BTB) and structurally interacted with actin, N-cadherin, and another PCP/polarity protein Scribble. Vangl2 knockdown (KD) by RNA interference in Sertoli cells cultured in vitro with an established tight junction-permeability barrier led to BTB tightening, whereas its overexpression using a full-length cDNA construct perturbed the barrier function. These changes were mediated through an alteration on the organization actin microfilaments at the ES in Sertoli cells, involving actin-regulatory proteins, epidermal growth factor receptor pathway substrate 8, actin-related protein 3, and Scribble, which in turn affected the function of adhesion protein complexes at the ES during the epithelial cycle of spermatogenesis. Using Polyplus in vivo-jetPEI reagent as a transfection medium to silence Vangl2 in the testis in vivo by RNA interference with high efficacy, Vangl2 KD led to changes in F-actin organization at the ES in the epithelium, impeding spermatid and phagosome transport and spermatid polarity, meiosis, and BTB dynamics. For instance, step 19 spermatids remained embedded in the epithelium alongside with step 9 and 10 spermatids in stages IX-X tubules. In summary, the PCP protein Vangl2 is an ES regulator through its effects on actin microfilaments in the testis. PMID:26990065

  3. Ligand-specific, transient interaction between integrins and calreticulin during cell adhesion to extracellular matrix proteins is dependent upon phosphorylation/dephosphorylation events.

    PubMed Central

    Coppolino, M G; Dedhar, S

    1999-01-01

    As transmembrane heterodimers, integrins bind to both extracellular ligands and intracellular proteins. We are currently investigating the interaction between integrins and the intracellular protein calreticulin. A prostatic carcinoma cell line (PC-3) was used to demonstrate that calreticulin can be found in the alpha3 immunoprecipitates of cells plated on collagen type IV, but not when plated on vitronectin. Conversely, alphav immunoprecipitates contained calreticulin only when cells were plated on vitronectin, i. e. not when plated on collagen IV. The interactions between these integrins and calreticulin were independent of actin cytoskeleton assembly and were transient, being maximal approx. 10-30 min after the cells came into contact with the substrates prior to complete cell spreading and formation of firm adhesive contacts. We demonstrate that okadaic acid, an inhibitor of intracellular serine/threonine protein phosphatases, inhibited the alpha3beta1-mediated adhesion of PC-3 cells to collagen IV and the alpha2beta1-mediated attachment of Jurkat cells to collagen I. This inhibition by okadaic acid was accompanied by inhibition of the ligand-specific interaction of calreticulin with the respective integrins in the two cell types. Additionally, we found that pharmacological inhibition of mitogen-activated protein kinase kinase (MEK) resulted in prolongation of the calreticulin-integrin interaction, and enhancement of PC-3 cell attachment to collagen IV. We conclude that calreticulin interacts transiently with integrins during cell attachment and spreading. This interaction depends on receptor occupation, is ligand-specific, and can be modulated by protein phosphatase and MEK activity. PMID:10229657

  4. Orphan G protein-coupled receptor GPR56 plays a role in cell transformation and tumorigenesis involving the cell adhesion pathway.

    PubMed

    Ke, Ning; Sundaram, Roshni; Liu, Guohong; Chionis, John; Fan, Wufang; Rogers, Cheryl; Awad, Tarif; Grifman, Mirta; Yu, Dehua; Wong-Staal, Flossie; Li, Qi-Xiang

    2007-06-01

    GPR56 is an orphan G protein - coupled receptor, mutations of which have recently been associated with bilateral frontoparietal polymicrogyria, a rare neurologic disease that has implications in brain development. However, no phenotype beyond central nervous system has yet been described for the GPR56-null mutations despite abundant GPR56 expression in many non - central nervous system adult tissues. In the present study, we show that higher GPR56 expression is correlated with the cellular transformation phenotypes of several cancer tissues compared with their normal counterparts, implying a potential oncogenic function. RNA interference-mediated GPR56 silencing results in apoptosis induction and reduced anchorage-independent growth of cancer cells via increased anoikis, whereas cDNA overexpression resulted in increased foci formation in mouse fibroblast NIH3T3 cell line. When GPR56 silencing was induced in vivo in several xenograft tumor models, significant tumor responses (including regression) were observed, suggesting the potential of targeting GPR56 in the development of tumor therapies. The expression profiling of GPR56-silenced A2058 melanoma cell line revealed several genes whose expression was affected by GPR56 silencing, particularly those in the integrin-mediated signaling and cell adhesion pathways. The potential role of GPR56 in cancer cell adhesion was further confirmed by the observation that GPR56 silencing also reduced cell adhesion to the extracellular matrix, which is consistent with the observed increase in anoikis and reduction in anchorage-independent growth phenotypes. The oncogenic potential and apparent absence of physiologic defects in adult human tissues lacking GPR56, as well as the targetable nature of G protein - coupled receptor by small molecule or antibody, make GPR56 an attractive drug target for the development of cancer therapies. PMID:17575113

  5. The conserved dual phosphorylation sites of the Candida albicans Hog1 protein are crucial for white-opaque switching, mating, and pheromone-stimulated cell adhesion.

    PubMed

    Chang, Wen-Han; Liang, Shen-Huan; Deng, Fu-Sheng; Lin, Ching-Hsuan

    2016-08-01

    Candida albicans is an opportunistic human pathogen capable of causing life-threatening infections in immunocompromised patients. C. albicans has a unique morphological transition between white and opaque phases. These two cells differ in virulence, mating capability, biofilm formation, and host-cell interaction. Previous studies revealed that deletion of the SSK2, PBS2, or HOG1 gene resulted in 100% opaque cell formation and suppressed the mating response. Thr-174 and Tyr-176 of the Hog1 protein are important phosphoacceptors and can be activated in response to stimuli. In this study, we first demonstrated the importance of two conserved phosphorylation sites in white-opaque switching, mating, and pheromone-stimulated cell adhesion. Six Hog1 point-mutated strains were generated, including nonphosphorylated strains (Hog1(T174A), Hog1(Y176F), and Hog1(T174A,Y176F)) and negatively charged phosphorylated strains (Hog1(T174D), Hog1(Y176D), and Hog1(T174D,Y176D)). Point mutation on Thr-174, Tyr-176 or in combination with the Hog1 protein in C. albicans MTL homozygous strains stimulated opaque cell formation at a frequency of 100%. Furthermore, mating projections of point-mutated strains were significantly shorter and their mating efficiencies and pheromone-stimulated cell adhesive numbers were lower than those of the wild-type. By investigating the effects of Hog1 phosphorylation in ssk1Δ and sln1Δ, we also demonstrate that the phosphorylation intensity of Hog1p is directly involved in the white-opaque switching. Taken together, the results of our study demonstrate that dual phosphorylation sites of C. albicans are crucial for white-opaque transition, sexual mating, and pheromone-induced cell adhesion. PMID:27118797

  6. The membrane-cytoskeletal protein 4.1N is involved in the process of cell adhesion, migration and invasion of breast cancer cells.

    PubMed

    Ji, Zhenyu; Shi, Xiaofang; Liu, Xin; Shi, Yu; Zhou, Qingqing; Liu, Xilong; Li, Li; Ji, Xiang; Gao, Yanfeng; Qi, Yuanming; Kang, Qiaozhen

    2012-10-01

    Protein 4.1N belongs to the protein 4.1 superfamily that links transmembrane proteins to the actin cytoskeleton. Recent evidence has shown that protein 4.1 is important in tumor suppression. However, the functions of 4.1N in the metastasis of breast cancer are largely unknown. In the present study, MCF-7, T-47D and MDA-MB-231 breast cancer cell lines with various metastatic abilities were employed. Protein 4.1N was found to be expressed in poorly metastatic MCF-7 and middle metastatic T-47D cell lines, and was predominantly associated with cell-cell junctions. However, no 4.1N expression was detected in the highly metastatic MDA-MB-231 cells. Moreover, re-expression of 4.1N in MDA-MB-231 cells inhibited cell adhesion, migration and invasion. The results suggest that protein 4.1N is a negative regulator of cell metastasis in breast cancer. PMID:23170136

  7. The membrane-cytoskeletal protein 4.1N is involved in the process of cell adhesion, migration and invasion of breast cancer cells

    PubMed Central

    JI, ZHENYU; SHI, XIAOFANG; LIU, XIN; SHI, YU; ZHOU, QINGQING; LIU, XILONG; LI, LI; JI, XIANG; GAO, YANFENG; QI, YUANMING; KANG, QIAOZHEN

    2012-01-01

    Protein 4.1N belongs to the protein 4.1 superfamily that links transmembrane proteins to the actin cytoskeleton. Recent evidence has shown that protein 4.1 is important in tumor suppression. However, the functions of 4.1N in the metastasis of breast cancer are largely unknown. In the present study, MCF-7, T-47D and MDA-MB-231 breast cancer cell lines with various metastatic abilities were employed. Protein 4.1N was found to be expressed in poorly metastatic MCF-7 and middle metastatic T-47D cell lines, and was predominantly associated with cell-cell junctions. However, no 4.1N expression was detected in the highly metastatic MDA-MB-231 cells. Moreover, re-expression of 4.1N in MDA-MB-231 cells inhibited cell adhesion, migration and invasion. The results suggest that protein 4.1N is a negative regulator of cell metastasis in breast cancer. PMID:23170136

  8. The Src Homology 3 Domain Is Required for Junctional Adhesion Molecule Binding to the Third PDZ Domain of the Scaffolding Protein ZO-1

    SciTech Connect

    Nomme, Julian; Fanning, Alan S.; Caffrey, Michael; Lye, Ming F.; Anderson, James M.; Lavie, Arnon

    2012-01-20

    Tight junctions are cell-cell contacts that regulate the paracellular flux of solutes and prevent pathogen entry across cell layers. The assembly and permeability of this barrier are dependent on the zonula occludens (ZO) membrane-associated guanylate kinase (MAGUK) proteins ZO-1, -2, and -3. MAGUK proteins are characterized by a core motif of protein-binding domains that include a PDZ domain, a Src homology 3 (SH3) domain, and a region of homology to guanylate kinase (GUK); the structure of this core motif has never been determined for any MAGUK. To better understand how ZO proteins organize the assembly of protein complexes we have crystallized the entire PDZ3-SH3-GUK core motif of ZO-1. We have also crystallized this core motif in complex with the cytoplasmic tail of the ZO-1 PDZ3 ligand, junctional adhesion molecule A (JAM-A) to determine how the activity of different domains is coordinated. Our study shows a new feature for PDZ class II ligand binding that implicates the two highly conserved Phe{sup -2} and Ser{sup -3} residues of JAM. Our x-ray structures and NMR experiments also show for the first time a role for adjacent domains in the binding of ligands to PDZ domains in the MAGUK proteins family.

  9. α2-Macroglobulin Can Crosslink Multiple Plasmodium falciparum Erythrocyte Membrane Protein 1 (PfEMP1) Molecules and May Facilitate Adhesion of Parasitized Erythrocytes

    PubMed Central

    Stevenson, Liz; Laursen, Erik; Cowan, Graeme J.; Bandoh, Betty; Barfod, Lea; Cavanagh, David R.; Andersen, Gregers R.; Hviid, Lars

    2015-01-01

    Rosetting, the adhesion of Plasmodium falciparum-infected erythrocytes to uninfected erythrocytes, involves clonal variants of the parasite protein P. falciparum erythrocyte membrane protein 1 (PfEMP1) and soluble serum factors. While rosetting is a well-known phenotypic marker of parasites associated with severe malaria, the reason for this association remains unclear, as do the molecular details of the interaction between the infected erythrocyte (IE) and the adhering erythrocytes. Here, we identify for the first time a single serum factor, the abundant serum protease inhibitor α2-macroglobulin (α2M), which is both required and sufficient for rosetting mediated by the PfEMP1 protein HB3VAR06 and some other rosette-mediating PfEMP1 proteins. We map the α2M binding site to the C terminal end of HB3VAR06, and demonstrate that α2M can bind at least four HB3VAR06 proteins, plausibly augmenting their combined avidity for host receptors. IgM has previously been identified as a rosette-facilitating soluble factor that acts in a similar way, but it cannot induce rosetting on its own. This is in contrast to α2M and probably due to the more limited cross-linking potential of IgM. Nevertheless, we show that IgM works synergistically with α2M and markedly lowers the concentration of α2M required for rosetting. Finally, HB3VAR06+ IEs share the capacity to bind α2M with subsets of genotypically distinct P. falciparum isolates forming rosettes in vitro and of patient parasite isolates ex vivo. Together, our results are evidence that P. falciparum parasites exploit α2M (and IgM) to expand the repertoire of host receptors available for PfEMP1-mediated IE adhesion, such as the erythrocyte carbohydrate moieties that lead to formation of rosettes. It is likely that this mechanism also affects IE adhesion to receptors on vascular endothelium. The study opens opportunities for broad-ranging immunological interventions targeting the α2M—(and IgM-) binding domains of PfEMP1

  10. Outer Membrane Protein U (OmpU) Mediates Adhesion of Vibrio mimicus to Host Cells via Two Novel N-Terminal Motifs

    PubMed Central

    Liu, Xueqin; Gao, Huihui; Xiao, Nin; Liu, Yan; Li, Jinnian; Li, Lin

    2015-01-01

    Vibrio mimicus (V.mimicus) is a causative agent of ascites disease in aquatic animals. Our previous studies have demonstrated that the outer membrane protein U (OmpU) from V.mimicus is an immunoprotective antigen with six immunodominant linear B-cell epitopes. Although the N-terminus of OmpU contains potential binding motifs, it remained unclear whether OmpU possesses adhesion function. Here, the adhesive capacity of recombinant OmpU and V.mimicus to epithelioma papulosum cyprinid (EPC) cells was determined by immunofluorescence and adherence assay. The results showed that after co-incubated with rOmpU, an obvious visible green fluorescence could be observed on the EPC cell surface and the nuclei exhibited blue fluorescence; while the control cell surface did not show any signal, only nuclei exhibited blue fluorescence. The average number of wild-type strain adhered to each cell was 32.3 ± 4.5. The average adhesion number of OmpU gene deletion mutant was significantly reduced to 10.8 ± 0.5 (P < 0.01) and restored to 31.3 ± 2.8 by complement strain (P >0.05). Pretreatment of cells with rOmpU reduced the average adhesion number of wild-type strain to 9.7 ± 2.9 (P < 0.01). Likewise, binding was significantly decreased to 8.8 ± 3.2 (P < 0.01) due to blocking role of OmpU antibodies. To determine binding motifs of OmpU, six immunodominant B-cell epitope peptides labeled with FITC were employed in flow cytometry-based binding assay. Two FITC-labeled epitope peptides (aa90-101 and aa173-192) showed strong binding to EPC cells (the fluorescence positive cell rate was 99 ± 0.6% and 98 ± 0.3%, respectively), which could be specifically competed by excess corresponding unlabeled peptides, whereas the remaining four showed a low level of background binding. This is the first demonstration that OmpU possesses adhesion function and its N terminal 90–101 and 173–192 amino acid regions are critical sites for cell surface binding. PMID:25742659

  11. Molecular modelling and experimental studies of mutation and cell-adhesion sites in the fibronectin type III and whey acidic protein domains of human anosmin-1.

    PubMed Central

    Robertson, A; MacColl, G S; Nash, J A; Boehm, M K; Perkins, S J; Bouloux, P M

    2001-01-01

    Anosmin-1, the gene product of the KAL gene, is implicated in the pathogenesis of X-linked Kallmann's syndrome. Anosmin-1 protein expression is restricted to the basement membrane and interstitial matrix of tissues affected in this syndrome during development. The anosmin-1 sequence indicates an N-terminal cysteine-rich domain, a whey acidic protein (WAP) domain, four fibronectin type III (FnIII) domains and a C-terminal histidine-rich region, and shows similarity with cell-adhesion molecules, such as neural cell-adhesion molecule, TAG-1 and L1. We investigated the structural and functional significance of three loss-of-function missense mutations of anosmin-1 using comparative modelling of the four FnIII and the WAP domains based on known NMR and crystal structures. Three missense mutation-encoded amino acid substitutions, N267K, E514K and F517L, were mapped to structurally defined positions on the GFCC' beta-sheet face of the first and third FnIII domains. Electrostatic maps demonstrated large basic surfaces containing clusters of conserved predicted heparan sulphate-binding residues adjacent to these mutation sites. To examine these modelling results anosmin-1 was expressed in insect cells. The incorporation of the three mutations into recombinant anosmin-1 had no effect on its secretion. The removal of two dibasic motifs that may constitute potential physiological cleavage sites for anosmin-1 had no effect on cleavage. Peptides based on the anosmin-1 sequences R254--K285 and P504--K527 were then synthesized in order to assess the effect of the three mutations on cellular adhesion, using cell lines that represented potential functional targets of anosmin-1. Peptides (10 microg/ml) incorporating the N267K and E514K substitutions promoted enhanced adhesion to 13.S.1.24 rat olfactory epithelial cells and canine MDCK1 kidney epithelial cells (P<0.01) compared with the wild-type peptides. This result was attributed to the introduction of a lysine residue adjacent to

  12. Regulation of promyogenic signal transduction by cell-cell contact and adhesion

    SciTech Connect

    Krauss, Robert S.

    2010-11-01

    Skeletal myoblast differentiation involves acquisition of the muscle-specific transcriptional program and morphological changes, including fusion into multinucleated myofibers. Differentiation is regulated by extracellular signaling cues, including cell-cell contact and adhesion. Cadherin and Ig adhesion receptors have been implicated in distinct but overlapping stages of myogenesis. N-cadherin signals through the Ig receptor Cdo to activate p38 MAP kinase, while the Ig receptor neogenin signals to activate FAK; both processes promote muscle-specific gene expression and myoblast fusion. M-cadherin activates Rac1 to enhance fusion. Specific Ig receptors (Kirre and Sns) are essential for myoblast fusion in Drosophila, also signaling through Rac, and vertebrate orthologs of Kirre and Sns have partially conserved function. Mice lacking specific cytoplasmic signaling factors activated by multiple receptors (e.g., Rac1) have strong muscle phenotypes in vivo. In contrast, mice lacking individual adhesion receptors that lie upstream of these factors have modest phenotypes. Redundancy among receptors may account for this. Many of the mammalian Ig receptors and cadherins associate with each other, and multivalent interactions within these complexes may require removal of multiple components to reveal dramatic defects in vivo. Nevertheless, it is possible that the murine adhesion receptors rate-limiting in vivo have not yet been identified or fully assessed.

  13. Regulation of promyogenic signal transduction by cell-cell contact and adhesion

    PubMed Central

    Krauss, Robert S.

    2010-01-01

    Skeletal myoblast differentiation involves acquisition of the muscle-specific transcriptional program and morphological changes, including fusion into multinucleated myofibers. Differentiation is regulated by extracellular signaling cues, including cell-cell contact and adhesion. Cadherin and Ig adhesion receptors have been implicated in distinct but overlapping stages of myogenesis. N-cadherin signals through the Ig receptor Cdo to activate p38 MAP kinase, while the Ig receptor neogenin signals to activate FAK; both processes promote muscle-specific gene expression and myoblast fusion. M-cadherin activates Rac1 to enhance fusion. Specific Ig receptors (Kirre, Sns) are essential for myoblast fusion in Drosophila, also signaling through Rac, and vertebrate orthologs of Kirre and Sns have partially conserved function. Mice lacking specific cytoplasmic signaling factors activated by multiple receptors (e.g., Rac1) have strong muscle phenotypes in vivo. In contrast, mice lacking individual adhesion receptors that lie upstream of these factors have modest phenotypes. Redundancy among receptors may account for this. Many of the mammalian Ig receptors and cadherins associate with each other, and multivalent interactions within these complexes may require removal of multiple components to reveal dramatic defects in vivo. Nevertheless, it is possible that the murine adhesion receptors rate-limiting in vivo have not yet been identified or fully assessed. PMID:20471976

  14. CPNA-1, a copine domain protein, is located at integrin adhesion sites and is required for myofilament stability in Caenorhabditis elegans.

    PubMed

    Warner, Adam; Xiong, Ge; Qadota, Hiroshi; Rogalski, Teresa; Vogl, A Wayne; Moerman, Donald G; Benian, Guy M

    2013-03-01

    We identify cpna-1 (F31D5.3) as a novel essential muscle gene in the nematode Caenorhabditis elegans. Antibodies specific to copine domain protein atypical-1 (CPNA-1), as well as a yellow fluorescent protein translational fusion, are localized to integrin attachment sites (M-lines and dense bodies) in the body-wall muscle of C. elegans. CPNA-1 contains an N-terminal predicted transmembrane domain and a C-terminal copine domain and binds to the M-line/dense body protein PAT-6 (actopaxin) and the M-line proteins UNC-89 (obscurin), LIM-9 (FHL), SCPL-1 (SCP), and UNC-96. Proper CPNA-1 localization is dependent upon PAT-6 in embryonic and adult muscle. Nematodes lacking cpna-1 arrest elongation at the twofold stage of embryogenesis and display disruption of the myofilament lattice. The thick-filament component myosin heavy chain MYO-3 and the M-line component UNC-89 are initially localized properly in cpna-1-null embryos. However, in these embryos, when contraction begins, MYO-3 and UNC-89 become mislocalized into large foci and animals die. We propose that CPNA-1 acts as a linker between an integrin-associated protein, PAT-6, and membrane-distal components of integrin adhesion complexes in the muscle of C. elegans. PMID:23283987

  15. Effect of the knockdown of death-associated protein 1 expression on cell adhesion, growth and migration in breast cancer cells.

    PubMed

    Wazir, Umar; Sanders, Andrew J; Wazir, Ali; Baig, Ruqia Mehmood; Jiang, Wen G; Ster, Irina C; Sharma, Anup K; Mokbel, Kefah

    2015-03-01

    Death-associated protein 1 (DAP1) is a highly conserved phosphoprotein involved in the regulation of autophagy. A previous clinical study by our group suggested an association between low DAP1 expression and clinicopathological parameters of human breast cancer. In the present study, we aimed to determine the role of DAP1 in cancer cell behaviour in the context of human breast cancer. We developed knockdown sublines of MCF7 and MDA-MB‑231, and performed growth, adhesion and invasion assays and electric cell-substrate impedance sensing (ECIS) studies of the post-wound migration of cells. In addition, we studied the mRNA expression of caspase 8 and 9, DELE, IPS1, cyclin D1 and p21 in the control and knockdown sublines. Knockdown was associated with increased adhesion and migration, significantly so in the MDA-MB-231DAP1kd cell subline (p=0.029 and p=0.001, respectively). Growth in MCF7 cells showed a significant suppression on day 3 (p=0.029), followed by an increase in growth matching the controls on day 5. While no change in the apoptotic response to serum starvation could be attributed to DAP1 knockdown, the expression of known components of the apoptosis pathway (caspase 8) and cell cycle (p21) was significantly reduced in the MCF7DAP1kd cell subline (p≤0.05), while in MDA-MB-231DAP1kd the expression of a pro-apoptotic molecule, IPS1, was suppressed (p≤0.05). DAP1 may have an important role in cell adhesion, migration and growth in the context of breast cancer and has significant associations with the apoptosis pathway. Furthermore, we believe that delayed increase in growth observed in the MCF7DAP1kd cell subline may indicate activation of a strongly pro-oncogenic pathway downstream of DAP1. PMID:25530065

  16. A 130-kDa Protein 4.1B Regulates Cell Adhesion, Spreading, and Migration of Mouse Embryo Fibroblasts by Influencing Actin Cytoskeleton Organization*

    PubMed Central

    Wang, Jie; Song, Jinlei; An, Chao; Dong, Wenji; Zhang, Jingxin; Yin, Changcheng; Hale, John; Baines, Anthony J.; Mohandas, Narla; An, Xiuli

    2014-01-01

    Protein 4.1B is a member of protein 4.1 family, adaptor proteins at the interface of membranes and the cytoskeleton. It is expressed in most mammalian tissues and is known to be required in formation of nervous and cardiac systems; it is also a tumor suppressor with a role in metastasis. Here, we explore functions of 4.1B using primary mouse embryonic fibroblasts (MEF) derived from wild type and 4.1B knock-out mice. MEF cells express two 4.1B isoforms: 130 and 60-kDa. 130-kDa 4.1B was absent from 4.1B knock-out MEF cells, but 60-kDa 4.1B remained, suggesting incomplete knock-out. Although the 130-kDa isoform was predominantly located at the plasma membrane, the 60-kDa isoform was enriched in nuclei. 130-kDa-deficient 4.1B MEF cells exhibited impaired cell adhesion, spreading, and migration; they also failed to form actin stress fibers. Impaired cell spreading and stress fiber formation were rescued by re-expression of the 130-kDa 4.1B but not the 60-kDa 4.1B. Our findings document novel, isoform-selective roles for 130-kDa 4.1B in adhesion, spreading, and migration of MEF cells by affecting actin organization, giving new insight into 4.1B functions in normal tissues as well as its role in cancer. PMID:24381168

  17. MUC16/CA125 in the Context of Modular Proteins with an Annotated Role in Adhesion-Related Processes: In Silico Analysis

    PubMed Central

    Jankovic, Miroslava; Mitic, Ninoslav

    2012-01-01

    Mucin 16 (MUC16) is a type I transmembrane protein, the extracellular portion of which is shed after proteolytic degradation and is denoted as CA125 antigen, a well known tumor marker for ovarian cancer. Regarding its polypeptide and glycan structures, as yet there is no detailed insight into their heterogeneity and ligand properties, which may greatly influence its function and biomarker potential. This study was aimed at obtaining further insight into the biological capacity of MUC16/CA125, using in silico analysis of corresponding mucin sequences, including similarity searches as well as GO (gene ontology)-based function prediction. The results obtained pointed to the similarities within extracellular serine/threonine rich regions of MUC16 to sequences of proteins expressed in evolutionary distant taxa, all having in common an annotated role in adhesion-related processes. Specifically, a homology to conserved domains from the family of herpesvirus major outer envelope protein (BLLF1) was found. In addition, the possible involvement of MUC16/CA125 in carbohydrate-binding interactions or cellular transport of protein/ion was suggested. PMID:22949868

  18. Haloarchaeal myovirus φCh1 harbours a phase variation system for the production of protein variants with distinct cell surface adhesion specificities.

    PubMed

    Klein, R; Rössler, N; Iro, M; Scholz, H; Witte, A

    2012-01-01

    The φCh1 myovirus, which infects the haloalkaliphilic archaeon Natrialba magadii, contains an invertible region that comprises the convergent open reading frames (ORFs) 34 and 36, which code for the putative tail fibre proteins gp34 and gp36 respectively. The inversion leads to an exchange of the C-termini of these proteins, thereby creating different types of tail fibres. Gene expression experiments revealed that only ORF34 is transcribed, indicating that φCh1 produces tail fibre proteins exclusively from this particular ORF. Only one of the two types of tail fibres encoded by ORF34 is able to bind to Nab. magadii in vitro. This is reflected by the observation that during the early phases of the infection cycle, the lysogenic strain L11 carries its invertible region exclusively in the orientation that produces that specific type of tail fibre. Obviously, Nab. magadii can only be infected by viruses carrying this particular type of tail fibre. By mutational analysis, the binding domain of gp34 was localized to the C-terminal part of the protein, particularly to a galactose-binding domain. The involvement of galactose residues in cell adhesion was supported by the observation that the addition of α-D-galactose to purified gp34 or whole virions prevented their attachment to Nab. magadii. PMID:22111759

  19. The Pmt2p-Mediated Protein O-Mannosylation Is Required for Morphogenesis, Adhesive Properties, Cell Wall Integrity and Full Virulence of Magnaporthe oryzae

    PubMed Central

    Guo, Min; Tan, Leyong; Nie, Xiang; Zhu, Xiaolei; Pan, Yuemin; Gao, Zhimou

    2016-01-01

    Protein O-mannosylation is a type of O-glycosylation that is characterized by the addition of mannose residues to target proteins, and is initially catalyzed by evolutionarily conserved protein O-mannosyltransferases (PMTs). In this study, three members of PMT were identified in Magnaporthe oryzae, and the pathogenic roles of MoPmt2, a member of PMT2 subfamily, were analyzed. We found that MoPmt2 is a homolog of Saccharomyces cerevisiae Pmt2 and could complement yeast Pmt2 function in resistance to CFW. Quantitative RT–PCR revealed that MoPmt2 is highly expressed during conidiation, and targeted disruption of MoPmt2 resulted in defects in conidiation and conidia morphology. The MoPmt2 mutants also showed a distinct reduction in fungal growth, which was associated with severe alterations in hyphal polarity. In addition, we found that the MoPmt2 mutants severely reduced virulence on both rice plants and barley leaves. The subsequent examination revealed that the fungal adhesion, conidial germination, CWI and invasive hyphae growth in host cells are responsible for defects on appressorium mediated penetration, and thus attenuated the pathogenicity of MoPmt2 mutants. Taken together, our results suggest that protein O-mannosyltransferase MoPmt2 plays essential roles in fungal growth and development, and is required for the full pathogenicity of M. oryzae. PMID:27199956

  20. The Pmt2p-Mediated Protein O-Mannosylation Is Required for Morphogenesis, Adhesive Properties, Cell Wall Integrity and Full Virulence of Magnaporthe oryzae.

    PubMed

    Guo, Min; Tan, Leyong; Nie, Xiang; Zhu, Xiaolei; Pan, Yuemin; Gao, Zhimou

    2016-01-01

    Protein O-mannosylation is a type of O-glycosylation that is characterized by the addition of mannose residues to target proteins, and is initially catalyzed by evolutionarily conserved protein O-mannosyltransferases (PMTs). In this study, three members of PMT were identified in Magnaporthe oryzae, and the pathogenic roles of MoPmt2, a member of PMT2 subfamily, were analyzed. We found that MoPmt2 is a homolog of Saccharomyces cerevisiae Pmt2 and could complement yeast Pmt2 function in resistance to CFW. Quantitative RT-PCR revealed that MoPmt2 is highly expressed during conidiation, and targeted disruption of MoPmt2 resulted in defects in conidiation and conidia morphology. The MoPmt2 mutants also showed a distinct reduction in fungal growth, which was associated with severe alterations in hyphal polarity. In addition, we found that the MoPmt2 mutants severely reduced virulence on both rice plants and barley leaves. The subsequent examination revealed that the fungal adhesion, conidial germination, CWI and invasive hyphae growth in host cells are responsible for defects on appressorium mediated penetration, and thus attenuated the pathogenicity of MoPmt2 mutants. Taken together, our results suggest that protein O-mannosyltransferase MoPmt2 plays essential roles in fungal growth and development, and is required for the full pathogenicity of M. oryzae. PMID:27199956

  1. Polyimide adhesives

    NASA Technical Reports Server (NTRS)

    Progar, D. J.; Bell, V. L.; Saintclair, T. L. (Inventor)

    1974-01-01

    A process of preparing aromatic polyamide-acids for use as adhesives is described. An equimolar quantity of an aromatic dianhydride is added to a stirred solution of an aromatic diamine in a water or alcohol-miscible ether solvent to obtain a viscous polymer solution. The polymeric-acid intermediate polymer does not become insoluble but directly forms a smooth viscous polymer solution. These polyamic-acid polymers are converted, by heating in the range of 200-300 C and with pressure, to form polyimides with excellent adhesive properties.

  2. Estrogen and pure antiestrogen fulvestrant (ICI 182 780) augment cell–matrigel adhesion of MCF-7 breast cancer cells through a novel G protein coupled estrogen receptor (GPR30)-to-calpain signaling axis

    SciTech Connect

    Chen, Yan; Li, Zheng; He, Yan; Shang, Dandan; Pan, Jigang; Wang, Hongmei; Chen, Huamei; Zhu, Zhuxia; Wang, Xudong

    2014-03-01

    Fulvestrant (ICI 182 780, ICI) has been used in treating patients with hormone-sensitive breast cancer, yet initial or acquired resistance to endocrine therapies frequently arises and, in particular, cancer recurs as metastasis. We demonstrate here that both 17-beta-estradiol (E2) and ICI enhance cell adhesion to matrigel in MCF-7 breast cancer cells, with increased autolysis of calpain 1 (large subunit) and proteolysis of focal adhesion kinase (FAK), indicating calpain activation. Additionally, either E2 or ICI induced down-regulation of estrogen receptor α without affecting G protein coupled estrogen receptor 30 (GPR30) expression. Interestingly, GPR30 agonist G1 triggered calpain 1 autolysis but not calpain 2, whereas ER agonist diethylstilbestrol caused no apparent calpain autolysis. Furthermore, the actions of E2 and ICI on calpain and cell adhesion were tremendously suppressed by G15, or knockdown of GPR30. E2 and ICI also induced phosphorylation of extracellular regulated protein kinases 1 and 2 (ERK1/2), and suppression of ERK1/2 phosphorylation by U0126 profoundly impeded calpain activation triggered by estrogenic and antiestrogenic stimulations indicating implication of ERK1/2 in the GPR30-mediated action. Lastly, the E2- or ICI-induced cell adhesion was dramatically impaired by calpain-specific inhibitors, ALLN or calpeptin, suggesting requirement of calpain in the GPR30-associated action. These data show that enhanced cell adhesion by E2 and ICI occurs via a novel GPR30-ERK1/2-calpain pathway. Our results indicate that targeting the GPR30 signaling may be a potential strategy to reduce metastasis and improve the efficacy of antiestrogens in treatment of advanced breast cancer. - Highlights: • Estrogen and ICI augment adhesion to matrigel with calpain activation in MCF-7 cells. • GPR30 mediates cell–matrigel adhesion and calpain activation via ERK1/2. • Calpain is required in the cell–matrigel adhesion induced by E2 and ICI.

  3. The adaptor protein SAP directly associates with PECAM-1 and regulates PECAM-1-mediated-cell adhesion in T-like cell lines.

    PubMed

    Proust, Richard; Crouin, Catherine; Gandji, Leslie Yewakon; Bertoglio, Jacques; Gesbert, Franck

    2014-04-01

    SAP is a small cytosolic adaptor protein expressed in hematopoietic lineages whose main function is to regulate intracellular signaling pathways induced by the triggering of members of the SLAM receptor family. In this paper, we have identified the adhesion molecule PECAM-1 as a new partner for SAP in a conditional yeast two-hybrid screen. PECAM-1 is an immunoglobulin-like molecule expressed by endothelial cells and leukocytes, which possesses both pro- and anti-inflammatory properties. However, little is known about PECAM-1 functions in T cells. We show that SAP directly and specifically interacts with the cytosolic tyrosine 686 of PECAM-1. We generated different T-like cell lines in which SAP or PECAM-1 are expressed or down modulated and we demonstrate that a diminished SAP expression correlates with a diminished PECAM-1-mediated adhesion. Although SAP has mainly been shown to associate with SLAM receptors, we evidence here that SAP is a new actor downstream of PECAM-1. PMID:24388971

  4. Insulin-Like Growth Factor Binding Protein-2 Promotes Adhesion of Endothelial Progenitor Cells to Endothelial Cells via Integrin α5β1.

    PubMed

    Feng, Nianping; Zhang, Zhuo; Wang, Zhengfei; Zheng, Haihong; Qu, Fujun; He, Xijun; Wang, Chunlai

    2015-11-01

    The contribution of endothelial progenitor cells (EPCs) to new vessel formation has been studied in different physiological and pathological conditions for decades. As previously suggested, insulin-like growth factor binding protein-2 (IGFBP-2) may interact with integrins and promote cell migration. However, the role of IGFBP-2 in regulation of EPC functions remains largely unknown. In this present study, we found that overexpression of IGFBP-2 in human umbilical vein endothelial cells (HUVECs) promoted EPC-endothelial adhesion. Conversely, siRNA-mediated depletion of IGFBP-2 inhibited oxygen-glucose deprivation (OGD)-induced EPC-endothelial adhesion. Further, we demonstrated that the arginine-glycine-aspartic acid (RGD) motif in its C-domain is required for interaction with integrin α5β1. In addition, treatment with IGFBP-2 significantly enhanced incorporation of EPCs into tubule networks formed by HUVECs. Thus, our findings suggest that exogenous administration of IGFBP-2 may facilitate neovascularization and improve treatment of ischemic conditions. PMID:26076738

  5. Hyaluronan-mediated cellular adhesion

    NASA Astrophysics Data System (ADS)

    Curtis, Jennifer

    2005-03-01

    Many cells surround themselves with a cushioning halo of polysaccharides that is further strengthened and organized by proteins. In fibroblasts and chrondrocytes, the primary component of this pericellular matrix is hyaluronan, a large linear polyanion. Hyaluronan production is linked to a variety of disease, developmental, and physiological processes. Cells manipulate the concentration of hyaluronan and hyaluronan receptors for numerous activities including modulation of cell adhesion, cell motility, and differentiation. Recent investigations by identify hyaluronan's role in mediating early-stage cell adhesion. An open question is how the cell removes the 0.5-10 micron thick pericellular matrix to allow for further mature adhesion events requiring nanometer scale separations. In this investigation, holographic optical tweezers are used to study the adhesion and viscoelastic properties of chondrocytes' pericellular matrix. Ultimately, we aim to shed further light on the spatial and temporal details of the dramatic transition from micron to nanometer gaps between the cell and its adhesive substrate.

  6. Integrated Proteomics Identified Up-Regulated Focal Adhesion-Mediated Proteins in Human Squamous Cell Carcinoma in an Orthotopic Murine Model

    PubMed Central

    Granato, Daniela C.; Zanetti, Mariana R.; Kawahara, Rebeca; Yokoo, Sami; Domingues, Romênia R.; Aragão, Annelize Z.; Agostini, Michelle; Carazzolle, Marcelo F.; Vidal, Ramon O.; Flores, Isadora L.; Korvala, Johanna; Cervigne, Nilva K.; Silva, Alan R. S.; Coletta, Ricardo D.; Graner, Edgard; Sherman, Nicholas E.; Leme, Adriana F. Paes

    2014-01-01

    Understanding the molecular mechanisms of oral carcinogenesis will yield important advances in diagnostics, prognostics, effective treatment, and outcome of oral cancer. Hence, in this study we have investigated the proteomic and peptidomic profiles by combining an orthotopic murine model of oral squamous cell carcinoma (OSCC), mass spectrometry-based proteomics and biological network analysis. Our results indicated the up-regulation of proteins involved in actin cytoskeleton organization and cell-cell junction assembly events and their expression was validated in human OSCC tissues. In addition, the functional relevance of talin-1 in OSCC adhesion, migration and invasion was demonstrated. Taken together, this study identified specific processes deregulated in oral cancer and provided novel refined OSCC-targeting molecules. PMID:24858105

  7. Natural Underwater Adhesives

    PubMed Central

    Stewart, Russell J.; Ransom, Todd C.; Hlady, Vladimir

    2011-01-01

    The general topic of this review is protein-based underwater adhesives produced by aquatic organisms. The focus is on mechanisms of interfacial adhesion to native surfaces and controlled underwater solidification of natural water-borne adhesives. Four genera that exemplify the broad range of function, general mechanistic features, and unique adaptations are discussed in detail: blue mussels, acorn barnacles, sandcastle worms, and freshwater caddisfly larva. Aquatic surfaces in nature are charged and in equilibrium with their environment, populated by an electrical double layer of ions as well as adsorbed natural polyelectrolytes and microbial biofilms. Surface adsorption of underwater bioadhesives likely occurs by exchange of surface bound ligands by amino acid sidechains, driven primarily by relative affinities and effective concentrations of polymeric functional groups. Most aquatic organisms exploit modified amino acid sidechains, in particular phosphorylated serines and hydroxylated tyrosines (dopa), with high-surface affinity that form coordinative surface complexes. After delivery to the surfaces as a fluid, permanent natural adhesives solidify to bear sustained loads. Mussel plaques are assembled in a manner superficially reminiscent of in vitro layer-by-layer strategies, with sequentially delivered layers associated through Fe(dopa)3 coordination bonds. The adhesives of sandcastle worms, caddisfly larva, and barnacles may be delivered in a form somewhat similar to in vitro complex coacervation. Marine adhesives are secreted, or excreted, into seawater that has a significantly higher pH and ionic strength than the internal environment. Empirical evidence suggests these environment triggers could provide minimalistic, fail-safe timing mechanisms to prevent premature solidification (insolubilization) of the glue within the secretory system, yet allow rapid solidification after secretion. Underwater bioadhesives are further strengthened by secondary covalent

  8. A novel O-linked glycan modulates Campylobacter jejuni major outer membrane protein-mediated adhesion to human histo-blood group antigens and chicken colonization

    PubMed Central

    Mahdavi, Jafar; Pirinccioglu, Necmettin; Oldfield, Neil J.; Carlsohn, Elisabet; Stoof, Jeroen; Aslam, Akhmed; Self, Tim; Cawthraw, Shaun A.; Petrovska, Liljana; Colborne, Natalie; Sihlbom, Carina; Borén, Thomas; Wooldridge, Karl G.; Ala'Aldeen, Dlawer A. A.

    2014-01-01

    Campylobacter jejuni is an important cause of human foodborne gastroenteritis; strategies to prevent infection are hampered by a poor understanding of the complex interactions between host and pathogen. Previous work showed that C. jejuni could bind human histo-blood group antigens (BgAgs) in vitro and that BgAgs could inhibit the binding of C. jejuni to human intestinal mucosa ex vivo. Here, the major flagella subunit protein (FlaA) and the major outer membrane protein (MOMP) were identified as BgAg-binding adhesins in C. jejuni NCTC11168. Significantly, the MOMP was shown to be O-glycosylated at Thr268; previously only flagellin proteins were known to be O-glycosylated in C. jejuni. Substitution of MOMP Thr268 led to significantly reduced binding to BgAgs. The O-glycan moiety was characterized as Gal(β1–3)-GalNAc(β1–4)-GalNAc(β1–4)-GalNAcα1-Thr268; modelling suggested that O-glycosylation has a notable effect on the conformation of MOMP and this modulates BgAg-binding capacity. Glycosylation of MOMP at Thr268 promoted cell-to-cell binding, biofilm formation and adhesion to Caco-2 cells, and was required for the optimal colonization of chickens by C. jejuni, confirming the significance of this O-glycosylation in pathogenesis. PMID:24451549

  9. In vitro and in silico studies on cell adhesion protein peroxinectin from Fenneropenaeus indicus and screening of heme blockers against activity.

    PubMed

    Sivakamavalli, Jeyachandran; Selvaraj, Chandrabose; Singh, Sanjeev Kumar; Vaseeharan, Baskaralingam

    2016-05-01

    In invertebrates, the prophenoloxidase (proPO) pathway is involved in the phenol-like antioxidant production against invading pathogens. Overproduction of melanin and phenolic substances leads to the disruption of hemocytes (own host cells); therefore, there is a prerequisite to regulate the antioxidant production, which is performed by the proteases and proPO-associated cell adhesion protein peroxinectin (PX). PX is a macromolecular structure consisting of protein involved in the proPO pathway, which is a potential target in the regulatory mechanism in crustaceans. In the proPO cascade, pattern recognition proteins initiate the proPO cascade by the consequent reaction, and PX is involved in the key step in the regulatory mechanism of phenoloxidase enzyme synthesis. In the present study, Indian white shrimp Fenneropenaeus indicus PX (Fein-PX) gene sequence was used. Upregulation of Fein-PX was determined using immunostimulants β-glucan (agonists) and examined its expression by quantitative RT-PCR. To find the downregulation or negative regulation of Fein-PX, inhibitors were screened, and its 3D model provides molecular insights into the rationale inhibitor design for developing an effective molecule against Fein-PX. PMID:26686319

  10. Cbf11 and Cbf12, the fission yeast CSL proteins, play opposing roles in cell adhesion and coordination of cell and nuclear division

    SciTech Connect

    Prevorovsky, Martin; Grousl, Tomas; Stanurova, Jana; Rynes, Jan; Nellen, Wolfgang; Puta, Frantisek; Folk, Petr

    2009-05-01

    The CSL (CBF1/RBP-J{kappa}/Suppressor of Hairless/LAG-1) family is comprised of transcription factors essential for metazoan development, mostly due to their involvement in the Notch receptor signaling pathway. Recently, we identified two novel classes of CSL genes in the genomes of several fungal species, organisms lacking the Notch pathway. In this study, we characterized experimentally cbf11{sup +} and cbf12{sup +}, the two CSL genes of Schizosaccharomyces pombe, in order to elucidate the CSL function in fungi. We provide evidence supporting their identity as genuine CSL genes. Both cbf11{sup +} and cbf12{sup +} are non-essential; they have distinct expression profiles and code for nuclear proteins with transcription activation potential. Significantly, we demonstrated that Cbf11 recognizes specifically the canonical CSL response element GTG{sup A}/{sub G}GAA in vitro. The deletion of cbf11{sup +} is associated with growth phenotypes and altered colony morphology. Furthermore, we found that Cbf11 and Cbf12 play opposite roles in cell adhesion, nuclear and cell division and their coordination. Disturbed balance of the two CSL proteins leads to cell separation defects (sep phenotype), cut phenotype, and high-frequency diploidization in heterothallic strains. Our data show that CSL proteins operate in an organism predating the Notch pathway, which should be of relevance to the understanding of (Notch-independent) CSL functions in metazoans.

  11. Photoperiod-Dependent Effects of 4-tert-Octylphenol on Adherens and Gap Junction Proteins in Bank Vole Seminiferous Tubules

    PubMed Central

    Kuras, Paulina; Lydka-Zarzycka, Marta; Bilinska, Barbara

    2013-01-01

    In the present study we evaluated in vivo and in vitro effects of 4-tert-octylphenol (OP) on the expression and distribution of adherens and gap junction proteins, N-cadherin, β-catenin, and connexin 43 (Cx43), in testes of seasonally breeding rodents, bank voles. We found that in bank vole testes expression and distribution of N-cadherin, β-catenin, and Cx43 were photoperiod dependent. Long-term treatment with OP (200 mg/kg b.w.) resulted in the reduction of junction proteins expressions (P < 0.05, P < 0.01) and their delocalization in the testes of males kept in long photoperiod, whereas in short-day animals slight increase of Cx43 (P < 0.05), N-cadherin, and β-catenin (statistically nonsignificant) levels was observed. Effects of OP appeared to be independent of FSH and were maintained during in vitro organ culture, indicating that OP acts directly on adherens and gap junction proteins in the testes. An experiment performed using an antiestrogen ICI 182,780 demonstrated that the biological effects of OP on β-catenin and Cx43 involve an estrogen receptor-mediated response. Taken together, in bank vole organization of adherens and gap junctions and their susceptibility to OP are related to the length of photoperiod. Alterations in cadherin/catenin and Cx43-based junction may partially result from activation of estrogen receptor α and/or β signaling pathway. PMID:23737770

  12. Marine Bioinspired Underwater Contact Adhesion.

    PubMed

    Clancy, Sean K; Sodano, Antonio; Cunningham, Dylan J; Huang, Sharon S; Zalicki, Piotr J; Shin, Seunghan; Ahn, B Kollbe

    2016-05-01

    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. PMID:27046671

  13. The C. elegans EMAP-like protein, ELP-1 is required for touch sensation and associates with microtubules and adhesion complexes

    PubMed Central

    Hueston, Jennifer L; Herren, Gina Purinton; Cueva, Juan G; Buechner, Matthew; Lundquist, Erik A; Goodman, Miriam B; Suprenant, Kathy A

    2008-01-01

    Background The founding member of the EMAP-like protein family is the Echinoderm Microtubule-Associated Protein (EMAP), so-named for its abundance in sea urchin, starfish, and sand dollar eggs. The EMAP-like protein family has five members in mammals (EML1 through EML5) and only one in both Drosophila (ELP-1) and C. elegans (ELP-1). Biochemical studies of sea urchin EMAP and vertebrate EMLs implicate these proteins in the regulation of microtubule stability. So far, however, the physiological function of this protein family remains unknown. Results We examined the expression pattern of C. elegans ELP-1 by means of transgenic gene expression in living embryos and adults, and by immunolocalization with an ELP-1-specific antibody in fixed tissues. In embryos, ELP-1 is expressed in the hypodermis. In larvae and adults, ELP-1 is expressed in the body wall, spermatheca and vulval muscles, intestine, and hypodermal seam cells. In muscle, ELP-1 is associated with adhesion complexes near the cell surface and is bound to a criss-crossing network of microtubules in the cytoplasm. ELP-1 is also expressed in a subset of mechanoreceptor neurons, including the ray neurons in the male tail, microtubule-rich touch receptor neurons, and the six ciliated IL1 neurons. This restricted localization in the nervous system implies that ELP-1 plays a role in mechanotransmission. Consistent with this idea, decreasing ELP-1 expression decreases sensitivity to gentle touch applied to the body wall. Conclusion These data imply that ELP-1 may play an important role during the transmission of forces and signals between the body surface and both muscle cells and touch receptor neurons. PMID:19014691

  14. Exploring natural silk protein sericin for regenerative medicine: an injectable, photoluminescent, cell-adhesive 3D hydrogel

    NASA Astrophysics Data System (ADS)

    Wang, Zheng; Zhang, Yeshun; Zhang, Jinxiang; Huang, Lei; Liu, Jia; Li, Yongkui; Zhang, Guozheng; Kundu, Subhas C.; Wang, Lin

    2014-11-01

    Sericin, a major component of silk, has a long history of being discarded as a waste during silk processing. The value of sericin for tissue engineering is underestimated and its potential application in regenerative medicine has just begun to be explored. Here we report the successful fabrication and characterization of a covalently-crosslinked 3D pure sericin hydrogel for delivery of cells and drugs. This hydrogel is injectable, permitting its implantation through minimally invasive approaches. Notably, this hydrogel is found to exhibit photoluminescence, enabling bioimaging and in vivo tracking. Moreover, this hydrogel system possesses excellent cell-adhesive capability, effectively promoting cell attachment, proliferation and long-term survival of various types of cells. Further, the sericin hydrogel releases bioactive reagents in a sustained manner. Additionally, this hydrogel demonstrates good elasticity, high porosity, and pH-dependent degradation dynamics, which are advantageous for this sericin hydrogel to serve as a delivery vehicle for cells and therapeutic drugs. With all these unique features, it is expected that this sericin hydrogel will have wide utility in the areas of tissue engineering and regenerative medicine.

  15. Exploring natural silk protein sericin for regenerative medicine: an injectable, photoluminescent, cell-adhesive 3D hydrogel

    PubMed Central

    Wang, Zheng; Zhang, Yeshun; Zhang, Jinxiang; Huang, Lei; Liu, Jia; Li, Yongkui; Zhang, Guozheng; Kundu, Subhas C.; Wang, Lin

    2014-01-01

    Sericin, a major component of silk, has a long history of being discarded as a waste during silk processing. The value of sericin for tissue engineering is underestimated and its potential application in regenerative medicine has just begun to be explored. Here we report the successful fabrication and characterization of a covalently-crosslinked 3D pure sericin hydrogel for delivery of cells and drugs. This hydrogel is injectable, permitting its implantation through minimally invasive approaches. Notably, this hydrogel is found to exhibit photoluminescence, enabling bioimaging and in vivo tracking. Moreover, this hydrogel system possesses excellent cell-adhesive capability, effectively promoting cell attachment, proliferation and long-term survival of various types of cells. Further, the sericin hydrogel releases bioactive reagents in a sustained manner. Additionally, this hydrogel demonstrates good elasticity, high porosity, and pH-dependent degradation dynamics, which are advantageous for this sericin hydrogel to serve as a delivery vehicle for cells and therapeutic drugs. With all these unique features, it is expected that this sericin hydrogel will have wide utility in the areas of tissue engineering and regenerative medicine. PMID:25412301

  16. Exploring natural silk protein sericin for regenerative medicine: an injectable, photoluminescent, cell-adhesive 3D hydrogel.

    PubMed

    Wang, Zheng; Zhang, Yeshun; Zhang, Jinxiang; Huang, Lei; Liu, Jia; Li, Yongkui; Zhang, Guozheng; Kundu, Subhas C; Wang, Lin

    2014-01-01

    Sericin, a major component of silk, has a long history of being discarded as a waste during silk processing. The value of sericin for tissue engineering is underestimated and its potential application in regenerative medicine has just begun to be explored. Here we report the successful fabrication and characterization of a covalently-crosslinked 3D pure sericin hydrogel for delivery of cells and drugs. This hydrogel is injectable, permitting its implantation through minimally invasive approaches. Notably, this hydrogel is found to exhibit photoluminescence, enabling bioimaging and in vivo tracking. Moreover, this hydrogel system possesses excellent cell-adhesive capability, effectively promoting cell attachment, proliferation and long-term survival of various types of cells. Further, the sericin hydrogel releases bioactive reagents in a sustained manner. Additionally, this hydrogel demonstrates good elasticity, high porosity, and pH-dependent degradation dynamics, which are advantageous for this sericin hydrogel to serve as a delivery vehicle for cells and therapeutic drugs. With all these unique features, it is expected that this sericin hydrogel will have wide utility in the areas of tissue engineering and regenerative medicine. PMID:25412301

  17. Arginine-glycine-aspartic acid- and fibrinogen gamma-chain carboxyterminal peptides inhibit platelet adherence to arterial subendothelium at high wall shear rates. An effect dissociable from interference with adhesive protein binding.

    PubMed Central

    Lawrence, J B; Kramer, W S; McKeown, L P; Williams, S B; Gralnick, H R

    1990-01-01

    Arg-Gly-Asp (RGD)- and fibrinogen gamma-chain carboxyterminal (GQQHHLGGAKQAGDV) peptides inhibit fibrinogen, fibronectin (Fn), vitronectin, and von Willebrand factor (vWF) binding to the platelet glycoprotein IIb-IIIa complex (GP IIb-IIIa). GP IIb-IIIa, vWF, and Fn are essential for normal platelet adherence to subendothelium. We added peptides to normal citrated whole blood before perfusion over human umbilical artery subendothelium and evaluated platelet adherence morphometrically at high (2,600 s-1) and low (800 s-1) wall shear rates. We also examined the effects of the peptides on platelet adhesion to collagen in a static system. At the high wall shear rate, RGDS and GQQHHLGGAKQAGDV caused dose-dependent reduction in the surface coverage with spread and adherent platelets. Amino acid transposition and conservative substitutions of RGD peptides and the AGDV peptide significantly inhibited platelet adherence at 2,600 s-1. By contrast, the modified RGD peptides and AGDV do not affect adhesive protein binding to platelets. None of the native or modified RGD- or fibrinogen gamma-chain peptides significantly inhibited either platelet adherence to subendothelium at 800 s-1 or platelet adhesion to collagen. Our findings demonstrate that peptides that interfere with adhesive protein binding to GP IIb-IIIa inhibit platelet adherence to vascular subendothelium with flowing blood only at high wall shear rates. Platelet adherence to subendothelium at high wall shear rates appears to be mediated by different recognition specificities from those required for fluid-phase adhesive protein binding or static platelet adhesion. PMID:2243140

  18. Semicircular canal morphogenesis in the zebrafish inner ear requires the function of gpr126 (lauscher), an adhesion class G protein-coupled receptor gene

    PubMed Central

    Geng, Fan-Suo; Abbas, Leila; Baxendale, Sarah; Holdsworth, Celia J.; Swanson, A. George; Slanchev, Krasimir; Hammerschmidt, Matthias; Topczewski, Jacek; Whitfield, Tanya T.

    2013-01-01

    Morphogenesis of the semicircular canal ducts in the vertebrate inner ear is a dramatic example of epithelial remodelling in the embryo, and failure of normal canal development results in vestibular dysfunction. In zebrafish and Xenopus, semicircular canal ducts develop when projections of epithelium, driven by extracellular matrix production, push into the otic vesicle and fuse to form pillars. We show that in the zebrafish, extracellular matrix gene expression is high during projection outgrowth and then rapidly downregulated after fusion. Enzymatic disruption of hyaluronan in the projections leads to their collapse and a failure to form pillars: as a result, the ears swell. We have cloned a zebrafish mutant, lauscher (lau), identified by its swollen ear phenotype. The primary defect in the ear is abnormal projection outgrowth and a failure of fusion to form the semicircular canal pillars. Otic expression of extracellular matrix components is highly disrupted: several genes fail to become downregulated and remain expressed at abnormally high levels into late larval stages. The lau mutations disrupt gpr126, an adhesion class G protein-coupled receptor gene. Expression of gpr126 is similar to that of sox10, an ear and neural crest marker, and is partially dependent on sox10 activity. Fusion of canal projections and downregulation of otic versican expression in a hypomorphic lau allele can be restored by cAMP agonists. We propose that Gpr126 acts through a cAMP-mediated pathway to control the outgrowth and adhesion of canal projections in the zebrafish ear via the regulation of extracellular matrix gene expression. PMID:24067352

  19. The intermediate filament protein vimentin binds specifically to a recombinant integrin {alpha}2/{beta}1 cytoplasmic tail complex and co-localizes with native {alpha}2/{beta}1 in endothelial cell focal adhesions

    SciTech Connect

    Kreis, Stephanie; Schoenfeld, Hans-Joachim; Melchior, Chantal; Steiner, Beat; Kieffer, Nelly . E-mail: kieffer@cu.lu

    2005-04-15

    Integrin receptors are crucial players in cell adhesion and migration. Identification and characterization of cellular proteins that interact with their short {alpha} and {beta} cytoplasmic tails will help to elucidate the molecular mechanisms by which integrins mediate bi-directional signaling across the plasma membrane. Integrin {alpha}2{beta}1 is a major collagen receptor but to date, only few proteins have been shown to interact with the {alpha}2 cytoplasmic tail or with the {alpha}2{beta}1 complex. In order to identify novel binding partners of a {alpha}2{beta}1cytoplasmic domain complex, we have generated recombinant GST-fusion proteins, incorporating the leucine zipper heterodimerization cassettes of Jun and Fos. To ascertain proper functionality of the recombinant proteins, interaction with natural binding partners was tested. GST-{alpha}2 and GST-Jun {alpha}2 bound His-tagged calreticulin while GST-{beta}1 and GST-Fos {beta}1 proteins bound talin. In screening assays for novel binding partners, the immobilized GST-Jun {alpha}2/GST-Fos {beta}1 heterodimeric complex, but not the single subunits, interacted specifically with endothelial cell-derived vimentin. Vimentin, an abundant intermediate filament protein, has previously been shown to co-localize with {alpha}v{beta}3-positive focal contacts. Here, we provide evidence that this interaction also occurs with {alpha}2{beta}1-enriched focal adhesions and we further show that this association is lost after prolonged adhesion of endothelial cells to collagen.

  20. Heterologous expression of newly identified galectin-8 from sea urchin embryos produces recombinant protein with lactose binding specificity and anti-adhesive activity

    PubMed Central

    Karakostis, Kostantinos; Costa, Caterina; Zito, Francesca; Matranga, Valeria

    2015-01-01

    Galectin family members specifically bind beta-galactoside derivatives and are involved in different cellular events, including cell communication, signalling, apoptosis, and immune responses. Here, we report a tandem-repeat type galectin from the Paracentrotus lividus sea urchin embryo, referred to as Pl-GAL-8. The 933nt sequence encodes a protein of 34.73 kDa, containing the conserved HFNPRF and WGxExR motifs in the two highly similar carbohydrate-recognition domains (CRD). The three-dimensional protein structure model of the N-CRD confirms the high evolutionary conservation of carbohydrate binding sites. The temporal gene expression is regulated during development and transcripts localize at the tip of the archenteron at gastrula stage, in a subset of the secondary mesenchyme cells that differentiate into blastocoelar (immune) cells. Functional studies using a recombinant Pl-GAL-8 expressed in bacteria demonstrate its hemo-agglutinating activity on human red blood cells through the binding to lactose, as well as its ability in inhibiting the adhesion of human Hep-G2 cells to the substrate. The recent implications in autoimmune diseases and inflammatory disorders make Gal-8 an attractive candidate for therapeutic purposes. Our results offer a solid basis for addressing the use of the new Pl-GAL-8 in functional and applicative studies, respectively in the developmental and biomedical fields. PMID:26640155

  1. Identification of a ligand-binding site in an immunoglobulin fold domain of the Saccharomyces cerevisiae adhesion protein alpha-agglutinin.

    PubMed Central

    de Nobel, H; Lipke, P N; Kurjan, J

    1996-01-01

    The Saccharomyces cerevisiae adhesion protein alpha-agglutinin (Ag alpha 1p) is expressed by alpha cells and binds to the complementary a-agglutinin expressed by a cells. The N-terminal half of alpha-agglutinin is sufficient for ligand binding and has been proposed to contain an immunoglobulin (Ig) fold domain. Based on a structural homology model for this domain and a previously identified critical residue (His292), we made Ag alpha 1p mutations in three discontinuous patches of the domain that are predicted to be in close proximity to His292 in the model. Residues in each of the three patches were identified that are important for activity and therefore define a putative ligand binding site, whereas mutations in distant loops had no effect on activity. This putative binding site is on a different surface of the Ig fold than the defined binding sites of immunoglobulins and other members of the Ig superfamily. Comparison of protein interaction sites by structural and mutational analysis has indicated that the area of surface contact is larger than the functional binding site identified by mutagenesis. The putative alpha-agglutinin binding site is therefore likely to identify residues that contribute to the functional binding site within a larger area that contacts a-agglutinin. Images PMID:8741846

  2. Increased erythrocyte adhesion to VCAM-1 during pulsatile flow: Application of a microfluidic flow adhesion bioassay

    PubMed Central

    White, Jennell; Lancelot, Moira; Sarnaik, Sharada; Hines, Patrick

    2015-01-01

    Abstract Sickle cell disease (SCD) is characterized by microvascular occlusion mediated by adhesive interactions of sickle erythrocytes (SSRBCs) to the endothelium. Most in vitro flow adhesion assays measure SSRBC adhesion during continuous flow, although in vivo SSRBC adhesive interactions occur during pulsatile flow. Using a well-plate microfluidic flow adhesion system, we demonstrate that isolated SSRBCs adhere to vascular cell adhesion molecule (VCAM-1) at greater levels during pulsatile versus continuous flow. A significant increase in adhesive interactions was observed between all pulse frequencies 1 Hz to 2 Hz (60–120 beats/min) when compared to non-pulsatile flow. Adhesion of isolated SSRBCs and whole blood during pulsatile flow was unaffected by protein kinase A (PKA) inhibition, and exposure of SSRBCs to pulsatile flow did not affect the intrinsic adhesive properties of SSRBCs. The cell type responsible for increased adhesion of whole blood varied from patient to patient. We conclude that low flow periods of the pulse cycle allow more adhesive interactions between sickle erythrocytes and VCAM-1, and sickle erythrocyte adhesion in the context of whole blood may better reflect physiologic cellular interactions. The microfluidic flow adhesion bioassay used in this study may have applications for clinical assessment of sickle erythrocyte adhesion during pulsatile flow. PMID:24898561

  3. Inhibition of Adhesion of Enteropathogenic Escherichia coli to HEp-2 Cells by Binding of a Novel Peptide to EspB Protein.

    PubMed

    Li, Duoyun; Chen, Zhong; Cheng, Hang; Zheng, Jin-Xin; Pan, Wei-Guang; Yang, Wei-Zhi; Yu, Zhi-Jian; Deng, Qi-Wen

    2016-09-01

    Enteropathogenic Escherichia coli (EPEC) is a major cause of infantile diarrhea in developing countries. The translocator EspB is a key virulence factor in the process of the attaching and effacing effect of EPEC and plays a critical role in the pathogenesis of the bacteria. In this study, we aimed to select the peptides binding to EspB protein by phage display library and further investigate whether these peptides can decrease the extent of invasion and virulence of EPEC on host cells by targeting to EspB protein. The expression and purification of EspB protein from E. coli was demonstrated by Western blotting. The Ph.D. 12-mer peptide phage display library was used to screen the candidate peptides binding specifically to EspB protein. Furthermore, the affinity of these candidate peptides bound to EspB was identified by enzyme-linked immunosorbent assay (ELISA). Moreover, we investigated whether these screened peptides could decrease the adherence ratio of EPEC to HEp-2 cells with increasing concentration. Successful purification of EspB protein from pET21b-EspB-transformed E. coli was identified by Western blotting. Then, the candidate peptides including phages 6, 7, 8, and 12 were screened by the Ph.D. 12-mer peptide phage display library and ELISA test demonstrated that their affinity binding to EspB protein was high compared with the control. Functional analysis indicated that synthetic peptide-6 (YFPYSHTSPRQP) significantly decreased the adherence ratio of EPEC to HEp-2 cells with increasing concentration (P < 0.01). Peptide-6 (100 µg/mL) could lead to a 40 % decrease in the adherence ratio of EPEC to HEp-2 cells compared with control (P < 0.01). However, the other three peptides at different concentrations showed only a slight ability to block the adherence of EPEC to host cells. Our data provided a potential strategy to inhibit the adhesion of EPEC to epithelial cells by a candidate peptide targeted toward EspB protein. PMID:27246497

  4. Characterization of MspA, an immunogenic autotransporter protein that mediates adhesion to epithelial and endothelial cells in Neisseria meningitidis.

    PubMed

    Turner, D P J; Marietou, A G; Johnston, L; Ho, K K L; Rogers, A J; Wooldridge, K G; Ala'Aldeen, D A A

    2006-05-01

    A novel putative autotransporter protein (NMB1998) was identified in the available genomic sequence of meningococcal strain MC58 (ET-5; ST-32). The mspA gene is absent from the genomic sequences of meningococcal strain Z2491 (ET-IV; ST-4) and the gonococcal strain FA1090. An orthologue is present in the meningococcal strain FAM18 (ET-37; ST-11), but the sequence contains a premature stop codon, suggesting that the protein may not be expressed in this strain. MspA is predicted to be a 157-kDa protein with low cysteine content, and it exhibits 36 and 33% identity to the meningococcal autotransporter proteins immunoglobulin A1 (IgA1) protease and App, respectively. Search of the Pfam database predicts the presence of IgA1 protease and autotransporter beta-barrel domains. MspA was cloned, and a recombinant protein of the expected size was expressed and after being affinity purified was used to raise rabbit polyclonal monospecific antiserum. Immunoblot studies showed that ca. 125- and 95-kDa fragments of MspA are secreted in meningococcal strain MC58, which are absent from the isogenic mutant. Secretion of MspA was shown to be modified in an AspA isogenic mutant. A strain survey showed that MspA is expressed by all ST-32 and ST-41/44 (lineage 3) strains, but none of the ST-8 (A4) strains examined. Sera from patients convalescing from meningococcal disease were shown to contain MspA-specific antibodies. In bactericidal assays, anti-MspA serum was shown to kill the homologous strain (MC58) and another ST-32 strain. Escherichia coli-expressing recombinant MspA was shown to adhere to both human bronchial epithelial cells and brain microvascular endothelial cells. PMID:16622234

  5. Phenotypic, Morphological and Adhesive Differences of Human Hematopoietic Progenitor Cells Cultured on Murine versus Human Mesenchymal Stromal Cells

    PubMed Central

    Reichert, Doreen; Friedrichs, Jens; Ritter, Steffi; Käubler, Theresa; Werner, Carsten; Bornhäuser, Martin; Corbeil, Denis

    2015-01-01

    Xenogenic transplantation models have been developed to study human hematopoiesis in immunocompromised murine recipients. They still have limitations and therefore it is important to delineate all players within the bone marrow that could account for species-specific differences. Here, we evaluated the proliferative capacity, morphological and physical characteristics of human CD34+ hematopoietic stem and progenitor cells (HSPCs) after co-culture on murine or human bone marrow-derived mesenchymal stromal cells (MSCs). After seven days, human CD34+CD133– HSPCs expanded to similar extents on both feeder layers while cellular subsets comprising primitive CD34+CD133+ and CD133+CD34– phenotypes are reduced fivefold on murine MSCs. The number of migrating HSPCs was also reduced on murine cells suggesting that MSC adhesion influences cellular polarization of HSPC. We used atomic force microscopy-based single-cell force spectroscopy to quantify their adhesive interactions. We found threefold higher detachment forces of human HSPCs from murine MSCs compared to human ones. This difference is related to the N-cadherin expression level on murine MSCs since its knockdown abolished their differential adhesion properties with human HSPCs. Our observations highlight phenotypic, morphological and adhesive differences of human HSPCs when cultured on murine or human MSCs, which raise some caution in data interpretation when xenogenic transplantation models are used. PMID:26498381

  6. Adhesive Performance of Biomimetic Adhesive-Coated Biologic Scaffolds

    PubMed Central

    Murphy, John L.; Vollenweider, Laura; Xu, Fangmin; Lee, Bruce P.

    2010-01-01

    Surgical repair of a discontinuity in traumatized or degenerated soft tissues is traditionally accomplished using sutures. A current trend is to reinforce this primary repair with surgical grafts, meshes, or patches secured with perforating mechanical devices (i.e., sutures, staples, or tacks). These fixation methods frequently lead to chronic pain and mesh detachment. We developed a series of biodegradable adhesive polymers that are synthetic mimics of mussel adhesive proteins (MAPs), composed of 3,4-dihydroxyphenylalanine (DOPA)-derivatives, polyethylene glycol (PEG), and polycaprolactone (PCL). These polymers can be cast into films, and their mechanical properties, extent of swelling, and degradation rate can be tailored through the composition of the polymers as well as blending with additives. When coated onto a biologic mesh used for hernia repair, these adhesive constructs demonstrated adhesive strengths significantly higher than fibrin glue. With further development, a pre-coated bioadhesive mesh may represent a new surgical option for soft tissue repair. PMID:20919699

  7. Focal adhesion kinases in adhesion structures and disease.

    PubMed

    Eleniste, Pierre P; Bruzzaniti, Angela

    2012-01-01

    Cell adhesion to the extracellular matrix (ECM) is essential for cell migration, proliferation, and embryonic development. Cells can contact the ECM through a wide range of matrix contact structures such as focal adhesions, podosomes, and invadopodia. Although they are different in structural design and basic function, they share common remodeling proteins such as integrins, talin, paxillin, and the tyrosine kinases FAK, Pyk2, and Src. In this paper, we compare and contrast the basic organization and role of focal adhesions, podosomes, and invadopodia in different cells. In addition, we discuss the role of the tyrosine kinases, FAK, Pyk2, and Src, which are critical for the function of the different adhesion structures. Finally, we discuss the essential role of these tyrosine kinases from the perspective of human diseases. PMID:22888421

  8. Focal Adhesion Kinases in Adhesion Structures and Disease

    PubMed Central

    Eleniste, Pierre P.; Bruzzaniti, Angela

    2012-01-01

    Cell adhesion to the extracellular matrix (ECM) is essential for cell migration, proliferation, and embryonic development. Cells can contact the ECM through a wide range of matrix contact structures such as focal adhesions, podosomes, and invadopodia. Although they are different in structural design and basic function, they share common remodeling proteins such as integrins, talin, paxillin, and the tyrosine kinases FAK, Pyk2, and Src. In this paper, we compare and contrast the basic organization and role of focal adhesions, podosomes, and invadopodia in different cells. In addition, we discuss the role of the tyrosine kinases, FAK, Pyk2, and Src, which are critical for the function of the different adhesion structures. Finally, we discuss the essential role of these tyrosine kinases from the perspective of human diseases. PMID:22888421

  9. Cadherin-Dependent Cell Morphology in an Epithelium: Constructing a Quantitative Dynamical Model

    PubMed Central

    Gemp, Ian M.; Carthew, Richard W.; Hilgenfeldt, Sascha

    2011-01-01

    Cells in the Drosophila retina have well-defined morphologies that are attained during tissue morphogenesis. We present a computer simulation of the epithelial tissue in which the global interfacial energy between cells is minimized. Experimental data for both normal cells and mutant cells either lacking or misexpressing the adhesion protein N-cadherin can be explained by a simple model incorporating salient features of morphogenesis that include the timing of N-cadherin expression in cells and its temporal relationship to the remodeling of cell-cell contacts. The simulations reproduce the geometries of wild-type and mutant cells, distinguish features of cadherin dynamics, and emphasize the importance of adhesion protein biogenesis and its timing with respect to cell remodeling. The simulations also indicate that N-cadherin protein is recycled from inactive interfaces to active interfaces, thereby modulating adhesion strengths between cells. PMID:21814505

  10. Cadherin-dependent cell morphology in an epithelium: constructing a quantitative dynamical model.

    PubMed

    Gemp, Ian M; Carthew, Richard W; Hilgenfeldt, Sascha

    2011-07-01

    Cells in the Drosophila retina have well-defined morphologies that are attained during tissue morphogenesis. We present a computer simulation of the epithelial tissue in which the global interfacial energy between cells is minimized. Experimental data for both normal cells and mutant cells either lacking or misexpressing the adhesion protein N-cadherin can be explained by a simple model incorporating salient features of morphogenesis that include the timing of N-cadherin expression in cells and its temporal relationship to the remodeling of cell-cell contacts. The simulations reproduce the geometries of wild-type and mutant cells, distinguish features of cadherin dynamics, and emphasize the importance of adhesion protein biogenesis and its timing with respect to cell remodeling. The simulations also indicate that N-cadherin protein is recycled from inactive interfaces to active interfaces, thereby modulating adhesion strengths between cells. PMID:21814505

  11. Adhesive mechanisms in cephalopods: a review.

    PubMed

    von Byern, Janek; Klepal, Waltraud

    2006-01-01

    Several genera of cephalopods (Nautilus, Sepia, Euprymna and Idiosepius) produce adhesive secretions, which are used for attachment to the substratum, for mating and to capture prey. These adhesive structures are located in different parts of the body, viz. in the digital tentacles (Nautilus), in the ventral surface of the mantle and fourth arm pair (Sepia), in the dorsal epidermis (Euprymna), or in the dorsal mantle side and partly on the fins (Idiosepius). Adhesion in Sepia is induced by suction of dermal structures on the mantle, while for Nautilus, Euprymna and Idiosepius adhesion is probably achieved by chemical substances. Histochemical studies indicate that in Nautilus and Idiosepius secretory cells that appear to be involved in adhesion stain for carbohydrates and protein, whilst in Euprymna only carbohydrates are detectable. De-adhesion is either achieved by muscle contraction of the tentacles and mantle (Nautilus and Sepia) or by secretion of substances (Euprymna). The de-adhesive mechanism used by Idiosepius remains unknown. PMID:17110356

  12. Interaction between Endothelial Protein C Receptor and Intercellular Adhesion Molecule 1 to Mediate Binding of Plasmodium falciparum-Infected Erythrocytes to Endothelial Cells

    PubMed Central

    Avril, Marion; Bernabeu, Maria; Benjamin, Maxwell; Brazier, Andrew Jay

    2016-01-01

    ABSTRACT Intercellular adhesion molecule 1 (ICAM-1) and the endothelial protein C receptor (EPCR) are candidate receptors for the deadly complication cerebral malaria. However, it remains unclear if Plasmodium falciparum parasites with dual binding specificity are involved in cytoadhesion or different parasite subpopulations bind in brain microvessels. Here, we investigated this issue by studying different subtypes of ICAM-1-binding parasite lines. We show that two parasite lines expressing domain cassette 13 (DC13) of the P. falciparum erythrocyte membrane protein 1 (PfEMP1) family have dual binding specificity for EPCR and ICAM-1 and further mapped ICAM-1 binding to the first DBLβ domain following the PfEMP1 head structure in both proteins. As PfEMP1 head structures have diverged between group A (EPCR binders) and groups B and C (CD36 binders), we also investigated how ICAM-1-binding parasites with different coreceptor binding traits influence P. falciparum-infected erythrocyte binding to endothelial cells. Whereas levels of binding to tumor necrosis factor alpha (TNF-α)-stimulated endothelial cells from the lung and brain by all ICAM-1-binding parasite lines increased, group A (EPCR and ICAM-1) was less dependent than group B (CD36 and ICAM-1) on ICAM-1 upregulation. Furthermore, both group A DC13 parasite lines had higher binding levels to brain endothelial cells (a microvascular niche with limited CD36 expression). This study shows that ICAM-1 is a coreceptor for a subset of EPCR-binding parasites and provides the first evidence of how EPCR and ICAM-1 interact to mediate parasite binding to both resting and TNF-α-activated primary brain and lung endothelial cells. PMID:27406562

  13. Expression of the cell adhesion proteins BEN/SC1/DM-GRASP and TAG-1 defines early steps of axonogenesis in the human spinal cord.

    PubMed

    Karagogeos, D; Pourquié, C; Kyriakopoulou, K; Tavian, M; Stallcup, W; Péault, B; Pourquié, O

    1997-03-17

    We have studied the expression pattern of two cell adhesion proteins of the immunoglobin (Ig) superfamily, BEN/SC1/DM-GRASP (BEN) and the transient axonal glycoprotein TAG-1, during the development of the human nervous system. This study was performed by immunocytochemistry on sections of human embryos ranging from 4 to 13 weeks postconception. The overall distribution of the two proteins during development is very similar to that reported in other vertebrate species, but several important differences have been observed. Both proteins exhibit a transient expression on selected neuronal populations, which include the motor and the sensory neurons. In addition, BEN was also detected on virtually all neurons derived from the neural crest as well as in nonneuronal tissues. A major difference of expression with the chick embryo is that, in the motor neurons, BEN expression was not observed at early stages of development, thus arguing against a role of this molecule in pathfinding and fasciculation. BEN was observed to be restricted to subsets of motor neurons, such as the medial column at the upper limb level. Expression was also detected in a laterodorsal population of the ventral horn cells, which are likely to correspond to migrating preganglionic neurons that originate from the motor pool at the thoracic level. TAG-1 was found on commissural neurons and weakly on the sympathetic neurons; it was also detected on restricted nonneuronal populations. In addition, we observed TAG-1 expression in fibers that could correspond either to subsets of dorsal root ganglia (DRGs) central afferences (including the Ia fibers) or to the axons of association interneurons and in scattered motoneurons likely to correspond either to preganglionic neurons, to gamma-motoneurons, or to late-born motoneurons. Therefore, our results indicate that the molecular strategies used to establish the axonal scaffolding of the nervous system in humans are extremely conserved among the different

  14. 3,4-Methylenedioxy-β-nitrostyrene inhibits adhesion and migration of human triple-negative breast cancer cells by suppressing β1 integrin function and surface protein disulfide isomerase.

    PubMed

    Chen, I-Hua; Chang, Fang-Rong; Wu, Yang-Chang; Kung, Po-Hsiung; Wu, Chin-Chung

    2015-03-01

    Triple negative breast cancer (TNBC) exhibits an aggressive clinical course by high metastatic potential. It is known that integrin-mediated cell adhesion and migration are important for cancer metastasis. In the present study, a synthetic compound, 3, 4-methyenedioxy-β-nitrostyrene (MNS), significantly inhibited adhesion of TNBC cell lines to different extracellular matrix (ECM) components. The antimetastatic capacity of MNS was also observed through reducing TNBC cells migration and invasion without affecting cell viability. Confocal microscopy revealed that MNS disrupted the formation of focal adhesion complex and actin stress fiber networks. Consistent with this finding, MNS inhibited phosphorylation of focal adhesion kinase (FAK) and paxillin as detected by Western blot analysis. In exploring the underlying mechanism, we found that MNS inhibited phosphorylation of FAK as a result of reducing β1 integrin activation and clustering. A cell-impermeable dithiol reagent, 2, 3-dimercaptopropane-1-sulfonic acid abrogated all of MNS's actions, indicating that MNS may react with thiol groups of cell surface proteins that are involved in regulation of β1 integrin function as well as cell adhesion and migration. Cell surface protein disulfide isomerase (PDI) has been reported to be essential for the affinity modulation of β integrins. We also demonstrated that MNS inhibited PDI activity both in a pure enzyme system and in intact cancer cells. Taken together, our results suggest that MNS inhibits in vitro metastatic properties of TNBC cells through suppression of β1 integrin activation and focal adhesion signaling. Moreover, inhibition of surface PDI may contribute, at least in part, to the actions of MNS. These results suggest that MNS has a potential to be developed as an anticancer agent for treatment of TNBC. PMID:25593085

  15. Protein-mediated adhesion of Lactobacillus acidophilus BG2FO4 on human enterocyte and mucus-secreting cell lines in culture.

    PubMed Central

    Coconnier, M H; Klaenhammer, T R; Kernéis, S; Bernet, M F; Servin, A L

    1992-01-01

    The adhesion of Lactobacillus acidophilus BG2FO4, a human stool isolate, to two human enterocytelike cell lines (Caco-2 and HT-29) and to the mucus secreted by a subpopulation of mucus-secreting HT29-MTX cells was investigated. Scanning electron microscopy revealed that the bacteria interacted with the well-defined apical microvilli of Caco-2 cells without cell damage and with the mucus secreted by the subpopulation of HT29-MTX cells. The adhesion to Caco-2 cells did not require calcium and involved an adhesion-promoting factor that was present in the spent supernatant of L. acidophilus cultures. This factor promoted adhesion of poorly adhering human Lactobacillus casei GG but did not promote adhesion of L. casei CNRZ 387, a strain of dairy origin. The adherence components on the bacterial cells and in the spent supernatant were partially characterized. Carbohydrates on the bacterial cell wall appeared to be partly responsible for the interaction between the bacteria and the extracellular adhesion-promoting factor. The adhesion-promoting factor was proteinaceous, since trypsin treatment dramatically decreased the adhesion of the L. acidophilus strain. The adhesion-promoting factor may be an important component of Lactobacillus species that colonize the gastrointestinal tract. Images PMID:1622282

  16. Nanog Increases Focal Adhesion Kinase (FAK) Promoter Activity and Expression and Directly Binds to FAK Protein to Be Phosphorylated*

    PubMed Central

    Ho, Baotran; Olson, Gretchen; Figel, Sheila; Gelman, Irwin; Cance, William G.; Golubovskaya, Vita M.

    2012-01-01

    Nanog and FAK were shown to be overexpressed in cancer cells. In this report, the Nanog overexpression increased FAK expression in 293, SW480, and SW620 cancer cells. Nanog binds the FAK promoter and up-regulates its activity, whereas Nanog siRNA decreases FAK promoter activity and FAK mRNA. The FAK promoter contains four Nanog-binding sites. The site-directed mutagenesis of these sites significantly decreased up-regulation of FAK promoter activity by Nanog. EMSA showed the specific binding of Nanog to each of the four sites, and binding was confirmed by ChIP assay. Nanog directly binds the FAK protein by pulldown and immunoprecipitation assays, and proteins co-localize by confocal microscopy. Nanog binds the N-terminal domain of FAK. In addition, FAK directly phosphorylates Nanog in a dose-dependent manner by in vitro kinase assay and in cancer cells in vivo. The site-directed mutagenesis of Nanog tyrosines, Y35F and Y174F, blocked phosphorylation and binding by FAK. Moreover, overexpression of wild type Nanog increased filopodia/lamellipodia formation, whereas mutant Y35F and Y174F Nanog did not. The wild type Nanog increased cell invasion that was inhibited by the FAK inhibitor and increased by FAK more significantly than with the mutants Y35F and Y174F Nanog. Down-regulation of Nanog with siRNA decreased cell growth reversed by FAK overexpression. Thus, these data demonstrate the regulation of the FAK promoter by Nanog, the direct binding of the proteins, the phosphorylation of Nanog by FAK, and the effect of FAK and Nanog cross-regulation on cancer cell morphology, invasion, and growth that plays a significant role in carcinogenesis. PMID:22493428

  17. Nanog increases focal adhesion kinase (FAK) promoter activity and expression and directly binds to FAK protein to be phosphorylated.

    PubMed

    Ho, Baotran; Olson, Gretchen; Figel, Sheila; Gelman, Irwin; Cance, William G; Golubovskaya, Vita M

    2012-05-25

    Nanog and FAK were shown to be overexpressed in cancer cells. In this report, the Nanog overexpression increased FAK expression in 293, SW480, and SW620 cancer cells. Nanog binds the FAK promoter and up-regulates its activity, whereas Nanog siRNA decreases FAK promoter activity and FAK mRNA. The FAK promoter contains four Nanog-binding sites. The site-directed mutagenesis of these sites significantly decreased up-regulation of FAK promoter activity by Nanog. EMSA showed the specific binding of Nanog to each of the four sites, and binding was confirmed by ChIP assay. Nanog directly binds the FAK protein by pulldown and immunoprecipitation assays, and proteins co-localize by confocal microscopy. Nanog binds the N-terminal domain of FAK. In addition, FAK directly phosphorylates Nanog in a dose-dependent manner by in vitro kinase assay and in cancer cells in vivo. The site-directed mutagenesis of Nanog tyrosines, Y35F and Y174F, blocked phosphorylation and binding by FAK. Moreover, overexpression of wild type Nanog increased filopodia/lamellipodia formation, whereas mutant Y35F and Y174F Nanog did not. The wild type Nanog increased cell invasion that was inhibited by the FAK inhibitor and increased by FAK more significantly than with the mutants Y35F and Y174F Nanog. Down-regulation of Nanog with siRNA decreased cell growth reversed by FAK overexpression. Thus, these data demonstrate the regulation of the FAK promoter by Nanog, the direct binding of the proteins, the phosphorylation of Nanog by FAK, and the effect of FAK and Nanog cross-regulation on cancer cell morphology, invasion, and growth that plays a significant role in carcinogenesis. PMID:22493428

  18. Exploiting the superior protein resistance of polymer brushes to control single cell adhesion and polarisation at the micron scale

    PubMed Central

    Gautrot, Julien E.; Trappmann, Britta; Oceguera-Yanez, Fabian; Connelly, John; He, Ximin; Watt, Fiona M.; Huck, Wilhelm T.S.

    2010-01-01

    The control of the cell microenvironment on model patterned substrates allows the systematic study of cell biology in well defined conditions, potentially using automated systems. The extreme protein resistance of poly(oligo(ethylene glycol methacrylate)) (POEGMA) brushes is exploited to achieve high fidelity patterning of single cells. These coatings can be patterned by soft lithography on large areas (a microscope slide) and scale (substrates were typically prepared in batches of 200). The present protocol relies on the adsorption of extra-cellular matrix (ECM) proteins on unprotected areas using simple incubation and washing steps. The stability of POEGMA brushes, as examined via ellipsometry and SPR, is found to be excellent, both during storage and cell culture. The impact of substrate treatment, brush thickness and incubation protocol on ECM deposition, both for ultra-thin gold and glass substrates, is investigated via fluorescence microscopy and AFM. Optimised conditions result in high quality ECM patterns at the micron scale, even on glass substrates, that are suitable for controlling cell spreading and polarisation. These patterns are compatible with state-of-the-art technologies (fluorescence microscopy, FRET) used for live cell imaging. This technology, combined with single cell analysis methods, provides a platform for exploring the mechanisms that regulate cell behaviour. PMID:20347135

  19. Immunogenicity of orally administrated recombinant Lactobacillus casei Zhang expressing Cryptosporidium parvum surface adhesion protein P23 in mice.

    PubMed

    Geriletu; Xu, Rihua; Jia, Honglin; Terkawi, Mohamad Alaa; Xuan, Xuenan; Zhang, Heping

    2011-05-01

    Cryptosporidium parvum, an intestinal apicomplexan parasite, is a significant cause of diarrheal diseases in both humans and animals. What is more, there is no promising strategy for controlling cryptosporidiosis. In this study, the P23 immunodominant surface protein of C. parvum sporozoites was stably expressed in the Lactobacillus casei Zhang strain and its immunogenicity was evaluated in a mouse model. The molecular weight (23 kDa) and immunogenicity of p23 gene expressed by L. casei Zhang were similar to that of the native P23 protein. Oral immunization with control L. casei Zhang and recombinant L. casei Zhang-p23 activated the mucosal immune system to elicit serum immunoglobulin G (IgG) and mucosal IgA in mice. Furthermore, the expression of cytokines such as IL-4, IL-6, and IFN-γ in splenocytes of mice was detected by real-time PCR after oral immunization. P23-specific immunocyte activation was also verified. These findings indicate that the live L. casei Zhang vector may be a new tool for the production of mucosal vaccines against cryptosporidiosis in animals. PMID:21336991

  20. Type IV collagen is an activating ligand for the adhesion G protein-coupled receptor GPR126.

    PubMed

    Paavola, Kevin J; Sidik, Harwin; Zuchero, J Bradley; Eckart, Michael; Talbot, William S

    2014-08-12

    GPR126 is an orphan heterotrimeric guanine nucleotide-binding protein (G protein)-coupled receptor (GPCR) that is essential for the development of diverse organs. We found that type IV collagen, a major constituent of the basement membrane, binds to Gpr126 and activates its signaling function. Type IV collagen stimulated the production of cyclic adenosine monophosphate in rodent Schwann cells, which require Gpr126 activity to differentiate, and in human embryonic kidney (HEK) 293 cells expressing exogenous Gpr126. Type IV collagen specifically bound to the extracellular amino-terminal region of Gpr126 containing the CUB (complement, Uegf, Bmp1) and pentraxin domains. Gpr126 derivatives lacking the entire amino-terminal region were constitutively active, suggesting that this region inhibits signaling and that ligand binding relieves this inhibition to stimulate receptor activity. A new zebrafish mutation that truncates Gpr126 after the CUB and pentraxin domains disrupted development of peripheral nerves and the inner ear. Thus, our findings identify type IV collagen as an activating ligand for GPR126, define its mechanism of activation, and highlight a previously unrecognized signaling function of type IV collagen in basement membranes. PMID:25118328

  1. A c-di-GMP Effector System Controls Cell Adhesion by Inside-Out Signaling and Surface Protein Cleavage

    PubMed Central

    Newell, Peter D.; Boyd, Chelsea D.; Sondermann, Holger; O'Toole, George A.

    2011-01-01

    In Pseudomonas fluorescens Pf0-1 the availability of inorganic phosphate (Pi) is an environmental signal that controls biofilm formation through a cyclic dimeric GMP (c-di-GMP) signaling pathway. In low Pi conditions, a c-di-GMP phosphodiesterase (PDE) RapA is expressed, depleting cellular c-di-GMP and causing the loss of a critical outer-membrane adhesin LapA from the cell surface. This response involves an inner membrane protein LapD, which binds c-di-GMP in the cytoplasm and exerts a periplasmic output promoting LapA maintenance on the cell surface. Here we report how LapD differentially controls maintenance and release of LapA: c-di-GMP binding to LapD promotes interaction with and inhibition of the periplasmic protease LapG, which targets the N-terminus of LapA. We identify conserved amino acids in LapA required for cleavage by LapG. Mutating these residues in chromosomal lapA inhibits LapG activity in vivo, leading to retention of the adhesin on the cell surface. Mutations with defined effects on LapD's ability to control LapA localization in vivo show concomitant effects on c-di-GMP-dependent LapG inhibition in vitro. To establish the physiological importance of the LapD-LapG effector system, we track cell attachment and LapA protein localization during Pi starvation. Under this condition, the LapA adhesin is released from the surface of cells and biofilms detach from the substratum. This response requires c-di-GMP depletion by RapA, signaling through LapD, and proteolytic cleavage of LapA by LapG. These data, in combination with the companion study by Navarro et al. presenting a structural analysis of LapD's signaling mechanism, give a detailed description of a complete c-di-GMP control circuit—from environmental signal to molecular output. They describe a novel paradigm in bacterial signal transduction: regulation of a periplasmic enzyme by an inner membrane signaling protein that binds a cytoplasmic second messenger. PMID:21304920