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Sample records for adhesion protein n-cadherin

  1. Mechanical coupling between transsynaptic N-cadherin adhesions and actin flow stabilizes dendritic spines

    PubMed Central

    Chazeau, Anal; Garcia, Mikael; Czndr, Katalin; Perrais, David; Tessier, Batrice; Giannone, Grgory; Thoumine, Olivier

    2015-01-01

    The morphology of neuronal dendritic spines is a critical indicator of synaptic function. It is regulated by several factors, including the intracellular actin/myosin cytoskeleton and transcellular N-cadherin adhesions. To examine the mechanical relationship between these molecular components, we performed quantitative live-imaging experiments in primary hippocampal neurons. We found that actin turnover and structural motility were lower in dendritic spines than in immature filopodia and increased upon expression of a nonadhesive N-cadherin mutant, resulting in an inverse relationship between spine motility and actin enrichment. Furthermore, the pharmacological stimulation of myosin II induced the rearward motion of actin structures in spines, showing that myosin II exerts tension on the actin network. Strikingly, the formation of stable, spine-like structures enriched in actin was induced at contacts between dendritic filopodia and N-cadherincoated beads or micropatterns. Finally, computer simulations of actin dynamics mimicked various experimental conditions, pointing to the actin flow rate as an important parameter controlling actin enrichment in dendritic spines. Together these data demonstrate that a clutch-like mechanism between N-cadherin adhesions and the actin flow underlies the stabilization of dendritic filopodia into mature spines, a mechanism that may have important implications in synapse initiation, maturation, and plasticity in the developing brain. PMID:25568337

  2. ?-Catenin Localization and Sarcomere Self-Organization on N-Cadherin Adhesive Patterns Are Myocyte Contractility Driven

    PubMed Central

    Shieh, Adrian C.; A. Janmey, Paul; Kresh, J. Yasha

    2012-01-01

    The N-cadherin (N-cad) complex plays a crucial role in cardiac cell structure and function. Cadherins are adhesion proteins linking adjacent cardiac cells and, like integrin adhesions, are sensitive to force transmission. Forces through these adhesions are capable of eliciting structural and functional changes in myocytes. Compared to integrins, the mechanisms of force transduction through cadherins are less explored. ?-catenin is a major component of the cadherin-catenin complex, thought to provide a link to the cell actin cytoskeleton. Using N-cad micropatterned substrates in an adhesion constrainment model, the results from this study show that ?-catenin localizes to regions of highest internal stress in myocytes. This localization suggests that ?-catenin acts as an adaptor protein associated with the cadherin mechanosensory apparatus, which is distinct from mechanosensing through integrins. Myosin inhibition in cells bound by integrins to fibronectin-coated patterns disrupts myofibiril organization, whereas on N-cad coated patterns, myosin inhibition leads to better organized myofibrils. This result indicates that the two adhesion systems provide independent mechanisms for regulating myocyte structural organization. PMID:23077648

  3. 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

  4. Recapitulating cell-cell adhesion using N-cadherin biologically tethered to substrates.

    PubMed

    Vega L, Johana C M; Lee, Min Kyung; Jeong, Jae Hyun; Smith, Cartney E; Lee, Kwan Young; Chung, Hee Jung; Leckband, Deborah E; Kong, Hyunjoon

    2014-06-01

    Intercellular adhesion modulated by cadherin molecules plays an important role in diverse cellular functions including tissue morphogenesis, regeneration, and pathogenesis. However, it is a challenging task to decipher the effects of cell-cell adhesion in vitro because of difficulty in controlling the extent and numbers of cell-cell contacts. In this study, we hypothesize that tethering recombinant extracellular domains of neural cadherin with a C-terminal immunoglobulin Fc domain (N-Cad-Fc) to a substrate with an immobilized anti-Fc antibody (Fc-antibody) and a bifunctional polymer, which is reactive to both protein and substrate, would allow us to recapitulate cell-cell adhesion, independent of the number of cells plated on the substrate. To examine this hypothesis, we first immobilized Fc-antibody to a polyacrylamide hydrogel and a methacrylate-substituted glass using poly(amino-2-hydroxyethyl-co-2-methacryloxyethyl aspartamide)-g-poly(ethylene glycol)-N-hydroxysuccinimide ester (PHMAA-g-PEGNHS) and then incubated the gel in medium containing defined concentrations of the recombinant N-Cad-Fc. The resulting N-Cad-conjugated substrate enabled us to modulate adhesion of bone marrow stromal cells to the gel surface by varying the surface density of N-Cad-Fc. In contrast, direct chemical conjugation of N-Cad-Fc to the gel surface did not support cell adhesion. Additionally, the glass substrate biologically tethered with N-Cad-Fc promoted neuronal adhesion significantly more than substrates coated with poly-l-lysine. We suggest that this novel biological tethering method could be broadly applicable for modifying substrates with a variety of classical cadherins to enable the systematic study of the effects of cadherin-modulated cell-cell adhesion on cellular activities. PMID:24773064

  5. Soluble N-cadherin in human biological fluids.

    PubMed

    Derycke, Lara; De Wever, Olivier; Stove, Veronique; Vanhoecke, Barbara; Delanghe, Joris; Depypere, Herman; Bracke, Marc

    2006-12-15

    Classical cadherins such as E-, P- and N-cadherin are transmembrane proteins that mediate cell-cell adhesion, and are important in embryogenesis, maintenance of tissue integrity and cancer. Proteolytic shedding of the extracellular domain results in the generation of soluble E-, P- or N-cadherin ectodomains. Circulating soluble E- and P-cadherin have been described in the serum, and elevated levels were detected in cancer patients when compared with healthy persons. Here we report the presence of soluble N-cadherin, a 90-kD protein fragment, in the serum of both healthy persons and cancer patients, using a direct ELISA and immunoprecipitation. A correlation was found between prostate specific antigen and soluble N-cadherin, and significantly elevated levels were detected in prostate cancer follow-up patients. The N-cadherin protein is neo-expressed by carcinomas of the prostate, and is responsible for epithelial to fibroblastic transition. This is reflected by the higher concentrations of soluble N-cadherin in prostate cancer patients than in healthy persons. PMID:16998833

  6. Expression of N-cadherin and alpha-catenin in astrocytomas and glioblastomas.

    PubMed Central

    Shinoura, N.; Paradies, N. E.; Warnick, R. E.; Chen, H.; Larson, J. J.; Tew, J. J.; Simon, M.; Lynch, R. A.; Kanai, Y.; Hirohashi, S.

    1995-01-01

    We examined levels of mRNA and protein for N-cadherin, the predominant cadherin in neural tissues, and mRNA levels for the cadherin-associated protein, alpha-catenin, in a series of gliomas and in glioblastoma cell lines. mRNA levels for N-cadherin and alpha-catenin were significantly higher in glioblastomas than in low-grade astrocytomas or normal brain, while the levels of intact N-cadherin protein were similar in glioblastomas, low-grade astrocytomas and brain. In addition, there was no consistent relationship between invasiveness and expression of N-cadherin and alpha-catenin in highly invasive vs minimally invasive tumours within the same histopathological grade. To assess further the relationship between cadherin expression and neural tumour invasion, we measured N-cadherin expression, calcium-dependent cell adhesion and motility of several glioblastoma cell lines. While all N-cadherin-expressing lines were adhesive, no correlation was seen between the level of N-cadherin expression and cell motility. Together, these findings imply that, in contrast to the role played by E-cadherin in carcinomas, N-cadherin does not restrict the invasion of glioblastomas. Images Figure 1 Figure 2 Figure 3 Figure 4 PMID:7669572

  7. N-cadherin prodomain processing regulates synaptogenesis.

    PubMed

    Reins, Anala; Bernier, Louis-Philippe; McAdam, Robyn; Belkaid, Wiam; Shan, Weisong; Koch, Alexander W; Sgula, 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

  8. N-cadherin coordinates AMP kinase-mediated lung vascular repair.

    PubMed

    Jian, Ming-Yuan; Liu, Yanping; Li, Qian; Wolkowicz, Paul; Alexeyev, Mikhail; Zmijewski, Jaroslaw; Creighton, Judy

    2016-01-01

    Injury to the pulmonary circulation compromises endothelial barrier function and increases lung edema. Resolution of lung damage involves restoring barrier integrity, a process requiring reestablishment of endothelial cell-cell adhesions. However, mechanisms underlying repair in lung endothelium are poorly understood. In pulmonary microvascular endothelium, AMP kinase α1 (AMPKα1) stimulation enhances recovery of the endothelial barrier after LPS-induced vascular damage. AMPKα1 colocalizes to a discrete membrane compartment with the adhesion protein neuronal cadherin (N-cadherin). This study sought to determine N-cadherin's role in the repair process. Short-hairpin RNA against full-length N-cadherin or a C-terminally truncated N-cadherin, designed to disrupt the cadherin's interactions with intracellular proteins, were expressed in lung endothelium. Disruption of N-cadherin's intracellular domain caused translocation of AMPK away from the membrane and attenuated AMPK-mediated restoration of barrier function in LPS-treated endothelium. AMPK activity measurements indicated that lower basal AMPK activity in cells expressing the truncated N-cadherin compared with controls. Moreover, the AMPK stimulator 5-aminoimidazole-4-carboxamide ribonucleotide (AICAR) failed to increase AMPK activity in cells expressing the modified N-cadherin, indicating uncoupling of a functional association between AMPK and the cadherin. Isolated lung studies confirmed a physiologic role for this pathway in vivo. AMPK activation reversed LPS-induced increase in permeability, whereas N-cadherin inhibition hindered AMPK-mediated repair. Thus N-cadherin coordinates the vascular protective actions of AMPK through a functional link with the kinase. This study provides insight into intrinsic repair mechanisms in the lung and supports AMPK stimulation as a modality for treating vascular disease. PMID:26545901

  9. Two-tiered coupling between flowing actin and immobilized N-cadherin/catenin complexes in neuronal growth cones

    PubMed Central

    Garcia, Mikael; Leduc, Ccile; Lagardre, Matthieu; Argento, Amlie; Sibarita, Jean-Baptiste; Thoumine, Olivier

    2015-01-01

    Neuronal growth cones move forward by dynamically connecting actin-based motility to substrate adhesion, but the mechanisms at the individual molecular level remain unclear. We cultured primary neurons on N-cadherincoated micropatterned substrates, and imaged adhesion and cytoskeletal proteins at the ventral surface of growth cones using single particle tracking combined to photoactivated localization microscopy (sptPALM). We demonstrate transient interactions in the second time scale between flowing actin filaments and immobilized N-cadherin/catenin complexes, translating into a local reduction of the actin retrograde flow. Normal actin flow on micropatterns was rescued by expression of a dominant negative N-cadherin construct competing for the coupling between actin and endogenous N-cadherin. Fluorescence recovery after photobleaching (FRAP) experiments confirmed the differential kinetics of actin and N-cadherin, and further revealed a 20% actin population confined at N-cadherin micropatterns, contributing to local actin accumulation. Computer simulations with relevant kinetic parameters modeled N-cadherin and actin turnover well, validating this mechanism. Such a combination of short- and long-lived interactions between the motile actin network and spatially restricted adhesive complexes represents a two-tiered clutch mechanism likely to sustain dynamic environment sensing and provide the force necessary for growth cone migration. PMID:26038554

  10. Two-tiered coupling between flowing actin and immobilized N-cadherin/catenin complexes in neuronal growth cones.

    PubMed

    Garcia, Mikael; Leduc, Ccile; Lagardre, Matthieu; Argento, Amlie; Sibarita, Jean-Baptiste; Thoumine, Olivier

    2015-06-01

    Neuronal growth cones move forward by dynamically connecting actin-based motility to substrate adhesion, but the mechanisms at the individual molecular level remain unclear. We cultured primary neurons on N-cadherin-coated micropatterned substrates, and imaged adhesion and cytoskeletal proteins at the ventral surface of growth cones using single particle tracking combined to photoactivated localization microscopy (sptPALM). We demonstrate transient interactions in the second time scale between flowing actin filaments and immobilized N-cadherin/catenin complexes, translating into a local reduction of the actin retrograde flow. Normal actin flow on micropatterns was rescued by expression of a dominant negative N-cadherin construct competing for the coupling between actin and endogenous N-cadherin. Fluorescence recovery after photobleaching (FRAP) experiments confirmed the differential kinetics of actin and N-cadherin, and further revealed a 20% actin population confined at N-cadherin micropatterns, contributing to local actin accumulation. Computer simulations with relevant kinetic parameters modeled N-cadherin and actin turnover well, validating this mechanism. Such a combination of short- and long-lived interactions between the motile actin network and spatially restricted adhesive complexes represents a two-tiered clutch mechanism likely to sustain dynamic environment sensing and provide the force necessary for growth cone migration. PMID:26038554

  11. C-reactive protein inhibits expression of N-cadherin and ZEB-1 in murine colon adenocarcinoma.

    PubMed

    Kudo, Satoshi; Saito, Hajime; Motoyama, Satoru; Sasaki, Tomohiko; Imai, Kazuhiro; Konno, Hayato; Takashima, Shinogu; Atari, Maiko; Sato, Yusuke; Minamiya, Yoshihiro

    2015-09-01

    Epithelial-to-mesenchymal transition (EMT) is thought to play a key role in cancer cell invasion and metastasis. We previously demonstrated that cancer cell migration is inhibited by C-reactive protein (CRP), which is widely used as a biomarker of inflammation, though its functions are not fully understood. In the present study, we evaluated the effect of CRP on cancer cell migration and expression of mesenchymal and epithelial markers of EMT and of related transcription factors. MCA-38 murine colon adenocarcinoma cells were subcutaneously inoculated into the backs of C57BL/6 mice, which also received 1 μg of recombinant mouse CRP or vehicle (phosphate-buffered saline) subcutaneously every 3 days for 4 weeks. Thereafter, the mice were sacrificed for evaluation using quantitative real-time polymerase chain reaction (PCR) and immunohistochemistry. There was no statistical difference in tumor size between the control and CRP groups, but CRP dose-dependently inhibited MCA-38 cell migration. PCR analysis confirmed that CRP suppresses expression of N-cadherin (p < 0.01), a mesenchymal marker of EMT, and ZEB-1, an EMT-related transcription factor (p < 0.01). These findings suggest that CRP inhibits EMT in a MCA-38 tumor-bearing mouse model. CRP may thus be a potentially useful tool for preventing cancer progression through suppression of EMT. PMID:25864110

  12. 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 ?ERM) or the cytoplasmic tail (IC2 ?TAIL) 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. Conclusions These results indicate that ICAM-2 regulates endothelial barrier function and permeability through a pathway involving N-Cadherin, ERMs and Rac-1. PMID:24593809

  13. The Arf-GEF Schizo/Loner regulates N-cadherin to induce fusion competence of Drosophila myoblasts.

    PubMed

    Dottermusch-Heidel, Christine; Groth, Verena; Beck, Lothar; nel, Susanne-Filiz

    2012-08-01

    Myoblast fusion is a key process in multinucleated muscle formation. Prior to fusion, myoblasts recognize and adhere to each other with the aid of cell-adhesion proteins integrated into the membrane. Their intracellular domains participate in signal transduction by binding to cytoplasmic proteins. Here we identified the calcium-dependent cell-adhesion protein N-cadherin as the binding partner of the guanine-nucleotide exchange factor Schizo/Loner in Drosophila melanogaster. N-cadherin was expressed in founder cells and fusion-competent myoblasts of Drosophila during the first fusion phase. Our genetic analyses demonstrated that the myoblast fusion defect of schizo/loner mutants is rescued in part by the loss-of-function mutation of N-cadherin, which suggests that Schizo/Loner is a negative regulator of N-cadherin. Based on our findings, we propose a model where N-cadherin must be removed from the myoblast membrane to induce a protein-free zone at the cell-cell contact point to permit fusion. PMID:22595515

  14. SMAD4 Regulates Cell Motility through Transcription of N-Cadherin in Human Pancreatic Ductal Epithelium

    PubMed Central

    Kang, Ya'an; Ling, Jianhua; Suzuki, Rei; Roife, David; Chopin-Laly, Xavier; Truty, Mark J.; Chatterjee, Deyali; Wang, Huamin; Thomas, Ryan M.; Katz, Matthew H.; Chiao, Paul J.; Fleming, Jason B.

    2014-01-01

    Expression of the cellular adhesion protein N-cadherin is a critical event during epithelial-mesenchymal transition (EMT). The SMAD4 protein has been identified as a mediator of transforming growth factor-? (TGF-?) superfamily signaling, which regulates EMT, but the mechanisms linking TGF-? signaling to N-cadherin expression remain unclear. When the TGF-? pathway is activated, SMAD proteins, including the common mediator SMAD4, are subsequently translocated into the nucleus, where they influence gene transcription via SMAD binding elements (SBEs). Here we describe a mechanism for control of CDH2, the gene encoding N-cadherin, through the canonical TGF?SMAD4 pathway. We first identified four previously undescribed SBEs within the CDH2 promoter. Using telomerase immortalized human pancreatic ductal epithelium, we found that TGF-? stimulation prompted specific SMAD4 binding to all four SBEs. Luciferase reporter and SMAD4-knockdown experiments demonstrated that specific SMAD4 binding to the SBE located at ?3790 bp to ?3795 bp within the promoter region of CDH2 was necessary for TGF-?-stimulated transcription. Expression of N-cadherin on the surface of epithelial cells facilitates motility and invasion, and we demonstrated that knockdown of SMAD4 causes decreased N-cadherin expression, which results in diminished migration and invasion of human pancreatic ductal epithelial cells. Similar reduction of cell motility was produced after CDH2 knockdown. Together, these findings suggest that SMAD4 is critical for the TGF-?-driven upregulation of N-cadherin and the resultant invasive phenotype of human pancreatic ductal epithelial cells during EMT. PMID:25264609

  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. E- and N-cadherin expression and distribution during luteinization in the rat ovary.

    PubMed

    Machell, N H; Farookhi, R

    2003-06-01

    Cadherins, a family of Ca(2+)-dependent cell adhesion molecules, play an important role in ovarian tissue remodelling processes. The aim of this study was to examine the expression pattern of E- and N-cadherin in rat preovulatory follicles, luteinizing follicles and corpora lutea. Immature female rats were treated with equine chorionic gonadotrophin (eCG) to promote preovulatory follicle development. At 48 h after eCG treatment, the rats were injected with an ovulatory dose of hCG. Ovaries were analysed by western blot analysis and immunofluorescence for E- and N-cadherin expression at 48 h after eCG injection, and at 24 and 72 h after hCG injection. Ovaries of cyclic adult rats were examined to assess whether the changes in the expression pattern of cadherin were in agreement with those of the gonadotrophin-treated rats. Finally, expression of E-cadherin in luteinizing granulosa cells in vitro was assessed by RT-PCR and western blot analysis. Immunofluorescence results indicate that E-cadherin is expressed in the theca-interstial cells surrounding preovulatory follicles. N-cadherin expression is prominent in the membrana granulosa of these follicles. The initiation of luteinization with hCG leads to a decreased expression of N-cadherin in the membrana granulosa, whereas expression of E-cadherin starts within the luteinizing follicle. Both cadherins are prominently expressed in the fully formed corpus luteum at 72 h after hCG treatment. Immunofluorescence results revealed that the patterns of E- and N-cadherin expression in the gonadotrophin-treated rats were similar to those of the cyclic adult rats. Western blot analysis reflected similar changes for N-cadherin in the ovaries of both the cyclic adults and gonadotrophin-treated rats; however, they were different in E-cadherin expression. The expression of E-cadherin mRNA and protein was induced in vitro in luteinized granulosa cells. These results support the hypothesis that modulation of cadherin expression is an integral component of remodelling processes, including corpus luteum formation, in the ovary. The results also indicate that expression of E- and N-cadherin in granulosa-lutein cells appear to be under hormonal control. PMID:12773101

  17. Quantitative Immunohistochemistry of Desmosomal Proteins (Plakoglobin, Desmoplakin and Plakophilin), Connexin-43, and N-cadherin in Arrhythmogenic Cardiomyopathy: An Autopsy Study

    PubMed Central

    Tavora, Fabio; Zhang, Mingchang; Cresswell, Nathaniel; Li, Ling; Fowler, David; Franco, Marcello; Burke, Allen

    2013-01-01

    Background: Arrhythmogenic right ventricular cardiomyopathy (ARVC) is a genetic disorder related to mutations in desmosomal proteins. The current study tests the hypothesis that immunohistochemical staining for desmosomal proteins is of diagnostic utility by studying autopsy-confirmed cases of ARVC. Methods and Results: We studied 23 hearts from patients dying suddenly with ARVC. Control subject tissues were 21 hearts from people dying from non-cardiac causes (n=15), dilated cardiomyopathy (n=3) and coronary artery disease (n=3). Areas free of fibrofatty change or scarring were assessed on 50 sections from ARVC (24 left ventricle, 26 right ventricle) and 28 sections from controls. Immunohistochemical stains against plakoglobin, plakophilin, desmoplakin, connexin-43, and N-cadherin were applied and area expression analyzed by computerized morphometry. Desmin was stained as a control for fixation and similarly analyzed. The mean area of desmin expression was similar in controls and ARVC (86% vs. 85%, p=0.6). Plakoglobin expression was 4.9% 0.3% in controls, vs. 4.6% 0.3% in ARVC (p=0.3). Plakophilin staining was 4.8% 0.3% in controls vs. 4.4% 03% in ARVC (p=0.3). Desmoplakin staining was 3.4% in controls vs. 3.2 0.2% in ARVC (p=0.6). There were no significant differences when staining was compared between right and left ventricles (all p > 0.1). For non-desmosomal proteins, the mean area of connexin-43 staining showed no significant difference by presence of disease. Conclusions: The small and insignificant decrease in junction protein expression in ARVC suggests that immunohistochemistry is not a useful tool for the diagnosis. PMID:23802019

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

    PubMed

    Bremmer, Felix; Schallenberg, Simon; Jarry, Hubertus; Kffer, Stefan; Kaulfuss, Silke; Burfeind, Peter; Strau, Arne; Thelen, Paul; Radzun, Heinz Joachim; Strbel, Philipp; Honecker, Friedemann; Behnes, Carl Ludwig

    2015-10-20

    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. 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

  20. N-Cadherin Promotes Recruitment and Migration of Neural Progenitor Cells from the SVZ Neural Stem Cell Niche into Demyelinated Lesions

    PubMed Central

    Klingener, Michael; Chavali, Manideep; Singh, Jagdeep; McMillan, Nadia; Coomes, Alexandra; Dempsey, Peter J.; Chen, Emily I.

    2014-01-01

    Discrete cellular microenvironments regulate stem cell pools and their development, as well as function in maintaining tissue homeostasis. Although the signaling elements modulating neural progenitor cells (NPCs) of the adult subventricular zone (SVZ) niche are fairly well understood, the pathways activated following injury and the resulting outcomes, are less clear. In the present study, we used mouse models of demyelination and proteomics analysis to identify molecular cues present in the adult SVZ niche during injury, and analyzed their role on NPCs in the context of promoting myelin repair. Proteomic analysis of SVZ tissue from mice with experimental demyelination identified several proteins that are known to play roles in NPC proliferation, adhesion, and migration. Among the proteins found to be upregulated were members of the N-cadherin signaling pathway. During the onset of demyelination in the subcortical white matter (SCWM), activation of epidermal growth factor receptor (EGFR) signaling in SVZ NPCs stimulates the interaction between N-cadherin and ADAM10. Upon cleavage and activation of N-cadherin signaling by ADAM10, NPCs undergo cytoskeletal rearrangement and polarization, leading to enhanced migration out of the SVZ into demyelinated lesions of the SCWM. Genetically disrupting either EGFR signaling or ADAM10 inhibits this pathway, preventing N-cadherin regulated NPC polarization and migration. Additionally, in vivo experiments using N-cadherin gain- and loss-of-function approaches demonstrated that N-cadherin enhances the recruitment of SVZ NPCs into demyelinated lesions. Our data revealed that EGFR-dependent N-cadherin signaling physically initiated by ADAM10 cleavage is the response of the SVZ niche to promote repair of the injured brain. PMID:25031401

  1. Rich regulates target specificity of photoreceptor cells and N-cadherin trafficking in the Drosophila visual system via Rab6.

    PubMed

    Tong, Chao; Ohyama, Tomoko; Tien, An-Chi; Rajan, Akhila; Haueter, Claire M; Bellen, Hugo J

    2011-08-11

    Neurons establish specific synaptic connections with their targets, a process that is highly regulated. Numerous cell adhesion molecules have been implicated in target recognition, but how these proteins are precisely trafficked and targeted is poorly understood. To identify components that affect synaptic specificity, we carried out a forward genetic screen in the Drosophila eye. We identified a gene, named ric1 homologue (rich), whose loss leads to synaptic specificity defects. Loss of rich leads to reduction of N-Cadherin in the photoreceptor cell synapses but not of other proteins implicated in target recognition, including Sec15, DLAR, Jelly belly, and PTP69D. The Rich protein binds to Rab6, and Rab6 mutants display very similar phenotypes as the rich mutants. The active form of Rab6 strongly suppresses the rich synaptic specificity defect, indicating that Rab6 is regulated by Rich. We propose that Rich activates Rab6 to regulate N-Cadherin trafficking and affects synaptic specificity. PMID:21835342

  2. 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

  3. 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

  4. Tuberin-deficiency downregulates N-cadherin and upregulates vimentin in kidney tumor of TSC patients

    PubMed Central

    Liang, Sitai; Salas, Tiffanie; Gencaslan, Emre; Li, Baojie; Habib, Samy L.

    2014-01-01

    Angiomyolipomas (AMLs) are associated with cell fibrosis in kidney of Tuberous Sclerosis Complex patients. The mechanism by which the fibrotic proteins accumulated in AMLs has not been explored. In the present study, we investigated the role of Akt/tuberin/mTOR pathway in the regulation cell fibrosis proteins. AML cells that expressed low levels of tuberin showed less expression of N-cadherin and higher of vimentin proteins compared to HEK293 cells. AML cells infected with Ad-tuberin showed a significant decrease in vimentin and an increase in N-cadherin protein expression. In addition, cells treated with rapamycin showed a significant increase in p-Akt and a decrease in p-p70S6K that was associated with a decrease expression of vimentin and a slight increase expression in N-cadherin. On the other hand, cells treated with Akt inhibitor revealed a significant decrease in p-Akt and p-p70S6K that was associated with a significant decrease in vimentin and an increase in N-cadherin expression. In addition, cells transfected with DN-Akt or DN-S6K show significant increase expression in N-cadherin and a decrease in vimentin. Moreover, cells transfected with siRNA against rictor or siRNA against raptor resulted in a decrease in vimentin and an increase N-cadherin expression. Kidney tumors from TSC patients showed significant decrease in N-cadherin and significant increased in vimentin protein expression compared to control kidney tissues. These data comprise the first report to provide the role of Akt/tuberin/mTORC1/2 in the regulation of N-cadherin and vimentin that are involved in the progression of fibrosis in kidney tumor of TSC patients. PMID:25149531

  5. Tuberin-deficiency downregulates N-cadherin and upregulates vimentin in kidney tumor of TSC patients.

    PubMed

    Liang, Sitai; Salas, Tiffanie; Gencaslan, Emre; Li, Baojie; Habib, Samy L

    2014-08-30

    Angiomyolipomas (AMLs) are associated with cell fibrosis in kidney of Tuberous Sclerosis Complex patients. The mechanism by which the fibrotic proteins accumulated in AMLs has not been explored. In the present study, we investigated the role of Akt/tuberin/mTOR pathway in the regulation cell fibrosis proteins. AML cells that expressed low levels of tuberin showed less expression of N-cadherin and higher of vimentin proteins compared to HEK293 cells. AML cells infected with Ad-tuberin showed a significant decrease in vimentin and an increase in N-cadherin protein expression. In addition, cells treated with rapamycin showed a significant increase in p-Akt and a decrease in p-p70S6K that was associated with a decrease expression of vimentin and a slight increase expression in N-cadherin. On the other hand, cells treated with Akt inhibitor revealed a significant decrease in p-Akt and p-p70S6K that was associated with a significant decrease in vimentin and an increase in N-cadherin expression. In addition, cells transfected with DN-Akt or DN-S6K show significant increase expression in N-cadherin and a decrease in vimentin. Moreover, cells transfected with siRNA against rictor or siRNA against raptor resulted in a decrease in vimentin and an increase N-cadherin expression. Kidney tumors from TSC patients showed significant decrease in N-cadherin and significant increased in vimentin protein expression compared to control kidney tissues. These data comprise the first report to provide the role of Akt/tuberin/mTORC1/2 in the regulation of N-cadherin and vimentin that are involved in the progression of fibrosis in kidney tumor of TSC patients. PMID:25149531

  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. Metformin represses cancer cells via alternate pathways in N-cadherin expressing vs. N-cadherin deficient cells

    PubMed Central

    Wu, Shulin; Zhuo, Yangjia; Otsetov, Aleksandar G.; Cai, Chao; Zhong, Weide; Wu, Chin-Lee; Olumi, Aria F.

    2015-01-01

    Metformin has emerged as a potential anticancer agent. Here, we demonstrate that metformin plays an anti-tumor role via repressing N-cadherin, independent of AMPK, in wild-type N-cadherin cancer cells. Ectopic-expression of N-cadherin develops metformin-resistant cancer cells, while suppression of N-cadherin sensitizes cancer to metformin. Manipulation of AMPK expression does not alter sensitivity of cancer to metformin. We show that NF-kappaB is a downstream molecule of N-cadherin and metformin regulates NF-kappaB signaling via suppressing N-cadherin. Moreover, we also suggest that TWIST1 is an upstream molecule of N-cadherin/NF-kappaB signaling and manipulation of TWIST1 expression changes the sensitivity of cancer cells to metformin. In contrast to the cells that express N-cadherin, in N-cadherin deficient cells, metformin plays an anti-tumor role via activation of AMPK. Ectopic expression of N-cadherin makes cancer more resistant to metformin. Therefore, we suggest that metformin's anti-cancer therapeutic effect is mediated through different molecular mechanism in wild-type vs. deficient N-cadherin cancer cells. At last, we selected 49 out of 984 patients samples with prostatic cancer after radical prostatectomy (selection criteria: Gleason score ? 7 and all patients taking metformin) and showed levels of N-cadherin, p65 and AMPK could predict post-surgical recurrence in prostate cancer after treatment of metformin. PMID:26359363

  8. Metformin represses cancer cells via alternate pathways in N-cadherin expressing vs. N-cadherin deficient cells.

    PubMed

    Ge, Rongbin; Wang, Zongwei; Wu, Shulin; Zhuo, Yangjia; Otsetov, Aleksandar G; Cai, Chao; Zhong, Weide; Wu, Chin-Lee; Olumi, Aria F

    2015-10-01

    Metformin has emerged as a potential anticancer agent. Here, we demonstrate that metformin plays an anti-tumor role via repressing N-cadherin, independent of AMPK, in wild-type N-cadherin cancer cells. Ectopic-expression of N-cadherin develops metformin-resistant cancer cells, while suppression of N-cadherin sensitizes cancer to metformin. Manipulation of AMPK expression does not alter sensitivity of cancer to metformin. We show that NF-kappaB is a downstream molecule of N-cadherin and metformin regulates NF-kappaB signaling via suppressing N-cadherin. Moreover, we also suggest that TWIST1 is an upstream molecule of N-cadherin/NF-kappaB signaling and manipulation of TWIST1 expression changes the sensitivity of cancer cells to metformin. In contrast to the cells that express N-cadherin, in N-cadherin deficient cells, metformin plays an anti-tumor role via activation of AMPK. Ectopic expression of N-cadherin makes cancer more resistant to metformin. Therefore, we suggest that metformin's anti-cancer therapeutic effect is mediated through different molecular mechanism in wild-type vs. deficient N-cadherin cancer cells. At last, we selected 49 out of 984 patients' samples with prostatic cancer after radical prostatectomy (selection criteria: Gleason score ? 7 and all patients taking metformin) and showed levels of N-cadherin, p65 and AMPK could predict post-surgical recurrence in prostate cancer after treatment of metformin. PMID:26359363

  9. Recruitment of ?-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

    ?-Catenin is a key component that connects transmembrane cadherin with the actin cytoskeleton at the cell-cell interface. However, the role of the ?-catenin/cadherin interaction in smooth muscle has not been well characterized. Here stimulation with acetylcholine promoted the recruitment of ?-catenin to N-cadherin in smooth muscle cells/tissues. Knockdown of ?-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 ?-catenin knockdown. In addition, the expression of the ?-catenin armadillo domain disrupted the recruitment of ?-catenin to N-cadherin. Force development, but not myosin light chain phosphorylation and actin polymerization, was reduced by the expression of the ?-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 ?-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 ?-catenin to N-cadherin, which may facilitate intercellular mechanotransduction. The association of ?-catenin with N-cadherin is regulated by actin polymerization during contractile activation. PMID:25713069

  10. N-cadherin/FGFR promotes metastasis through epithelial-to-mesenchymal transition and stem/progenitor cell-like properties

    PubMed Central

    Qian, X; Anzovino, A; Kim, S; Suyama, K; Yao, J; Hulit, J; Agiostratidou, G; Chandiramani, N; McDaid, HM; Nagi, C; Cohen, HW; Phillips, GR; Norton, L; Hazan, RB

    2014-01-01

    N-cadherin and HER2/neu were found to be co-expressed in invasive breast carcinomas. To test the contribution of N-cadherin and HER2 in mammary tumor metastasis, we targeted N-cadherin expression in the mammary epithelium of the MMTV-Neu mouse. In the context of ErbB2/Neu, N-cadherin stimulated carcinoma cell invasion, proliferation and metastasis. N-cadherin caused fibroblast growth factor receptor (FGFR) upmodulation, resulting in epithelial-to-mesenchymal transition (EMT) and stem/progenitor like properties, involving Snail and Slug upregulation, mammosphere formation and aldehyde dehydrogenase activity. N-cadherin potentiation of the FGFR stimulated extracellular signal regulated kinase (ERK) and protein kinase B (AKT) phosphorylation resulting in differential effects on metastasis. Although ERK inhibition suppressed cyclin D1 expression, cell proliferation and stem/progenitor cell properties, it did not affect invasion or EMT. Conversely, AKT inhibition suppressed invasion through Akt 2 attenuation, and EMT through Snail inhibition, but had no effect on cyclin D1 expression, cell proliferation or mammosphere formation. These findings suggest N-cadherin/FGFR has a pivotal role in promoting metastasis through differential regulation of ERK and AKT, and underscore the potential for targeting the FGFR in advanced ErbB2-amplified breast tumors. PMID:23975425

  11. Engineered N-cadherin and L1 biomimetic substrates concertedly promote neuronal differentiation, neurite extension and neuroprotection of human neural stem cells.

    PubMed

    Cherry, Jocie F; Bennett, Neal K; Schachner, Melitta; Moghe, Prabhas V

    2014-10-01

    We investigated the design of neurotrophic biomaterial constructs for human neural stem cells, guided by neural developmental cues of N-cadherin and L1 adhesion molecules. Polymer substrates fabricated either as two-dimensional (2-D) films or three-dimensional (3-D) microfibrous scaffolds were functionalized with fusion chimeras of N-cadherin-Fc alone and in combination with L1-Fc, and the effects on differentiation, neurite extension and survival of H9 human-embryonic-stem-cell-derived neural stem cells (H9-NSCs) were quantified. Combinations of N-cadherin and L1-Fc co-operatively enhanced neuronal differentiation profiles, indicating the critical nature of the two complementary developmental cues. Notably, substrates presenting low levels of N-cadherin-Fc concentrations, combined with proportionately higher L1-Fc concentration, most enhanced neurite outgrowth and the degree of MAP2+ and neurofilament-M+ H9-NSCs. Low N-cadherin-Fc alone promoted improved cell survival following oxidative stress, compared to higher concentrations of N-cadherin-Fc alone or combinations with L1-Fc. Pharmacological and antibody blockage studies revealed that substrates presenting low levels of N-cadherin are functionally competent so long as they elicit a threshold signal mediated by homophilic N-cadherin and fibroblast growth factor signaling. Overall, these studies highlight the ability of optimal combinations of N-cadherin and L1 to recapitulate a "neurotrophic" microenvironment that enhances human neural stem cell differentiation and neurite outgrowth. Additionally, 3-D fibrous scaffolds presenting low N-cadherin-Fc further enhanced the survival of H9-NSCs compared to equivalent 2-D films. This indicates that similar biofunctionalization approaches based on N-cadherin and L1 can be translated to 3-D "transplantable" scaffolds with enhanced neurotrophic behaviors. Thus, the insights from this study have fundamental and translational impacts for neural-stem-cell-based regenerative medicine. PMID:24914828

  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. N-cadherin negatively regulates collective Drosophila glial migration through actin cytoskeleton remodeling.

    PubMed

    Kumar, Arun; Gupta, Tripti; Berzsenyi, Sara; Giangrande, Angela

    2015-03-01

    Cell migration is an essential and highly regulated process. During development, glia cells and neurons migrate over long distances - in most cases collectively - to reach their final destination and build the sophisticated architecture of the nervous system, the most complex tissue of the body. Collective migration is highly stereotyped and efficient, defects in the process leading to severe human diseases that include mental retardation. This dynamic process entails extensive cell communication and coordination, hence, the real challenge is to analyze it in the entire organism and at cellular resolution. We here investigate the impact of the N-cadherin adhesion molecule on collective glial migration, by using the Drosophila developing wing and cell-type specific manipulation of gene expression. We show that N-cadherin timely accumulates in glial cells and that its levels affect migration efficiency. N-cadherin works as a molecular brake in a dosage-dependent manner, by negatively controlling actin nucleation and cytoskeleton remodeling through ?/? catenins. This is the first in vivo evidence for N-cadherin negatively and cell autonomously controlling collective migration. PMID:25593128

  14. 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.

  15. N-cadherin Restrains PTH Activation of Lrp6/?-catenin Signaling and Osteoanabolic Action

    PubMed Central

    Revollo, Leila; Kading, Jacqueline; Jeong, Sung Yeop; Li, Jiemin; Salazar, Valerie; Mbalaviele, Gabriel; Civitelli, Roberto

    2014-01-01

    Interaction between parathyroid hormone/parathyroid hormone-related peptide receptor 1 (PTHR1) and low density lipoprotein receptor-related protein 6 (Lrp6) is important for parathyroid hormone (PTH) signaling and anabolic action. Since N-cadherin has been shown to negatively regulate canonical Wnt/?-catenin signaling, we asked whether N-cadherin alters PTH signaling and stimulation of bone formation. Ablation of the N-cadherin gene (Cdh2) in primary osteogenic lineage cells resulted in increased Lrp6/PTHR1 interaction in response to PTH134, associated with enhanced PTH-induced PKA signaling and PKA-dependent ?-catenin C-terminus phosphorylation, which promotes ?-catenin transcriptional activity. ?-catenin C-terminus phosphorylation was abolished by Lrp6 knockdown. Accordingly, PTH134 stimulation of Tcf/Lef target genes, Lef1 and Axin2, was also significantly enhanced in Cdh2 deficient cells. This enhanced responsiveness to PTH extends to the osteo-anabolic effect of PTH, as mice with a conditional Cdh2 deletion in Osx+ cells treated with intermittent doses of PTH134 exhibited significantly larger gains in trabecular bone mass relative to control mice, the result of accentuated osteoblast activity. Therefore, N-cadherin modulates Lrp6/PTHR1 interaction, restraining the intensity of PTH-induced ?-catenin signaling, and ultimately influencing bone formation in response to intermittent PTH administration. PMID:25088803

  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. Cdon promotes neural crest migration by regulating N-cadherin localization.

    PubMed

    Powell, Davalyn R; Williams, Jason S; Hernandez-Lagunas, Laura; Salcedo, Ernesto; O'Brien, Jenean H; Artinger, Kristin Bruk

    2015-11-15

    Neural crest cells (NCCs) are essential embryonic progenitor cells that are unique to vertebrates and form a remarkably complex and coordinated system of highly motile cells. Migration of NCCs occurs along specific pathways within the embryo in response to both environmental cues and cell-cell interactions within the neural crest population. Here, we demonstrate a novel role for the putative Sonic hedgehog (Shh) receptor and cell adhesion regulator, cdon, in zebrafish neural crest migration. cdon is expressed in developing premigratory NCCs but is downregulated once the cells become migratory. Knockdown of cdon results in aberrant migration of trunk NCCs: crestin positive cells can emigrate out of the neural tube but stall shortly after the initiation of migration. Live cell imaging analysis demonstrates reduced directedness of migration, increased velocity and mispositioned cell protrusions. In addition, transplantation analysis suggests that cdon is required cell-autonomously for directed NCC migration in the trunk. Interestingly, N-cadherin is mislocalized following cdon knockdown suggesting that the role of cdon in NCCs is to regulate N-cadherin localization. Our results reveal a novel role for cdon in zebrafish neural crest migration, and suggest a mechanism by which Cdon is required to localize N-cadherin to the cell membrane in migratory NCCs for directed migration. PMID:26256768

  18. 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 invitro and invivo 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

  19. 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

  20. 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

  1. Adhesives from modified soy protein

    DOEpatents

    Sun, Susan (Manhattan, KS); Wang, Donghai (Manhattan, KS); Zhong, Zhikai (Manhattan, KS); Yang, Guang (Shanghai, CN)

    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.

  2. 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

  3. N-cadherin expression level as a critical indicator of invasion in non-epithelial tumors

    PubMed Central

    Péglion, Florent; Etienne-Manneville, Sandrine

    2012-01-01

    Cancer cell dissemination away from the primary tumor and their ability to form metastases remain the major causes of death from cancer. Understanding the molecular mechanisms triggering this event could lead to the design of new cancer treatments. The establishment and the maintenance of tissue architecture depend on the coordination of cell behavior within this tissue. Cell-cell interactions must form adhesive structures between neighboring cells while remaining highly dynamic to allow and control tissue renewal or remodeling. Among intercellular junctions, cadherin-based adherens junctions mediate strong physical interactions and transmit information from the cell microenvironment to the cytoplasm. Disruption of these cell-cell contacts perturbs the polarity of epithelial tissues leading to their disorganization and ultimately to aggressive carcinomas. In non-epithelial tissues, the role of cadherins in the development of cancer is still debated. We recently found that downregulation of N-cadherin in malignant glioma—the most frequent primary brain tumor—results in cell polarization defects leading to abnormal motile behavior with increased cell speed and decreased persistence in directionality. Re-expression of N-cadherin in glioma cells restores cell polarity and limits glioma cell migration, providing a potential therapeutic tool for diffuse glioma. PMID:22983195

  4. The N-cadherin cytoplasmic domain confers anchorage-independent growth and the loss of contact inhibition

    PubMed Central

    Ozawa, Masayuki

    2015-01-01

    Tumor growth is characterized by anchorage independence and the loss of contact inhibition. Previously, we showed that either a red fluorescent protein (DsRed)-tagged N-cadherin or E-cadherin cytoplasmic domain (DNCT or DECT) could function as a dominant negative inhibitor by blocking the cell surface localization of endogenous E-cadherin and inducing cell dissociation. Here, we show that expression of DNCT abrogated contact inhibition of proliferation and conferred anchorage-independent growth. DNCT expression induced the relocation of the tumor suppressor Merlin from the cell surface to intracellular compartments. Although DNCT expression induced redistribution of TAZ from the cytoplasm to the nucleus, YAP/TAZ signaling was not activated. An E-cadherin?-catenin chimera that functions as a ?-cateninindependent cell adhesion molecule restored contact inhibition and anchorage-dependency of growth. Addition of the SV40 large T antigen nuclear localization signal reversed the effects of DNCT expression, indicating that DNCT functioned outside of the nucleus. PMID:26481443

  5. The N-cadherin cytoplasmic domain confers anchorage-independent growth and the loss of contact inhibition.

    PubMed

    Ozawa, Masayuki

    2015-01-01

    Tumor growth is characterized by anchorage independence and the loss of contact inhibition. Previously, we showed that either a red fluorescent protein (DsRed)-tagged N-cadherin or E-cadherin cytoplasmic domain (DNCT or DECT) could function as a dominant negative inhibitor by blocking the cell surface localization of endogenous E-cadherin and inducing cell dissociation. Here, we show that expression of DNCT abrogated contact inhibition of proliferation and conferred anchorage-independent growth. DNCT expression induced the relocation of the tumor suppressor Merlin from the cell surface to intracellular compartments. Although DNCT expression induced redistribution of TAZ from the cytoplasm to the nucleus, YAP/TAZ signaling was not activated. An E-cadherin-?-catenin chimera that functions as a ?-catenin-independent cell adhesion molecule restored contact inhibition and anchorage-dependency of growth. Addition of the SV40 large T antigen nuclear localization signal reversed the effects of DNCT expression, indicating that DNCT functioned outside of the nucleus. PMID:26481443

  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. N-Cadherin and Keratinocyte Growth Factor Receptor Mediate the Functional Interplay between Ki-RASG12V and p53V143A in Promoting Pancreatic Cell Migration, Invasion, and Tissue Architecture Disruption

    PubMed Central

    Deramaudt, Therese B.; Takaoka, Munenori; Upadhyay, Rabi; Bowser, Mark J.; Porter, Jess; Lee, Amy; Rhoades, Ben; Johnstone, Cameron N.; Weissleder, Ralph; Hingorani, Sunil R.; Mahmood, Umar; Rustgi, Anil K.

    2006-01-01

    The genetic basis of pancreatic ductal adenocarcinoma, which constitutes the most common type of pancreatic malignancy, involves the sequential activation of oncogenes and inactivation of tumor suppressor genes. Among the pivotal genetic alterations are Ki-RAS oncogene activation and p53 tumor suppressor gene inactivation. We explain that the combination of these genetic events facilitates pancreatic carcinogenesis as revealed in novel three-dimensional cell (spheroid cyst) culture and in vivo subcutaneous and orthotopic xenotransplantation models. N-cadherin, a member of the classic cadherins important in the regulation of cell-cell adhesion, is induced in the presence of Ki-RAS mutation but subsequently downregulated with the acquisition of p53 mutation as revealed by gene microarrays and corroborated by reverse transcription-PCR and Western blotting. N-cadherin modulates the capacity of pancreatic ductal cells to migrate and invade, in part via complex formation with keratinocyte growth factor receptor and neural cell adhesion molecule and in part via interaction with p120-catenin. However, modulation of these complexes by Ki-RAS and p53 leads to enhanced cell migration and invasion. This preferentially induces the downstream effector AKT over mitogen-activated protein kinase to execute changes in cellular behavior. Thus, we are able to define molecules that in part are directly affected by Ki-RAS and p53 during pancreatic ductal carcinogenesis, and this provides a platform for potential new molecularly based therapeutic interventions. PMID:16705170

  8. Knock-down of ZBED6 in insulin-producing cells promotes N-cadherin junctions between beta-cells and neural crest stem cells in vitro

    PubMed Central

    Wang, Xuan; Xie, Beichen; Qi, Yu; Wallerman, Ola; Vasylovska, Svitlana; Andersson, Leif; Kozlova, Elena Nickolaevna; Welsh, Nils

    2016-01-01

    The role of the novel transcription factor ZBED6 for the adhesion/clustering of insulin-producing mouse MIN6 and βTC6 cells was investigated. Zbed6-silencing in the insulin producing cells resulted in increased three-dimensional cell-cell clustering and decreased adhesion to mouse laminin and human laminin 511. This was paralleled by a weaker focal adhesion kinase phosphorylation at laminin binding sites. Zbed6-silenced cells expressed less E-cadherin and more N-cadherin at cell-to-cell junctions. A strong ZBED6-binding site close to the N-cadherin gene transcription start site was observed. Three-dimensional clustering in Zbed6-silenced cells was prevented by an N-cadherin neutralizing antibody and by N-cadherin knockdown. Co-culture of neural crest stem cells (NCSCs) with Zbed6-silenced cells, but not with control cells, stimulated the outgrowth of NCSC processes. The cell-to-cell junctions between NCSCs and βTC6 cells stained more intensely for N-cadherin when Zbed6-silenced cells were co-cultured with NCSCs. We conclude that ZBED6 decreases the ratio between N- and E-cadherin. A lower N- to E-cadherin ratio may hamper the formation of three-dimensional beta-cell clusters and cell-to-cell junctions with NCSC, and instead promote efficient attachment to a laminin support and monolayer growth. Thus, by controlling beta-cell adhesion and cell-to-cell junctions, ZBED6 might play an important role in beta-cell differentiation, proliferation and survival. PMID:26750727

  9. The PX-RICS-14-3-3?/? Complex Couples N-cadherin-?-Catenin with Dynein-Dynactin to Mediate Its Export from the Endoplasmic Reticulum*

    PubMed Central

    Nakamura, Tsutomu; Hayashi, Tomoatsu; Mimori-Kiyosue, Yuko; Sakaue, Fumika; Matsuura, Ken; Iemura, Shun-ichiro; Natsume, Toru; Akiyama, Tetsu

    2010-01-01

    We have recently shown that ?-catenin-facilitated export of cadherins from the endoplasmic reticulum requires PX-RICS, a ?-catenin-interacting GTPase-activating protein for Cdc42. Here we show that PX-RICS interacts with isoforms of 14-3-3 and couples the N-cadherin-?-catenin complex to the microtubule-based molecular motor dynein-dynactin. Similar to knockdown of PX-RICS, knockdown of either 14-3-3? or -? resulted in the disappearance of N-cadherin and ?-catenin from the cell-cell boundaries. Furthermore, we found that PX-RICS and 14-3-3?/? are present in a large multiprotein complex that contains dynein-dynactin components as well as N-cadherin and ?-catenin. Both RNAi- and dynamitin-mediated inhibition of dynein-dynactin function also led to the absence of N-cadherin and ?-catenin at the cell-cell contact sites. Our results suggest that the PX-RICS-14-3-3?/? complex links the N-cadherin-?-catenin cargo with the dynein-dynactin motor and thereby mediates its endoplasmic reticulum export. PMID:20308060

  10. Identification of a novel intermediate filament-linked N-cadherin/gamma-catenin complex involved in the establishment of the cytoarchitecture of differentiated lens fiber cells.

    PubMed

    Leonard, Michelle; Chan, Yim; Menko, A Sue

    2008-07-15

    Tissue morphogenesis and maintenance of complex tissue architecture requires a variety of cell-cell junctions. Typically, cells adhere to one another through cadherin junctions, both adherens and desmosomal junctions, strengthened by association with cytoskeletal networks during development. Both beta- and gamma-catenins are reported to link classical cadherins to the actin cytoskeleton, but only gamma-catenin binds to the desmosomal cadherins, which links them to intermediate filaments through its association with desmoplakin. Here we provide the first biochemical evidence that, in vivo, gamma-catenin also mediates interactions between classical cadherins and the intermediate filament cytoskeleton, linked through desmoplakin. In the developing lens, which has no desmosomes, we discovered that vimentin became linked to N-cadherin complexes in a differentiation-state specific manner. This newly identified junctional complex was tissue specific but not unique to the lens. To determine whether in this junction N-cadherin was linked to vimentin through gamma-catenin or beta-catenin we developed an innovative "double" immunoprecipitation technique. This approach made possible, for the first time, the separation of N-cadherin/gamma-catenin from N-cadherin/beta-catenin complexes and the identification of multiple members of each of these isolated protein complexes. The study revealed that vimentin was associated exclusively with N-cadherin/gamma-catenin junctions. Assembly of this novel class of cadherin junctions was coincident with establishment of the unique cytoarchitecture of lens fiber cells. In addition, gamma-catenin had a distinctive localization to the vertices of these hexagonally shaped differentiating lens fiber cells, a region devoid of actin; while beta-catenin co-localized with actin at lateral cell interfaces. We believe this novel vimentin-linked N-cadherin/gamma-catenin junction provides the tensile strength necessary to establish and maintain structural integrity in tissues that lack desmosomes. PMID:18514185

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

    PubMed

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

    2013-11-01

    Mller glia function as retinal stem cells in adult zebrafish. In response to loss of retinal neurons, Mller 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 Mller glia, and that each Mller glial cell divides asymmetrically only once to produce an Alcama-negative, proliferating retinal progenitor. The initial mitotic division of Mller 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 Mller 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

  12. Unconventional Cadherin Localization in Honey Bee Gonads Revealed Through Domain-Specific Apis mellifera E- and N-Cadherin Antibodies Indicates Alternative Functions.

    PubMed

    Florecki, Mônica M; Hartfelder, Klaus

    2012-01-01

    As key factors in intercellular adhesion processes, cadherins play important roles in a plethora of developmental processes, including gametogenesis. In a previous study on cadherin localization in the gonads of honey bees, performed with heterologous pan-cadherin antibodies, we detected these proteins as (i) associated with cell membranes, (ii) as homogeneously distributed throughout the cytoplasm, and (iii) as nuclear foci in both somatic and germline cells, raising the possibility of alternative functions. To further investigate such unusual intracellular cadherin localization we produced specific antibodies against the N- and C-terminal domains of honey bee N- and E-cadherin. A 160 kDa protein was recognized by the E-cadherin antibodies as well as one of approximately 300 kDa from those raised against N-cadherin. In gonad preparations, both proteins were detected as dispersed throughout the cytoplasm and as nuclear foci in both germline and somatic cells of queen and worker ovarioles, as well as in the testioles of drones. This leads us to infer that cadherins may indeed be involved in certain signaling pathways and/or transcriptional regulation during gametogenesis. In late oogenesis stages, immunolabeling for both proteins was observed at the cell cortex, in conformity with a role in cell adhesion. In testioles, E-cadherin was seen in co-localization with fusomes, indicating a possible role in cyst organization. Taken together, the distribution of N- and E-cadherins in honey bee gonads is suggestive of alternative roles for cadherins in gametogenesis of both sexes. PMID:26466735

  13. Unconventional Cadherin Localization in Honey Bee Gonads Revealed Through Domain-Specific Apis mellifera E- and N-Cadherin Antibodies Indicates Alternative Functions

    PubMed Central

    Florecki, Mônica M.; Hartfelder, Klaus

    2012-01-01

    As key factors in intercellular adhesion processes, cadherins play important roles in a plethora of developmental processes, including gametogenesis. In a previous study on cadherin localization in the gonads of honey bees, performed with heterologous pan-cadherin antibodies, we detected these proteins as (i) associated with cell membranes, (ii) as homogeneously distributed throughout the cytoplasm, and (iii) as nuclear foci in both somatic and germline cells, raising the possibility of alternative functions. To further investigate such unusual intracellular cadherin localization we produced specific antibodies against the N- and C-terminal domains of honey bee N- and E-cadherin. A 160 kDa protein was recognized by the E-cadherin antibodies as well as one of approximately 300 kDa from those raised against N-cadherin. In gonad preparations, both proteins were detected as dispersed throughout the cytoplasm and as nuclear foci in both germline and somatic cells of queen and worker ovarioles, as well as in the testioles of drones. This leads us to infer that cadherins may indeed be involved in certain signaling pathways and/or transcriptional regulation during gametogenesis. In late oogenesis stages, immunolabeling for both proteins was observed at the cell cortex, in conformity with a role in cell adhesion. In testioles, E-cadherin was seen in co-localization with fusomes, indicating a possible role in cyst organization. Taken together, the distribution of N- and E-cadherins in honey bee gonads is suggestive of alternative roles for cadherins in gametogenesis of both sexes. PMID:26466735

  14. Shroom3 and a Pitx2-N-cadherin pathway function cooperatively to generate asymmetric cell shape changes during gut morphogenesis

    PubMed Central

    Plageman, Timothy F.; Zacharias, Amanda. L; Gage, Phillip J.; Lang., Richard A.

    2013-01-01

    The cytoskeletal protein Shroom3 is a potent inducer of epithelial cell shape change and is required for lens and neural plate morphogenesis. Analysis of gut morphogenesis in Shroom3 deficient mouse embryos revealed that the direction of gut rotation is also disrupted. It was recently established that Pitx2-dependent, asymmetrical cellular behaviors in the dorsal mesentery (DM) of the early mid-gut, a structure connecting the gut-tube to the rest of the embryo, contribute to the direction of gut rotation in chicken embryos by influencing the direction of the dorsal mesenteric tilt. Asymmetric cell shapes in the DM epithelium are hypothesized to contribute to the tilt, however, it is unclear what lies downstream of Pitx2 to alter epithelial cell shape. The cells of the left DM epithelium in either Pitx2 or Shroom3 deficient embryos are shorter and wider than those in control embryos and resemble the shape of those on the right, demonstrating that like Pitx2, Shroom3 is required for cell shape asymmetry and the leftward DM tilt. Because N-cadherin expression is specific to the left side and is Pitx2 dependent, we determined whether Shroom3 and N-cadherin function together to regulate cell shape in the left DM epithelium. Analysis of mouse embryos lacking one allele of both Shroom3 and N-cadherin revealed that they possess shorter and wider left epithelial DM cells when compared with Shroom3 or N-cadherin heterozygous embryos. This indicates a genetic interaction. Together these data provide evidence that Shroom3 and N-cadherin function cooperatively downstream of Pitx2 to directly regulate cell shape changes necessary for early gut tube morphogenesis. PMID:21726547

  15. Association of N-cadherin levels and downstream effectors of Rho GTPases with dendritic spine loss induced by chronic stress in rat hippocampal neurons.

    PubMed

    Castaeda, Patricia; Muoz, Mauricio; Garca-Rojo, Gonzalo; Ulloa, Jos L; Bravo, Javier A; Mrquez, Ruth; Garca-Prez, M Alexandra; Arancibia, Damaris; Araneda, Karina; Rojas, Paulina S; Mondaca-Ruff, David; Daz-Vliz, Gabriela; Mora, Sergio; Aliaga, Esteban; Fiedler, Jenny L

    2015-10-01

    Chronic stress promotes cognitive impairment and dendritic spine loss in hippocampal neurons. In this animal model of depression, spine loss probably involves a weakening of the interaction between pre- and postsynaptic cell adhesion molecules, such as N-cadherin, followed by disruption of the cytoskeleton. N-cadherin, in concert with catenin, stabilizes the cytoskeleton through Rho-family GTPases. Via their effector LIM kinase (LIMK), RhoA and ras-related C3 botulinum toxin substrate 1 (RAC) GTPases phosphorylate and inhibit cofilin, an actin-depolymerizing molecule, favoring spine growth. Additionally, RhoA, through Rho kinase (ROCK), inactivates myosin phosphatase through phosphorylation of the myosin-binding subunit (MYPT1), producing actomyosin contraction and probable spine loss. Some micro-RNAs negatively control the translation of specific mRNAs involved in Rho GTPase signaling. For example, miR-138 indirectly activates RhoA, and miR-134 reduces LIMK1 levels, resulting in spine shrinkage; in contrast, miR-132 activates RAC1, promoting spine formation. We evaluated whether N-cadherin/?-catenin and Rho signaling is sensitive to chronic restraint stress. Stressed rats exhibit anhedonia, impaired associative learning, and immobility in the forced swim test and reduction in N-cadherin levels but not ?-catenin in the hippocampus. We observed a reduction in spine number in the apical dendrites of CA1 pyramidal neurons, with no effect on the levels of miR-132 or miR-134. Although the stress did not modify the RAC-LIMK-cofilin signaling pathway, we observed increased phospho-MYPT1 levels, probably mediated by RhoA-ROCK activation. Furthermore, chronic stress raises the levels of miR-138 in accordance with the observed activation of the RhoA-ROCK pathway. Our findings suggest that a dysregulation of RhoA-ROCK activity by chronic stress could potentially underlie spine loss in hippocampal neurons. PMID:26010004

  16. VIP and VIP Gene Silencing Modulation of Differentiation Marker N-Cadherin and Cell Shape of Corneal Endothelium in Human Corneas Ex Vivo

    PubMed Central

    Koh, Shay-Whey M.; Chandrasekara, Krish; Abbondandolo, Cara J.; Coll, Timothy J.; Rutzen, Allan R.

    2008-01-01

    Purpose Vasoactive intestinal peptide (VIP) is expressed by corneal endothelial (CE) cells and is present in the aqueous humor, which bathes CE cells in vivo. This study demonstrated the role of CE cell VIP in maintaining the expression level of a CE differentiation marker, N-cadherin, and the hexagonal cell shape. Methods To determine the most effective VIP concentration, bovine corneoscleral explants were treated with 0 (control) and 10?12 to 10?6 M VIP. Paired human corneas (nine donors) from an eye bank were used as control; the other corneas were treated with VIP. To silence endogenous VIP, paired fresh human donor corneas (from seven cadavers) were transduced with VIP shRNA or the control lentiviral particles and then bisected/quartered for quantitative analysis by semiquantitative RT-PCR (for mRNA) and Western blot analysis/immunocytochemistry (for protein), whereas alizarin red S staining revealed CE cell shape. Results VIP concentration dependently increased bovine CE cell N-cadherin mRNA levels, with the maximal effect observed between 10?10 (1.47 0.06-fold; P = 0.002) and 10?8 M VIP (1.48 0.18-fold; P = 0.012). VIP (10?8 M) treatment increased N-cadherin protein levels in bovine and human CE cells to 1.98 0.28-fold (P = 0.005) and 1.17 0.10 (range, 0.91187)-fold (P = 0.050) of their respective controls. VIP antagonist (SN)VIPhyb diminished the VIP effect. VIP silencing resulted in deterioration of the hexagonal cell shape and decreased levels of VIP protein and mRNA, N-cadherin (but not connexin-43) mRNA and protein, and the antiapoptotic Bcl-2 protein. Conclusions Through its autocrine VIP, CE cells play an active role in maintaining the differentiated state and suppressing apoptosis in the corneal endothelium in situ. PMID:18441300

  17. Activity-dependent regulation of {beta}-catenin via {epsilon}-cleavage of N-cadherin

    SciTech Connect

    Uemura, Kengo; Kihara, Takeshi; Kuzuya, Akira; Okawa, Katsuya; Nishimoto, Takaaki; Bito, Haruhiko; Ninomiya, Haruaki; Sugimoto, Hachiro; Kinoshita, Ayae . E-mail: akinoshita@hs.med.kyoto-u.ac.jp; Shimohama, Shun

    2006-07-07

    N-cadherin is essential for excitatory synaptic contact in the hippocampus. Presenilin 1 (PS1) is located at sites of synaptic contact, forming a complex with N-cadherin and {beta}-catenin. Here, we report that human N-cadherin is cleaved by PS1/{gamma}-secretase in response to physiological concentration of glutamate (Glu) stimulation, yielding a fragment Ncad/CTF2. The expression of Ncad/CTF2 in neuronal cells led to its translocation to the nucleus, and caused a prominent enhancement of cytoplasmic and nuclear {beta}-catenin levels in a cell-cell contact dependent manner, via following mechanisms: 1, inhibition of {beta}-catenin phosphorylation; 2, transactivation of {beta}-catenin; and 3, inhibition of N-cadherin transcription, and finally enhanced {beta}-catenin nuclear signaling. Since the regulation of cellular {beta}-catenin level is essential for synaptic function, disruption in the cleavage of N-cadherin may be causally linked to the synaptic dysfunction associated with Alzheimer's disease (AD)

  18. 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.

  19. An adhesive protein capsule of Escherichia coli.

    PubMed Central

    Orskov, I; Birch-Andersen, A; Duguid, J P; Stenderup, J; Orskov, F

    1985-01-01

    The nature of the adhesive capacity of three hemagglutinating Escherichia coli strains that had earlier been described as nonfimbriated was studied. The strains that were isolated from human disease adhered to human buccal and urinary tract epithelial cells, an adhesion that was not inhibited by D-mannose. By crossed immunoelectrophoresis it was shown that the three strains produced a common antigen, Z1, developed after growth at 37 degrees C but not 18 degrees C. One of the strains produced an additional antigen, Z2, of almost the same electrophoretic mobility in crossed immunoelectrophoresis. A mutant of this strain deficient of its polysaccharide K antigen had maintained the adhesive capacity, indicating that the K antigen was not responsible for adhesion. A further mutant of the acapsular mutant produced a strongly reduced amount of the Z antigens and had lost the ability to adhere. The Z1 (and Z2?) antigens were therefore deemed to be responsible for adhesion. In sodium dodecyl sulfate-polyacrylamide gel electrophoresis of extracts of cells of the three strains, a heavy Coomassie-blue stained line was seen, indicating the presence of a protein subunit of molecular weight slightly above 14,400. By immunoblotting with absorbed antiserum, it was shown that this protein was the same as that detected by crossed immunoelectrophoresis. Protease from Streptomyces griseus, but not trypsin, digested the protein. Heating to 100 degrees C did not affect it. By immunoelectron microscopy of embedded and sectioned bacteria that had first been treated with specific antisera and ferritin-labeled antirabbit immunoglobulin, the protein adhesin-antibody complex was found to surround the bacteria as a heavy capsule. After negative staining with uranylacetate (pH approximately 4), the capsule appeared as a mesh of very fine filaments. The possible role of this capsule in the pathogenesis of disease is discussed. Images PMID:2856913

  20. Osteoblastic N-cadherin is not required for microenvironmental support and regulation of hematopoietic stem and progenitor cells

    PubMed Central

    Bromberg, Olga; Frisch, Benjamin J.; Weber, Jonathan M.; Porter, Rebecca L.; Civitelli, Roberto

    2012-01-01

    Hematopoietic stem cell (HSC) regulation is highly dependent on interactions with the marrow microenvironment. Controversy exists on N-cadherin's role in support of HSCs. Specifically, it is unknown whether microenvironmental N-cadherin is required for normal marrow microarchitecture and for hematopoiesis. To determine whether osteoblastic N-cadherin is required for HSC regulation, we used a genetic murine model in which deletion of Cdh2, the gene encoding N-cadherin, has been targeted to cells of the osteoblastic lineage. Targeted deletion of N-cadherin resulted in an age-dependent bone phenotype, ultimately characterized by decreased mineralized bone, but no difference in steady-state HSC numbers or function at any time tested, and normal recovery from myeloablative injury. Intermittent parathyroid hormone (PTH) treatment is well established as anabolic to bone and to increase marrow HSCs through microenvironmental interactions. Lack of osteoblastic N-cadherin did not block the bone anabolic or the HSC effects of PTH treatment. This report demonstrates that osteoblastic N-cadherin is not required for regulation of steady-state hematopoiesis, HSC response to myeloablation, or for rapid expansion of HSCs through intermittent treatment with PTH. PMID:22596259

  1. Cell adhesion to agrin presented as a nanopatterned substrate is consistent with an interaction with the extracellular matrix and not transmembrane adhesion molecules

    PubMed Central

    Wolfram, Tobias; Spatz, Joachim P; Burgess, Robert W

    2008-01-01

    Background Molecular spacing is important for cell adhesion in a number of ways, ranging from the ordered arrangement of matrix polymers extracellularly, to steric hindrance of adhesion/signaling complexes intracellularly. This has been demonstrated using nanopatterned RGD peptides, a canonical extracellular matrix ligand for integrin interactions. Cell adhesion was greatly reduced when the RGD-coated nanoparticles were separated by more than 60 nm, indicating a sharp spacing-dependent threshold for this form of cell adhesion. Results Here we show a similar dependence of cell adhesion on the spacing of agrin, a protein that exists as both a secreted, matrix-bound form and a type-2 transmembrane form in vivo. Agrin was presented as a substrate for cell adhesion assays by anchoring recombinant protein to gold nanoparticles that were arrayed at tunable distances onto glass coverslips. Cells adhered well to nanopatterned agrin, and when presented as uniformly coated substrates, adhesion to agrin was comparable to other well-studied adhesion molecules, including N-Cadherin. Adhesion of both mouse primary cortical neurons and rat B35 neuroblastoma cells showed a spacing-dependent threshold, with a sharp drop in adhesion when the space between agrin-coated nanoparticles increased from 60 to 90 nm. In contrast, adhesion to N-Cadherin decreased gradually over the entire range of distances tested (uniform, 30, 60, 90, and 160 nm). The spacing of the agrin nanopattern also influenced cell motility, and peptide competition suggested adhesion was partially integrin dependent. Finally, differences in cell adhesion to C-terminal agrin fragments of different lengths were detected using nanopatterned substrates, and these differences were not evident using uniformly coated substrates. Conclusion These results suggest nanopatterned substrates may provide a physiological presentation of adhesive substrates, and are consistent with cells adhering to agrin through a mechanism that more closely resembles an interaction with the extracellular matrix than a transmembrane adhesion molecule. PMID:19055842

  2. 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 higher engraftment of N-cad{sup +} LSK cells decreased subsequently in E15.5 and E18.5 FL. It is possible that N-cad expression conferred higher LTR activity to HSCs by facilitating interactions with the perisinusoidal niche, especially at E12.5. The down-regulation of N-cad during FL hematopoiesis may help us better understand the regulation and mobility of HSCs before migration into BM.

  3. Hydrogels that mimic developmentally relevant matrix and N-cadherin interactions enhance MSC chondrogenesis

    PubMed Central

    Bian, Liming; Guvendiren, Murat; Mauck, Robert L.; Burdick, Jason A.

    2013-01-01

    Methacrylated hyaluronic acid (HA) hydrogels provide a backbone polymer with which mesenchymal stem cells (MSCs) can interact through several cell surface receptors that are expressed by MSCs, including CD44 and CD168. Previous studies showed that this 3D hydrogel environment supports the chondrogenesis of MSCs, and here we demonstrate through functional blockade that these specific cell–material interactions play a role in this process. Beyond matrix interactions, cadherin molecules, a family of transmembrane glycoproteins, play a critical role in tissue development during embryogenesis, and N-cadherin is a key factor in mediating cell–cell interactions during mesenchymal condensation and chondrogenesis. In this study, we functionalized HA hydrogels with N-cadherin mimetic peptides and evaluated their role in regulating chondrogenesis and cartilage matrix deposition by encapsulated MSCs. Our results show that conjugation of cadherin peptides onto HA hydrogels promotes both early chondrogenesis of MSCs and cartilage-specific matrix production with culture, compared with unmodified controls or those with inclusion of a scrambled peptide domain. This enhanced chondrogenesis was abolished via treatment with N-cadherin–specific antibodies, confirming the contribution of these N-cadherin peptides to chondrogenesis. Subcutaneous implantation of MSC-seeded constructs also showed superior neocartilage formation in implants functionalized with N-cadherin mimetic peptides compared with controls. This study demonstrates the inherent biologic activity of HA-based hydrogels, as well as the promise of biofunctionalizing HA hydrogels to emulate the complexity of the natural cell microenvironment during embryogenesis, particularly in stem cell-based cartilage regeneration. PMID:23733927

  4. SPARC Fusion Protein Induces Cellular Adhesive Signaling

    PubMed Central

    Cheng, Lamei; Sage, E. Helene; Yan, Qi

    2013-01-01

    Secreted protein, acidic and rich in cysteine (SPARC) has been described as a counteradhesive matricellular protein with a diversity of biological functions associated with morphogenesis, remodeling, cellular migration, and proliferation. We have produced mouse SPARC with a FLAG-tag at the N-terminus of SPARC (Flag-SPARC, FSP) in a Bac-to-Bac baculoviral expression system. After affinity purification, this procedure yields SPARC of high purity, with an electrophoretic mobility of ?44 kDa under reducing conditions, and ?3839 kDa under non-reducing conditions. Unexpectedly, FSP adsorbed to plastic supported cell attachment and spreading, in a calcium-dependent manner. The adhesive activity of native FSP was inhibited by prior incubation with anti-SPARC IgG. Cell adhesion to FSP induced the formation of filopodia and lamellipodia but not focal adhesions that were prominent on cells that were attached to fibronectin. In addition, FSP induced the tyrosine phosphorylation of FAK and paxillin in attached epithelial cells. Erk1/2 and Rac were also activated in cells attached to FSP, but at a lower level in comparison to cells on fibronectin. This study provides new insight into the biological functions of SPARC, a matricellular protein with important roles in cell-extracellualr matrix interactions. PMID:23349702

  5. An Engineered N-Cadherin Substrate for Differentiation, Survival, and Selection of Pluripotent Stem Cell-Derived Neural Progenitors.

    PubMed

    Haque, Amranul; Adnan, Nihad; Motazedian, Ali; Akter, Farhima; Hossain, Sharif; Kutsuzawa, Koichi; Nag, Kakon; Kobatake, Eiry; Akaike, Toshihiro

    2015-01-01

    For stem cell-based treatment of neurodegenerative diseases a better understanding of key developmental signaling pathways and robust techniques for producing neurons with highest homogeneity are required. In this study, we demonstrate a method using N-cadherin-based biomimetic substrate to promote the differentiation of mouse embryonic stem cell (ESC)- and induced pluripotent stem cell (iPSC)-derived neural progenitor cells (NPCs) without exogenous neuro-inductive signals. We showed that substrate-dependent activation of N-cadherin reduces Rho/ROCK activation and ?-catenin expression, leading to the stimulation of neurite outgrowth and conversion into cells expressing neural/glial markers. Besides, plating dissociated cells on N-cadherin substrate can significantly increase the differentiation yield via suppression of dissociation-induced Rho/ROCK-mediated apoptosis. Because undifferentiated ESCs and iPSCs have low affinity to N-cadherin, plating dissociated cells on N-cadherin-coated substrate increase the homogeneity of differentiation by purging ESCs and iPSCs (~30%) from a mixture of undifferentiated cells with NPCs. Using this label-free cell selection approach we enriched differentiated NPCs plated as monolayer without ROCK inhibitor. Therefore, N-cadherin biomimetic substrate provide a powerful tool for basic study of cell-material interaction in a spatially defined and substrate-dependent manner. Collectively, our approach is efficient, robust and cost effective to produce large quantities of differentiated cells with highest homogeneity and applicable to use with other types of cells. PMID:26244942

  6. An Engineered N-Cadherin Substrate for Differentiation, Survival, and Selection of Pluripotent Stem Cell-Derived Neural Progenitors

    PubMed Central

    Haque, Amranul; Akter, Farhima; Hossain, Sharif; Kutsuzawa, Koichi; Nag, Kakon; Kobatake, Eiry; Akaike, Toshihiro

    2015-01-01

    For stem cell-based treatment of neurodegenerative diseases a better understanding of key developmental signaling pathways and robust techniques for producing neurons with highest homogeneity are required. In this study, we demonstrate a method using N-cadherin-based biomimetic substrate to promote the differentiation of mouse embryonic stem cell (ESC)- and induced pluripotent stem cell (iPSC)-derived neural progenitor cells (NPCs) without exogenous neuro-inductive signals. We showed that substrate-dependent activation of N-cadherin reduces Rho/ROCK activation and ?-catenin expression, leading to the stimulation of neurite outgrowth and conversion into cells expressing neural/glial markers. Besides, plating dissociated cells on N-cadherin substrate can significantly increase the differentiation yield via suppression of dissociation-induced Rho/ROCK-mediated apoptosis. Because undifferentiated ESCs and iPSCs have low affinity to N-cadherin, plating dissociated cells on N-cadherin-coated substrate increase the homogeneity of differentiation by purging ESCs and iPSCs (~30%) from a mixture of undifferentiated cells with NPCs. Using this label-free cell selection approach we enriched differentiated NPCs plated as monolayer without ROCK inhibitor. Therefore, N-cadherin biomimetic substrate provide a powerful tool for basic study of cellmaterial interaction in a spatially defined and substrate-dependent manner. Collectively, our approach is efficient, robust and cost effective to produce large quantities of differentiated cells with highest homogeneity and applicable to use with other types of cells. PMID:26244942

  7. Cell adhesion to unnatural ligands mediated by a bifunctional protein.

    PubMed

    Sánchez-Cortés, Juan; Bähr, Katinka; Mrksich, Milan

    2010-07-21

    This paper describes a molecular strategy to restore adhesion of cells to surfaces that otherwise do not present ligands that can mediate adhesion. The approach is based on a carbonic anhydrase fusion protein that binds benzenesulfonamides and that also includes the RGD peptide motif that can bind to cell-surface integrin adhesion receptors. In this way, the fusion protein can bind to a monolayer that presents the benzenesulfonamide ligand, thereby positioning the RGD peptide at the surface, where it can mediate the adhesion and spreading of cells. This strategy may provide a general method for promoting the adhesion of cells to non-natural surfaces or to defective biological matrices. PMID:20583796

  8. Vascular adhesion protein 1 in the eye.

    PubMed

    Luo, Wenting; Xie, Fang; Zhang, Zhongyu; Sun, Dawei

    2013-01-01

    Semicarbazide-sensitive amine oxidase/vascular adhesion protein-1 (SSAO/VAP-1), a dual-function molecule with adhesive and enzymatic properties, is expressed on the surface of vascular endothelial cells of mammals. It also exists as a soluble form (sVAP-1), which is implicated in oxidative stress via its enzymatic activity and can be a prognostic biomarker. Recent evidence suggests that VAP-1 is an important therapeutic target for several inflammation-related ocular diseases, such as uveitis, age-related macular degeneration (AMD), and diabetic retinopathy (DR), by involving in the recruitment of leukocytes at sites of inflammation. Furthermore, VAP-1 plays an important role in the pathogenesis of conjunctival inflammatory diseases such as pyogenic granulomas and the progression of conjunctival lymphoma. VAP-1 may be an alternative therapeutic target in ocular diseases. The in vivo imaging of inflammation using VAP-1 as a target molecule is a novel approach with a potential for early detection and characterization of inflammatory diseases. This paper reviews the critical roles of VAP-1 in ophthalmological diseases which may provide a novel research direction or a potent therapeutic strategy. PMID:23840939

  9. Vascular Adhesion Protein 1 in the Eye

    PubMed Central

    Luo, Wenting; Xie, Fang; Zhang, Zhongyu; Sun, Dawei

    2013-01-01

    Semicarbazide-sensitive amine oxidase/vascular adhesion protein-1 (SSAO/VAP-1), a dual-function molecule with adhesive and enzymatic properties, is expressed on the surface of vascular endothelial cells of mammals. It also exists as a soluble form (sVAP-1), which is implicated in oxidative stress via its enzymatic activity and can be a prognostic biomarker. Recent evidence suggests that VAP-1 is an important therapeutic target for several inflammation-related ocular diseases, such as uveitis, age-related macular degeneration (AMD), and diabetic retinopathy (DR), by involving in the recruitment of leukocytes at sites of inflammation. Furthermore, VAP-1 plays an important role in the pathogenesis of conjunctival inflammatory diseases such as pyogenic granulomas and the progression of conjunctival lymphoma. VAP-1 may be an alternative therapeutic target in ocular diseases. The in vivo imaging of inflammation using VAP-1 as a target molecule is a novel approach with a potential for early detection and characterization of inflammatory diseases. This paper reviews the critical roles of VAP-1 in ophthalmological diseases which may provide a novel research direction or a potent therapeutic strategy. PMID:23840939

  10. ADAM9 Up-Regulates N-Cadherin via miR-218 Suppression in Lung Adenocarcinoma Cells

    PubMed Central

    Sher, Yuh-Pyng; Wang, Li-Ju; Chuang, Li-Ling; Tsai, Mong-Hsun; Kuo, Ting-Ting; Huang, Cheng-Chung; Chuang, Eric Y.; Lai, Liang-Chuan

    2014-01-01

    Lung cancer is the leading cause of cancer death worldwide, and brain metastasis is a major cause of morbidity and mortality in lung cancer. CDH2 (N-cadherin, a mesenchymal marker of the epithelial-mesenchymal transition) and ADAM9 (a type I transmembrane protein) are related to lung cancer brain metastasis; however, it is unclear how they interact to mediate this metastasis. Because microRNAs regulate many biological functions and disease processes (e.g., cancer) by down-regulating their target genes, microRNA microarrays were used to identify ADAM9-regulated miRNAs that target CDH2 in aggressive lung cancer cells. Luciferase assays and western blot analysis showed that CDH2 is a target gene of miR-218. MiR-218 was generated from pri-mir-218-1, which is located in SLIT2, in non-invasive lung adenocarcinoma cells, whereas its expression was inhibited in aggressive lung adenocarcinoma. The down-regulation of ADAM9 up-regulated SLIT2 and miR-218, thus down-regulating CDH2 expression. This study revealed that ADAM9 activates CDH2 through the release of miR-218 inhibition on CDH2 in lung adenocarcinoma. PMID:24705471

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

    PubMed

    LaMora, A; Voigt, M M

    2009-03-31

    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:eGFP(sl1)), we have examined the involvement of cdh2 in the formation of sensory circuits by the peripheral nervous system. The tg(p2xr3.2:eGFP(sl1)) fish allows visualization of neurons comprising the trigeminal, facial, glossopharyngeal and vagal ganglia 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

  12. Dscam Proteins Direct Dendritic Targeting through Adhesion.

    PubMed

    Tadros, Wael; Xu, Shuwa; Akin, Orkun; Yi, Caroline H; Shin, Grace Ji-Eun; Millard, S Sean; Zipursky, S Lawrence

    2016-02-01

    Cell recognition molecules are key regulators of neural circuit assembly. The Dscam family of recognition molecules in Drosophila has been shown to regulate interactions between neurons through homophilic repulsion. This is exemplified by Dscam1 and Dscam2, which together repel dendrites of lamina neurons, L1 and L2, in the visual system. By contrast, here we show that Dscam2 directs dendritic targeting of another lamina neuron, L4, through homophilic adhesion. Through live imaging and genetic mosaics to dissect interactions between specific cells, we show that Dscam2 is required in L4 and its target cells for correct dendritic targeting. In a genetic screen, we identified Dscam4 as another regulator of L4 targeting which acts with Dscam2 in the same pathway to regulate this process. This ensures tiling of the lamina neuropil through heterotypic interactions. Thus, different combinations of Dscam proteins act through distinct mechanisms in closely related neurons to pattern neural circuits. PMID:26844831

  13. Aberrant methylation accounts for cell adhesion-related gene silencing during 3-methylcholanthrene and diethylnitrosamine induced multistep rat lung carcinogenesis associated with overexpression of DNA methyltransferases 1 and 3a

    SciTech Connect

    Liu Wenbin; Cui Zhihong; Ao Lin; Zhou Ziyuan; Zhou Yanhong; Yuan Xiaoyan; Xiang Yunlong; Liu Jinyi Cao Jia

    2011-02-15

    To evaluate the significance of alterations in cell adhesion-related genes methylation during lung multistep carcinogenesis induced by the genotoxic carcinogens 3-methylcholanthrene (MCA) and diethylnitrosamine (DEN), tissue samples microdissected from MCA/DEN-induced rat lung carcinogenesis model were subjected to methylation-specific PCR to evaluate the DNA methylation status of CADM1, TIMP3, E-cadherin and N-cadherin. Immunohistochemistry was used to determine protein expression of CADM1, TIMP3, N-cadherin and the DNA methyltransferases (DNMTs) 1, 3a and 3b. E-cadherin hypermethylation was not detected in any tissue. CADM1, TIMP3 and N-cadherin hypermethylation was correlated with the loss of their protein expression during the progression of pathologic lesions. The prevalence of DNA methylation of at least one gene and the average number of methylated genes increased with the histological progression. DNMT1 and DNMT3a protein expression increased progressively during the stages of lung carcinogenesis, whereas DNMT3b overexpression was only found in several samples. Furthermore, DNMT1 protein expression levels were correlated with CADM1 methylation, and DNMT3a protein expression levels were correlated with CADM1, TIMP3 and N-cadherin methylation. The average number of methylated genes during carcinogenesis was significantly correlated with DNMT1 and DNMT3a protein expression levels. Moreover, mRNA expression of CADM1 significantly increased after treatment with DNMT inhibitor 5-aza-2'-deoxycytidine in CADM1-methylated primary tumor cell lines. Our findings suggest that an accumulation of hypermethylation accounts for cell adhesion-related gene silencing is associated with dynamic changes in the progression of MCA/DEN-induced rat lung carcinogenesis. We suggest that DNMT1 and DNMT3a protein overexpression may be responsible for this aberrant DNA methylation.

  14. 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

  15. 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

  16. 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.

  17. 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...

  18. 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

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

    PubMed

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

    2016-03-01

    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 ?-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

  20. 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

  1. N-cadherin promotes epithelial-mesenchymal transition and cancer stem cell-like traits via ErbB signaling in prostate cancer cells.

    PubMed

    Wang, Min; Ren, Dong; Guo, Wei; Huang, Shuai; Wang, Zeyu; Li, Qiji; Du, Hong; Song, Libing; Peng, Xinsheng

    2016-02-01

    N-cadherin has been reported to be upregulated and associated with metastasis and poor prognosis in prostate cancer patients, however the underlying mechanism still remains puzzling. In the present study, we found that upregulation of N-cadherin enhanced, while downregulation of N-cadherin impaired the invasion, migration, and epithelial to mesenchymal transition (EMT) of prostate cancer (PCa) cells. Overexpression of N-cadherin increased the efficiency of colony and tumor spheroid formation and the stemness factor expression (including c-Myc, Klf4, Sox2 and Oct4), and vice versa. Furthermore, microarray analysis and western blot analysis mechanistically proved that N-cadherin activated ErbB signaling pathway by upregulating the expression of Grb2, pShc and pERK1/2. Importantly, the regulation of N-cadherin on EMT and stemness was counteracted by lapatinib, a specific ErbB signaling pathway inhibitor. Collectively, these findings demonstrate that N-cadherin regulates EMT and stemness of PCa cells via activating ErbB signaling pathway, which indicates the pivotal role of N-cadherin/ErbB axis in the metastasis of prostate cancer. PMID:26647992

  2. 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 properties to produce an environmentally responsible, formaldehyde-free adhesive. It is also imperative to study the adhesion between protein and wood.

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

    PubMed

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

    2016-02-01

    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

  4. 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

  5. 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.

  6. 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

  7. N-cadherin mediates plasticity-induced long-term spine stabilization

    PubMed Central

    Mendez, Pablo; De Roo, Mathias; Poglia, Lorenzo; Klauser, Paul

    2010-01-01

    Excitatory synapses on dendritic spines are dynamic structures whose stability can vary from hours to years. However, the molecular mechanisms regulating spine persistence remain essentially unknown. In this study, we combined repetitive imaging and a gain and loss of function approach to test the role of N-cadherin (NCad) on spine stability. Expression of mutant but not wild-type NCad promotes spine turnover and formation of immature spines and interferes with the stabilization of new spines. Similarly, the long-term stability of preexisting spines is reduced when mutant NCad is expressed but enhanced in spines expressing NCad-EGFP clusters. Activity and long-term potentiation (LTP) induction selectively promote formation of NCad clusters in stimulated spines. Although activity-mediated expression of NCad-EGFP switches synapses to a highly stable state, expression of mutant NCad or short hairpin RNAmediated knockdown of NCad prevents LTP-induced long-term stabilization of synapses. These results identify NCad as a key molecular component regulating long-term synapse persistence. PMID:20440002

  8. N-cadherin mediates plasticity-induced long-term spine stabilization.

    PubMed

    Mendez, Pablo; De Roo, Mathias; Poglia, Lorenzo; Klauser, Paul; Muller, Dominique

    2010-05-01

    Excitatory synapses on dendritic spines are dynamic structures whose stability can vary from hours to years. However, the molecular mechanisms regulating spine persistence remain essentially unknown. In this study, we combined repetitive imaging and a gain and loss of function approach to test the role of N-cadherin (NCad) on spine stability. Expression of mutant but not wild-type NCad promotes spine turnover and formation of immature spines and interferes with the stabilization of new spines. Similarly, the long-term stability of preexisting spines is reduced when mutant NCad is expressed but enhanced in spines expressing NCad-EGFP clusters. Activity and long-term potentiation (LTP) induction selectively promote formation of NCad clusters in stimulated spines. Although activity-mediated expression of NCad-EGFP switches synapses to a highly stable state, expression of mutant NCad or short hairpin RNA-mediated knockdown of NCad prevents LTP-induced long-term stabilization of synapses. These results identify NCad as a key molecular component regulating long-term synapse persistence. PMID:20440002

  9. 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.

  10. Emerging role for ERM proteins in cell adhesion and migration

    PubMed Central

    Chirivino, Dafne; Naba, Alexandra; Zwaenepoel, Ingrid

    2011-01-01

    The highly related ERM (Ezrin, Radixin, Moesin) proteins provide a regulated linkage between the membrane and the underlying actin cytoskeleton. They also provide a platform for the transmission of signals in responses to extracellular cues. Studies in different model organisms and in cultured cells have highlighted the importance of ERM proteins in the generation and maintenance of specific domains of the plasma membrane. A central question is how do ERM proteins coordinate actin filament organization and membrane protein transport/stability with signal transduction pathways to build up complex structures? Through their interaction with numerous partners including membrane proteins, actin cytoskeleton and signaling molecules, ERM proteins have the ability to organize multiprotein complexes in specific cellular compartments. Likewise, ERM proteins participate in diverse functions including cell morphogenesis, endocytosis/exocytosis, adhesion and migration. This review focuses on aspects still poorly understood related to the function of ERM proteins in epithelial cell adhesion and migration. PMID:21343695

  11. N-cadherin locks left-right asymmetry by ending the leftward movement of Hensen's node cells.

    PubMed

    Mendes, Raquel V; Martins, Gabriel G; Cristovo, Ana M; Sade, Leonor

    2014-08-11

    The stereotypic left-right (LR) asymmetric distribution of internal organs is due to an asymmetric molecular cascade in the lateral plate mesoderm (LPM) that is originated at the embryonic node. In chicken embryos, molecular asymmetries at Hensen's node are created by leftward cell movements that occur transiently. What terminates these movements, and, moreover, what is the impact of prolonging them on the LR asymmetry cascade? We show that leftward movements last longer when N-cadherin function is blocked and cease prematurely when N-cadherin is overexpressed on the right side of the node. The prolonged leftward movements lead to loss of asymmetric expression of fgf8 and nodal at the node region. This originates an abnormal expression of the asymmetric genes cer1 and snai1 in the LPM, resulting in mispositioned hearts. We conclude that N-cadherin stops the leftward cell movements and that this termination isan essential step in the establishment of LR asymmetry. PMID:25117685

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

    PubMed Central

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

    2012-01-01

    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 Kir2.1 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

  13. Nanoscale Adhesion Forces between Enamel Pellicle Proteins and Hydroxyapatite

    PubMed Central

    Vukosavljevic, D.; Hutter, J.L.; Helmerhorst, E.J.; Xiao, Y.; Custodio, W.; Zaidan, F.C.; Oppenheim, F.G.; Siqueira, W.L.

    2014-01-01

    The acquired enamel pellicle (AEP) is important for minimizing the abrasion caused by parafunctional conditions as they occur, for instance, during bruxism. It is a remarkable feature of the AEP that a protein/peptide film can provide enough protection in normofunction to prevent teeth from abrasion and wear. Despite its obvious critical role in the protection of tooth surfaces, the essential adhesion features of AEP proteins on the enamel surface are poorly characterized. The objective of this study was to measure the adhesion force between histatin 5, a primary AEP component, and hydroxyapatite (HA) surfaces. Both biotinylated histatin 5 and biotinylated human serum albumin were allowed to adsorb to streptavidin-coated silica microspheres attached to atomic force microscope (AFM) cantilevers. A multimode AFM with a Nanoscope IIIa controller was used to measure the adhesion force between protein-functionalized silica microspheres attached to cantilever tips and the HA surface. The imaging was performed in tapping mode with a Si3N4 AFM cantilever, while the adhesion forces were measured in AFM contact mode. A collection of force-distance curves (~3,000/replicate) was obtained to generate histograms from which the adhesion forces between histatin 5 or albumin and the HA surface were measured. We found that histatin 5 exhibited stronger adhesion forces (90% >1.830 nN) to the HA surface than did albumin (90% > 0.282 nN). This study presents an objective approach to adhesion force measurements between histatin 5 and HA, and provides the experimental basis for measuring the same parameters for other AEP constituents. Such knowledge will help in the design of synthetic proteins and peptides with preventive and therapeutic benefits for tooth enamel. PMID:24591293

  14. Hydrophobicity, Adhesion, and Surface-Exposed Proteins of Gliding Bacteria

    PubMed Central

    Sorongon, Maria L.; Bloodgood, Robert A.; Burchard, Robert P.

    1991-01-01

    The cell surface hydrophobicities of a variety of aquatic and terrestrial gliding bacteria were measured by an assay of bacterial adherence to hydrocarbons (BATH), hydrophobic interaction chromatography, and the salt aggregation test. The bacteria demonstrated a broad range of hydrophobicities. Results among the three hydrophobicity assays performed on very hydrophilic strains were quite consistent. Bacterial adhesion to glass did not correlate with any particular measure of surface hydrophobicity. Several adhesion-defective mutants of Cytophaga sp. strain U67 were found to be more hydrophilic than the wild type, particularly by the BATH assay and hydrophobic interaction chromatography. The very limited adhesion of these mutants correlated well with hydrophilicity as determined by the BATH assay. The hydrophobicities of several adhesion-competent revertants ranged between those of the wild type and the mutants. As measured by the BATH assay, starvation increased hydrophobicity of both the wild type and an adhesion-defective mutant. During filament fragmentation of Flexibacter sp. strain FS-1, marked changes in hydrophobicity and adhesion were accompanied by changes in the arrays of surface-exposed proteins as detected by an immobilized radioiodination procedure. Images PMID:16348583

  15. 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

  16. Vascular adhesion protein-1 blockade suppresses choroidal neovascularization

    PubMed Central

    Noda, Kousuke; She, Haicheng; Nakazawa, Toru; Hisatomi, Toshio; Nakao, Shintaro; Almulki, Lama; Zandi, Souska; Miyahara, Shinsuke; Ito, Yasuhiro; Thomas, Kennard L.; Garland, Rebecca C.; Miller, Joan W.; Gragoudas, Evangelos S.; Mashima, Yukihiko; Hafezi-Moghadam, Ali

    2008-01-01

    Vascular adhesion protein-1 (VAP-1) is an endothelial cell adhesion molecule involved in leukocyte recruitment. Leukocytes and, in particular, macrophages play an important role in the development of choroidal neovascularization (CNV), an integral component of age-related macular degeneration (AMD). Previously, we showed a role for VAP-1 in ocular inflammation. Here, we investigate the expression of VAP-1 in the choroid and its role in CNV development. VAP-1 was expressed in the choroid, exclusively in the vessels, and colocalized in the vessels of the CNV lesions. VAP-1 blockade with a novel and specific inhibitor significantly decreased CNV size, fluorescent angiographic leakage, and the accumulation of macrophages in the CNV lesions. Furthermore, VAP-1 blockade significantly reduced the expression of inflammation-associated molecules such as tumor necrosis factor (TNF) -?, monocyte chemoattractant protein (MCP) -1, and intercellular adhesion molecule (ICAM) -1. This work provides evidence for an important role of VAP-1 in the recruitment of macrophages to CNV lesions, establishing a novel link between VAP-1 and angiogenesis. Inhibition of VAP-1 may become a new therapeutic strategy in the treatment of AMD.Noda, K., She, H., Nakazawa, T., Hisatomi, T., Nakao, S., Almulki, L., Zandi, S., Miyahara, S., Ito, Y., Thomas, K. L., Garland, R. C., Miller, J. W., Gragoudas, E. S., Mashima, Y., Hafezi-Moghadam, A. Vascular adhesion protein-1 blockade suppresses choroidal neovascularization. PMID:18436961

  17. Blockade of vascular adhesion protein-1 attenuates choroidal neovascularization

    PubMed Central

    Yoshikawa, Nami; Ozawa, Yoko; Tsubota, Kazuo; Mashima, Yukihiko; Ishida, Susumu

    2012-01-01

    Purpose Vascular adhesion protein (VAP)-1 is an adhesion molecule elucidated as a mediator of the leukocyte recruitment cascade. The purpose of this study was to investigate the role of VAP-1 in ocular inflammatory neovascularization using a mouse laser-induced choroidal neovascularization (CNV) model. Methods CNV was induced with 532 nm laser irradiation in C57BL/6 mice, and production of VAP-1 protein in the retinal pigment epithelium (RPE) choroid during CNV formation was examined. CNV animals were treated with the specific VAP-1 inhibitor U-V002 or vehicle solution, and the volume of CNV tissue was evaluated with volumetric measurements. Macrophage infiltration into the CNV lesions was evaluated using two different techniques, flatmount staining and real-time polymerase chain reaction (PCR) for F4/80. The protein levels of intercellular adhesion molecule (ICAM)-1, monocyte chemoattractant protein (MCP)-1, P-selectin, and vascular endothelial growth factor (VEGF) in the RPE-choroid were measured with enzyme-linked immunosorbent assay (ELISA). Results VAP-1 inhibition significantly suppressed CNV formation in a dose-dependent manner and reduced macrophage infiltration into CNV lesions. Furthermore, VAP-1 blockade decreased the expression of ICAM-1 and MCP-1, both of which play a pivotal role in macrophage recruitment. Conclusions Our data suggest VAP-1 has an important role during ocular inflammatory neovascularization through leukocyte recruitment. VAP-1 inhibition may be a novel and potent therapeutic strategy in treating CNV formation. PMID:22419852

  18. 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 ?-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

  19. Protein trafficking and Alzheimer's disease.

    PubMed

    Uemura, Kengo; Kuzuya, Akira; Shimohama, Shun

    2004-02-01

    Mutations in presenilin 1 (PS1) cause early-onset familial Alzheimer;s disease (FAD). Although FAD accounts for less than 5% of all cases of Alzheimer;s disease (AD), extensive analyses of PS1 function have elucidated an important neuronal mechanism underling AD pathogenesis. PS1 is considered to be an essential component of gamma-secretase, which cleaves amyloid precursor protein (APP) at the transmembrane region and releases amyloid beta (Abeta) peptide. In addition to this well-documented function, a growing amount of evidence suggests that PS1 is involved in the intracellular trafficking of selected membrane proteins (i.e. APP, nicastrin, trkB, telencephalin). Recently, we have also shown that PS1 is involved in the trafficking of N-cadherin from the endoplasmic reticulum to the plasma membrane via the microtubule network. N-cadherin is localized at the synaptic junctional complex, providing an adhesive force across the synaptic cleft, and the its regulation is crucial for the neuron to exert its specific function, i.e. synaptic activity. In a mature neuron, polarized targeting of proteins from the cell body to the axonal and dendritic processes is essential for its proper function, especially, for the maintenance of synaptic function. Alterations in protein transport caused by a dysfunction in PS1 could lead to a disturbance in synaptic transmission and finally to neurodegeneration. This article will review the current knowledge of PS1 function in protein trafficking and discuss its potential role in AD pathogenesis. PMID:15975080

  20. Adhesion proteins expressed on human gamete surfaces and egg activation.

    PubMed

    Finaz, C; Hammami-Hamza, S

    2000-07-01

    The initiation and propagation of a Ca2+ signal through the egg seems to be the pivotal event in triggering of meiosis resumption. Over the past decade evidence has accumulated suggesting that sperm contact is essential for this phenomenon to occur in most physiological groups. Given their ability to transduce signals, adhesive proteins which are involved in various binding mechanisms such as cell migration, lymphocyte activation, phagocytosis and virus fusion may play a similar role in fertilization. They have been the subject of serious investigation in non-human mammals and some emerging data indicate that they are active in humans as well. Our goal is to review the presence of such molecules on human gametes and their relevant physiological role, i.e., integrins and their ligands, selectins, IgG Fc receptors and leucocyte differentiation markers. We will discuss how they might trigger egg activation through signaling pathways in light of their identified functions in other adhesion systems. The putative participation of specific human sperm proteins will also be evaluated. PMID:11043411

  1. Focal adhesion protein abnormalities in myelodysplastic mesenchymal stromal cells

    SciTech Connect

    Aanei, Carmen Mariana; Eloae, Florin Zugun; Flandrin-Gresta, Pascale; CNRS UMR 5239, Universite de Lyon, 42023, Saint-Etienne ; Tavernier, Emmanuelle; CNRS UMR 5239, Universite de Lyon, 42023, Saint-Etienne ; Carasevici, Eugen; Guyotat, Denis; CNRS UMR 5239, Universite de Lyon, 42023, Saint-Etienne ; Campos, Lydia; CNRS UMR 5239, Universite de Lyon, 42023, Saint-Etienne

    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.

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

    PubMed Central

    Haapasalmi, K.; Mkel, 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

  3. Cell-cell adhesion proteins in melanocytic pilomatrix carcinoma.

    PubMed

    Soler, Alejandro Peralta; Kindel, Susan E; McCloskey, Gayle; Burchette, James L

    2010-01-01

    Tumors of the matrix of rigid structures include matrical tumors of the hairs, nails, and teeth. These tumors share similar phenotypical and signaling features. Although benign matrical hair tumors are among the most common of these tumors, hair matrix tumors containing pigmented melanocytes are very rare. The malignant variant called melanocytic pilomatrix carcinoma contains benign colonizing dendritic melanocytes admixed with the carcinomatous follicular matrical cells.We studied the expression of cadherins and ?-catenin in melanocytic pilomatrix carcinoma because cadherin/catenin-dependent cell-cell adhesion and signals play a critical role in the development of hair and hair tumors. We examined the expression of E- and P-cadherin and the multifunctional protein ?-catenin in two cases of melanocytic pilomatrix carcinoma by immunohistochemistry. E- and P-cadherin are expressed at the cell membrane. In contrast, ?-catenin is distributed uniformly in the nucleus and cytoplasm of all tumor cells. The diffuse nuclear and cytoplasmic ?-catenin expression found in melanocytic pilomatrix carcinomas is indicative of transcriptional activation and ?-catenin-induced cell transformation.This is the first report of cadherin/catenin expression in melanocytic pilomatrix carcinoma. Although the study is limited by the number of these rare tumors, the data add information for the understanding of disease mechanisms in hair matrical tumors. Matrical tumors of the hairs share phenotypical features with other matrical tumors and show nuclear translocation of ?-catenin, suggesting a transcriptional activating rather than a cellcell adhesion function. PMID:21139959

  4. A new crystal form of human vascular adhesion protein 1

    PubMed Central

    Ernberg, Karin; McGrath, Aaron P.; Peat, Thomas S.; Adams, Timothy E.; Xiao, Xiaowen; Pham, Tam; Newman, Janet; McDonald, Ian A.; Collyer, Charles A.; Guss, J. Mitchell

    2010-01-01

    Human vascular adhesion protein 1 (VAP-1) is involved in lymphocyteendothelial cell adhesion and has been implicated in many human inflammatory diseases. VAP-1 is a member of the copper amine oxidase family of enzymes with a trihydroxyphenylalanine quinone (TPQ) cofactor. Previously characterized crystals of VAP-1 suffered from anisotropy and contained disordered regions; in addition, one form was consistently twinned. In an effort to grow crystals that diffracted to higher resolution for inhibitor-binding studies, a construct with an N-terminal deletion was made and expressed in the Chinese hamster ovary (CHO) glycosylation mutant cell line Lec8. Screening produced crystals that displayed some anisotropy and contained seven molecules per asymmetric unit. These crystals belonged to space group C2, with unit-cell parameters a = 394.5, b= 115.8, c = 179.3?, ? = 112.3. The structure was refined to a resolution of 2.9?, with R cryst and R free values of 0.250 and 0.286, respectively. PMID:21139198

  5. 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

  6. 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.

  7. Presenilin 1 and cadherins: stabilization of cell-cell adhesion and proteolysis-dependent regulation of transcription.

    PubMed

    Parisiadou, Loukia; Fassa, Angeliki; Fotinopoulou, Angeliki; Bethani, Ioanna; Efthimiopoulos, Spiros

    2004-01-01

    Presenilin-1 (PS1) has gained intensive attention in relation to Alzheimer's disease, since it has been shown that PS1 mutations are linked to familial Alzheimer's disease (FAD), and that PS1 is a member of the high molecular weight complex of gamma-secretase, which generates the carboxyl end of beta-amyloid peptide (gamma-cleavage). A parallel line of evidence suggests that upon formation of cell-cell contacts, presenilin colocalizes with cadherins at the cell surface and stabilizes the cadherin-based adhesion complex. Under conditions stimulating cell-cell dissociation, cadherins are processed by a PS1/gamma-secretase activity, promoting disassembly of adherens junctions, and resulting in the increase of cytosolic beta-catenin, which is an important regulator of the Wnt/Wingless signaling pathway. PS1 also controls the cleavage of a number of transmembrane proteins at the interface of their transmembrane and cytosolic domains (epsilon-cleavage), producing intracellular fragments with a putative transcriptional role. Remarkably, cleavage of N-cadherin by PS1 produces an intracellular fragment that downregulates CREB-mediated transcription, indicating a role of PS1 in gene expression. PS1 mutations associated with FAD abolish production of the N-cadherin intracellular fragment and thus fail to suppress CREB-dependent transcription. These findings suggest an alternative explanation for FAD that is separate from the widely accepted 'amyloid hypothesis': dysfunction in transcription regulatory mechanisms. PMID:16908988

  8. Expression of lncRNA-CCAT1, E-cadherin and N-cadherin in colorectal cancer and its clinical significance

    PubMed Central

    Ye, Zhenyu; Zhou, Ming; Tian, Bin; Wu, Bin; Li, Juncheng

    2015-01-01

    Objective: To explore the expression and clinical significance of IncRNA-CCAT and EMT related molecule E-cadherin and N-cadherin in colorectal cancer. Methods: The expression of IncRNA-CCAT1, E-cadherin and N-cadherin in 37 colorectal cancer tissue and para-carcinoma tissue was detected using qRT-PCR method, and the correlation of expression level with clinical and pathological features was studied. Results: The expression of IncRNA-CCAT1 in tumor tissue was significantly higher than that in normal para-carcinoma tissue (P < 0.001), and the expression level of CCAT1was significantly correlated with local infiltration depth (P < 0.001), tumor staging (P < 0.001), vascular invasion (P < 0.001) and CA19-9 level (P < 0.001); but not related with age, gender, location of tumor, tumor differentiation level, size of primary lesion and level of CEA (P > 0.05). The expression of E-cadherin in tumor tissues was significantly lower than in normal para-carcinoma tissues (P < 0.001), and the expression of N-cadherin was significantly higher than that in normal para-carcinoma tissues. The decrease in expression of E-cadherin and increase in expression of N-cadherin were significantly correlated with local infiltration depth (P < 0.001), tumor staging (P < 0.001), vascular invasion (P < 0.001), tumor differentiation level (P < 0.001) and CA19-9 level (P < 0.001), however not related with age, gender, tumor location, size of primary lesion and CEA level (P > 0.05). Conclusion: CCAT1 plays an important role in the genesis, development, invasion and metastasis; it mediates the EMT process of colorectal cancer; and its expected to be a new marker and treatment target in colorectal diagnosis and treatment. PMID:26064266

  9. 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

  10. Aberrant Glycosylation of Plasma Proteins in Severe Preeclampsia Promotes Monocyte Adhesion

    PubMed Central

    Kazanjian, Avedis A.; Tinnemore, Deborah; Gafken, Philip R.; Ogata, Yuko; Napolitano, Peter G.; Stallings, Jonathan D.; Ippolito, Danielle L.

    2014-01-01

    Glycosylation of plasma proteins increases during pregnancy. Our objectives were to investigate an anti-inflammatory role of these proteins in normal pregnancies and determine whether aberrant protein glycosylation promotes monocyte adhesion in preeclampsia. Plasma was prospectively collected from nonpregnant controls and nulliparous patients in all 3 trimesters. Patients were divided into cohorts based on the applicable postpartum diagnosis. U937 monocytes were preconditioned with enzymatically deglycosylated plasma, and monocyte adhesion to endothelial cell monolayers was quantified by spectrophotometry. Plasma from nonpregnant controls, first trimester normotensives, and first trimester patients with mild preeclampsia inhibited monocyteendothelial cell adhesion (P < .05), but plasma from first trimester patients with severe preeclampsia and second and third trimester normotensives did not. Deglycosylating plasma proteins significantly increased adhesion in all the cohorts. These results support a role of plasma glycoprotein interaction in monocyteendothelial cell adhesion and could suggest a novel therapeutic target for severe preeclampsia. PMID:23757314

  11. Adhesion G-Protein Coupled Receptors: Elusive Hybrids Come of Age

    PubMed Central

    Simundza, Julia; Cowin, Pamela

    2014-01-01

    Adhesion G-protein coupled receptors (GPCRs) are the most recently identified and least understood subfamily of GPCRs. Adhesion GPCRs are characterized by unusually long ectodomains with adhesion-related repeats that facilitate cell-cell and cell-cell matrix contact, as well as a proteolytic cleavage site-containing domain that is a structural hallmark of the family. Their unusual chimeric structure of adhesion-related ectodomain with a seven-pass transmembrane domain and cytoplasmic signaling makes these proteins highly versatile in mediating cellular signaling in response to extracellular adhesion or cell motility events. The ligand binding and cytoplasmic signaling modes for members of this family are beginning to be elucidated, and recent studies have demonstrated critical roles for Adhesion GPCRs in planar polarity and other important cell-cell and cell-matrix interactions during development and morphogenesis, as well as heritable diseases and cancer. PMID:24229322

  12. 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 cross-linking, the synergy of adhesive blends was evaluated through classical rule-of-mixture. The findings of this study warrants further investigation concerning other potential uses of recovered sludge protein, especially as food supplements and economic implications.

  13. Molecular Architecture of a Complex between an Adhesion Protein from the Malaria Parasite and Intracellular Adhesion Molecule 1*

    PubMed Central

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

    2013-01-01

    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 DBL? 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

  14. 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

  15. 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

  16. 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; Fernndez, M. Soledad; Arias, Jos Luis; Rutland, Mark W.; PaiPresent Address: CenterFunctional Nanomaterials, Brookhaven National Laboratory, 735 Brookhaven Avenue, Upton, New York 11973., Ranjith Krishna; Bergstrm, 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

  17. 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.

  18. G protein betagamma subunits regulate cell adhesion through Rap1a and its effector Radil.

    PubMed

    Ahmed, Syed M; Daulat, Avais M; Meunier, Alexandre; Angers, Stephane

    2010-02-26

    The activation of several G protein-coupled receptors is known to regulate the adhesive properties of cells in different contexts. Here, we reveal that Gbetagamma subunits of heterotrimeric G proteins regulate cell-matrix adhesiveness by activating Rap1a-dependent inside-out signals and integrin activation. We show that Gbetagamma subunits enter in a protein complex with activated Rap1a and its effector Radil and establish that this complex is required downstream of receptor stimulation for the activation of integrins and the positive modulation of cell-matrix adhesiveness. Moreover, we demonstrate that Gbetagamma and activated Rap1a promote the translocation of Radil to the plasma membrane at sites of cell-matrix contacts. These results add to the molecular understanding of how G protein-coupled receptors impinge on cell adhesion and suggest that the Gbetagamma x Rap1 x Radil complex plays important roles in this process. PMID:20048162

  19. 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.

  20. 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.

  1. A distinctive role for focal adhesion proteins in three-dimensional cell motility.

    PubMed

    Fraley, Stephanie I; Feng, Yunfeng; Krishnamurthy, Ranjini; Kim, Dong-Hwee; Celedon, Alfredo; Longmore, Gregory D; Wirtz, Denis

    2010-06-01

    Focal adhesions are large multi-protein assemblies that form at the basal surface of cells on planar dishes, and that mediate cell signalling, force transduction and adhesion to the substratum. Although much is known about focal adhesion components in two-dimensional (2D) systems, their role in migrating cells in a more physiological three-dimensional (3D) matrix is largely unknown. Live-cell microscopy shows that for cells fully embedded in a 3D matrix, focal adhesion proteins, including vinculin, paxillin, talin, alpha-actinin, zyxin, VASP, FAK and p130Cas, do not form aggregates but are diffusely distributed throughout the cytoplasm. Despite the absence of detectable focal adhesions, focal adhesion proteins still modulate cell motility, but in a manner distinct from cells on planar substrates. Rather, focal adhesion proteins in matrix-embedded cells regulate cell speed and persistence by affecting protrusion activity and matrix deformation, two processes that have no direct role in controlling 2D cell speed. This study shows that membrane protrusions constitute a critical motility/matrix-traction module that drives cell motility in a 3D matrix. PMID:20473295

  2. 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.

  3. Enterococcus faecalis surface proteins determine its adhesion mechanism to bile drain materials.

    PubMed

    Waar, Karola; van der Mei, Henny C; Harmsen, Hermie J M; Degener, John E; Busscher, Henk J

    2002-06-01

    An important step in infections associated with biliary drains is adhesion of micro-organisms to the surface. In this study the role of three surface proteins of Enterococcus faecalis (enterococcal surface protein, aggregation substances 1 and 373) in the adhesion to silicone rubber, fluoro-ethylene-propylene and polyethylene was examined. Four isogenic E. faecalis strains with and without aggregation substances and one strain expressing enterococcal surface protein were used. The kinetics of enterococcal adhesion to the materials was measured in situ in a parallel plate flow chamber. Initial deposition rates were similar for all strains, whereas the presence of surface proteins increased the total number of adhering bacteria. Nearest neighbour analysis demonstrated that enterococci expressing the whole sex-pheromone plasmid encoding aggregation substances 1 or 373 adhered in higher numbers through mechanisms of positive cooperativity, which means that adhesion of bacteria enhances the probability of adhesion of other bacteria near these bacteria. Enterococci with the enterococcal surface protein did not adhere through this mechanism. These findings indicate that the surface proteins of E. faecalis play a key role in the adhesion to bile drains and bile drain associated infections. PMID:12055306

  4. Adhesive Modular Proteins Occur in the Extracellular Mucilage of the Motile, Pennate Diatom Phaeodactylum tricornutum

    PubMed Central

    Dugdale, Tony M.; Willis, Anusuya; Wetherbee, Richard

    2006-01-01

    This Letter reports on adhesive modular proteins recorded by atomic force microscopy on live cells from the extracellular mucilage secreted from, and deposited around, the motile form of the pennate diatom Phaeodactylum tricornutum. This is the first report of modular proteins and their supramolecular assemblies, called adhesive nanofibers (ANFs), to be found on diatoms that use adhesives not only for substratum adhesion, but as a conduit for cell motility. The permanent adhesive pads secreted by Toxarium undulatum, a sessile centric diatom, were previously shown to possess ANFs with a modular protein backbone. Our results reported here suggest that modular proteins may be an important component of diatom adhesives in general, and that diatoms utilize the tensile strength, toughness, and flexibility of ANFs for multiple functions. Significantly, the genome of P. tricornutum has recently been sequenced; this will allow directed searches of the genome to be made for genes with modular protein homologs, and subsequent detailed studies of their molecular structure and function. PMID:16500978

  5. 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 greater amounts of vWF.

  6. 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.

  7. 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

  8. Plant protein interactions studied using AFM force spectroscopy: nanomechanical and adhesion properties.

    PubMed

    Fahs, Ahmad; Louarn, Guy

    2013-07-21

    The present work was focused on the nanomechanical and adhesion properties of the napin (2S albumin) and cruciferin (12S globulin) rapeseed (Brassica napus L.) proteins, respectively, a low and high molecular weight seed protein. Using chemically modified AFM tips, force spectroscopy experiments demonstrated notable differences in the tip-protein interaction strength with regard to the nature of the protein and pH of the aqueous environment. The results clearly underline the role of residence time and electrostatic interactions in the protein-protein adhesion force. Although the nanomechanical experiments concerned more than a single molecule, unfolding length and force characteristics of the rapeseed proteins have been statistically found to be sensitive to the structural properties of the protein. This study provides insight into the characterization of rapeseed proteins and then a better knowledge of their interaction and assembling at the nanoscale range. PMID:23732983

  9. Synergistic roles for lipids and proteins in the permanent adhesive of barnacle larvae.

    PubMed

    Gohad, Neeraj V; Aldred, Nick; Hartshorn, Christopher M; Jong Lee, Young; Cicerone, Marcus T; Orihuela, Beatriz; Clare, Anthony S; Rittschof, Dan; Mount, Andrew S

    2014-01-01

    Thoracian barnacles rely heavily upon their ability to adhere to surfaces and are environmentally and economically important as biofouling pests. Their adhesives have unique attributes that define them as targets for bio-inspired adhesive development. With the aid of multi-photon and broadband coherent anti-Stokes Raman scattering microscopies, we report that the larval adhesive of barnacle cyprids is a bi-phasic system containing lipids and phosphoproteins, working synergistically to maximize adhesion to diverse surfaces under hostile conditions. Lipids, secreted first, possibly displace water from the surface interface creating a conducive environment for introduction of phosphoproteins while simultaneously modulating the spreading of the protein phase and protecting the nascent adhesive plaque from bacterial biodegradation. The two distinct phases are contained within two different granules in the cyprid cement glands, implying far greater complexity than previously recognized. Knowledge of the lipidic contribution will hopefully inspire development of novel synthetic bioadhesives and environmentally benign antifouling coatings. PMID:25014570

  10. Attenuation of leukocyte adhesion by recombinant TNF-binding protein after hemorrhagic shock in the rat.

    PubMed

    Maier, Marcus; Strbele, Hubert; Voges, Jaqueline; Bauer, Clemens; Marzi, Ingo

    2003-05-01

    Ischemia/reperfusion injury involves a large number of humoral and cellular mediators that activate leukocytes that subsequently migrate to local tissues. Tumor necrosis factor (TNF)-alpha may be one of the most important mediators of this post-shock inflammatory response. In this study, we investigated the influence of a recombinant Type I (55 kDa) TNF-binding protein (TNF-BP) on leukocyte-endothelial interactions in the liver after hemorrhagic shock. Hemorrhagic shock was induced in female Sprague-Dawley rats (40 mmHg for 90 min) and a standardized resuscitation regimen was applied. At the time of resuscitation, animals were treated intravenously with either TNF-BP 4 mg/kg or placebo. The liver microcirculation was investigated using intravital fluorescence microscopy and immunohistochemistry at 5 h and 48 h after reperfusion. At 5 h, treatment with TNF-BP significantly reduced temporary leukocyte adhesion in the liver sinusoids as well as mean adhesion time of leukocytes in the hepatic central vein. In contrast, after 48 h, permanent leukocyte adhesion in the central hepatic vein was significantly reduced in the group receiving TNF-BP, whereas temporary leukocyte adhesion and mean adhesion time did not differ between the two groups. Both types of leukocyte adhesion, rolling adhesion after 5 h and firm adhesion after 48 h, were reduced in the group treated with TNF-BP, thereby suggesting a long-lasting anti-inflammatory effect. PMID:12744490

  11. Binding of the WASP/N-WASP-Interacting Protein WIP to Actin Regulates Focal Adhesion Assembly and Adhesion

    PubMed Central

    Massaad, Michel J.; Kumar, Lalit; Koduru, Suresh; Sasahara, Yoji; Anton, Ines; Bhasin, Manoj; Libermann, Towia

    2014-01-01

    The actin cytoskeleton is essential for cell adhesion and migration, functions important for tumor invasion. In addition to binding N-WASP/WASP, WIP binds and stabilizes F-actin. WIP?/? fibroblasts were used to test the role of WIP in F-actin function. WIP?/? cells had defective focal adhesion (FA), stress fiber assembly, and adherence to substrates, functions that were restored by transduction of wild-type WIP. Protein and mRNA levels of several FA constituents regulated by the myocardin-related transcription factor (MRTF)serum response factor (SRF) transcription factor complex were reduced in WIP?/? fibroblasts. The level of G-actin, which sequesters MRTF in the cytoplasm, was increased, and nuclear localization of MRTF-A and SRF was reduced, in WIP?/? fibroblasts. Transfection of an MRTF-A mutant that constitutively translocates to the nucleus or transfection of constitutively active SRF restored FA and stress fiber assembly. Fibroblasts from knock-in mice expressing a WIP mutant that fails to bind actin phenocopied WIP?/? fibroblasts. Thus, WIP is a novel regulator of FA assembly and cell adhesion. PMID:24797074

  12. Adhesive performance of sorghum protein extracted from sorghum DDGS and flour

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Distillers dried grains with solubles (DDGS) is the main co-product from grain-based ethanol production. The objective of this research was to compare the adhesive performance of three types of sorghum proteins: acetic acid-extracted sorghum protein from DDGS (PI), aqueous ethanol-extracted sorghum ...

  13. Effects of ECM Protein Mimetics on Adhesion and Proliferation of Chorion Derived Mesenchymal Stem Cells

    PubMed Central

    Kim, Ji-Hyun; Jekarl, Dong Wook; Kim, Myungshin; Oh, Eun-Jee; Kim, Yonggoo; Park, In Yang; Shin, Jong Chul

    2014-01-01

    Background: We evaluated the effects of fibronectin, collagen, cadherin, and laminin based extracellular matrix (ECM) protein mimetics coated with mussel derived adhesive protein (MAP) on adhesion and proliferation of chorionic mesenchymal stem cells (cMSCs). Methods: Human placental chorionic tissues from term third-trimester pregnancies (n=3) were used. The cMSCs were cultured on rationally designed ECM protein mimetics coated with MAP on plastic surfaces with the addition of reduced fetal bovine serum (0.5%, 1% FBS). Adhesion capabilities were monitored by a real time cell analysis system (RTCA) utilizing an impedance method. Proliferation capabilities were monitored by RTCA and MTS assay. Results: Of the ECM protein mimetics tested, GRGDSP(FN) coated surfaces exhibited the highest adhesion and proliferation capabilities on RTCA at FBS concentration of 0.5% and 1%. When 0.5% FBS was added to ECM protein mimetics during the MTS assay, GRGDSP(FN), REDV(FN), and collagen mimetics, GPKGAAGEPGKP(ColI) showed higher cMSCs proliferation compared with the control. When 1% FBS was added, GRGDSP(FN) and TAIPSCPEGTVPLYS(ColIV) showed significant cMSCs proliferation capacity. Conclusions: Fibronectin mimetics, GRGDSP(FN) amino acid sequence showed the highest adhesion and proliferation capabilities. In addition, results from RTCA assessment of cell viability correlated well with the tetrazolium-based MTS assay. PMID:24516355

  14. 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

  15. Rapid functional analysis in Xenopus oocytes of Po protein adhesive interactions.

    PubMed

    Yoshida, M; Colma, D R

    2001-06-01

    We have developed a coupled Xenopus oocyte expression system for evaluating the functional effects of mutations in known or suspected adhesion molecules, which allows for a very rapid assessment of intercellular adhesion. As a model protein, we first used Protein zero (Po), an adhesion molecule that mediates self-adhesion of the Schwann cell plasma membrane to form compact myelin in the mammalian PNS. A wide variety of mutations in Po cause certain human peripheral neuropathies, such as the Charcot-Marie-Tooth disease (CMT) type 1B and Dejerine-Sottas syndrome (DSS). After wild-type Po mRNA is injected, the protein is synthesized and correctly targeted to the oocyte cell surface. When two oocytes are paired, wild-type Po redistributes and concentrates at the cell-cell apposition region, and by electron microscopy, the oocyte pairs show close cell-cell appositions and are devoid of the microvilli that are observed in uninjected oocyte pairs. These are hallmark features of highly adhesive cell:cell interfaces. Several point mutations in Po were engineered, corresponding to the molecular defects in the CMT type 1B or DSS. The proteins encoded by these mutations reached the cell surface but failed to concentrate at the oocyte interface. Po carrying a point mutation that is found in DSS is not targeted on the plasma membrane and fail to accumulate at the cell-cell contact site. PMID:11519730

  16. c-Yes regulates cell adhesion at the blood-testis barrier and the apical ectoplasmic specialization in the seminiferous epithelium of rat testes.

    PubMed

    Xiao, Xiang; Mruk, Dolores D; Lee, Will M; Cheng, C Yan

    2011-04-01

    During spermatogenesis, extensive junction restructuring takes place at the blood-testis barrier (BTB) and the Sertoli cell-spermatid interface known as the apical ectoplasmic specialization (apical ES, a testis-specific adherens junction) in the seminiferous epithelium. However, the mechanism(s) that regulates these critical events in the testis remains unknown. Based on the current concept in the field, changes in the phosphorylation status of integral membrane proteins at these sites can induce alterations in protein endocytosis and recycling, causing junction restructuring. Herein, c-Yes, a non-receptor protein tyrosine kinase, was found to express abundantly at the BTB and apical ES stage-specifically, coinciding with junction restructuring events at these sites during the seminiferous epithelial cycle of spermatogenesis. c-Yes also structurally associated with adhesion proteins at the BTB (e.g., occludin and N-cadherin) and the apical ES (e.g., β1-integrin, laminins β3 and γ3), possibly to regulate phosphorylation status of proteins at these sites. SU6656, a selective c-Yes inhibitor, was shown to perturb the Sertoli cell tight junction-permeability barrier in vitro, which is mediated by changes in the distribution of occludin and N-cadherin at the cell-cell interface, moving from cell surface to cytosol, thereby destabilizing the tight junction-barrier. However, this disruptive effect of SU6656 on the barrier was blocked by testosterone. Furthermore, c-Yes is crucial to maintain the actin filament network in Sertoli cells since a blockade of c-Yes by SU6656 induced actin filament disorganization. In summary, c-Yes regulates BTB and apical ES integrity by maintaining proper distribution of integral membrane proteins and actin filament organization at these sites. PMID:21256972

  17. 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

  18. Proteomic analysis of ?4?1 integrin adhesion complexes reveals ?-subunit-dependent protein recruitment.

    PubMed

    Byron, Adam; Humphries, Jonathan D; Craig, Sue E; Knight, David; Humphries, Martin J

    2012-07-01

    Integrin adhesion receptors mediate cell-cell and cell-extracellular matrix interactions, which control cell morphology and migration, differentiation, and tissue integrity. Integrins recruit multimolecular adhesion complexes to their cytoplasmic domains, which provide structural and mechanosensitive signaling connections between the extracellular and intracellular milieux. The different functions of specific integrin heterodimers, such as ?4?1 and ?5?1, have been attributed to distinct signal transduction mechanisms that are initiated by selective recruitment of adhesion complex components to integrin cytoplasmic tails. Here, we report the isolation of ligand-induced adhesion complexes associated with wild-type ?4?1 integrin, an activated ?4?1 variant in the absence of the ? cytoplasmic domain (X4C0), and a chimeric ?4?1 variant with ?5 leg and cytoplasmic domains (?4P?5L), and the cataloguing of their proteomes by MS. Using hierarchical clustering and interaction network analyses, we detail the differential recruitment of proteins and highlight enrichment patterns of proteins to distinct adhesion complexes. We identify previously unreported components of integrin adhesion complexes and observe receptor-specific enrichment of molecules with previously reported links to cell migration and cell signaling processes. Furthermore, we demonstrate colocalization of MYO18A with active integrin in migrating cells. These datasets provide a resource for future studies of integrin receptor-specific signaling events. PMID:22623428

  19. Intraperitoneal administration of activated protein C prevents postsurgical adhesion band formation.

    PubMed

    Dinarvand, Peyman; Hassanian, Seyed Mahdi; Weiler, Hartmut; Rezaie, Alireza R

    2015-02-19

    Postsurgical peritoneal adhesion bands are the most important causes of intestinal obstruction, pelvic pain, and female infertility. In this study, we used a mouse model of adhesion and compared the protective effect of activated protein C (APC) to that of the Food and Drug Administration-approved antiadhesion agent, sodium hyaluronate/carboxymethylcellulose (Seprafilm) by intraperitoneal administration of either APC or Seprafilm to experimental animals. Pathological adhesion bands were graded on day 7, and peritoneal fluid concentrations of tissue plasminogen activator (tPA), d-dimer, thrombin-antithrombin complex, and cytokines (IL-1?, IL-6, interferon-?, tumor necrosis factor-?, transforming growth factor-?1) were evaluated. Inflammation scores were also measured based on histologic data obtained from peritoneal tissues. Relative to Seprafilm, intraperitoneal administration of human APC led to significantly higher reduction of postsurgical adhesion bands. Moreover, a markedly lower inflammation score was obtained in the adhesive tissues of the APC-treated group, which correlated with significantly reduced peritoneal concentrations of proinflammatory cytokines and an elevated tPA level. Further studies using variants of human APC with or without protease-activated receptor 1 (PAR1) signaling function and mutant mice deficient for either endothelial protein C receptor (EPCR) or PAR1 revealed that the EPCR-dependent signaling activity of APC is primarily responsible for its protective activity in this model. These results suggest APC has therapeutic potential for preventing postsurgical adhesion bands. PMID:25575539

  20. Protein-to-film adhesion as examined by amino analysis of protein binding to three different packaging films.

    PubMed

    Clardy, C B; Han, I Y; Acton, J C; Wardlaw, F B; Bridges, W B; Dawson, P L

    1998-05-01

    A weak protein solution extracted from chicken breast meat was exposed to three types of packaging materials. The crude myofibrillar protein solution (12.0 mg protein/mL buffer) was suspended in a 0.6M NaCl/NaPO4 buffer, then placed in bags made from either polyethylene (nonbinding film), a nylon blend (binding film), or Surlyn (binding film). Two separate experiments were conducted to determine the effects of exposure time at a constant temperature and varying endpoint exposure temperatures on the amount of bound protein by amino acid analysis. Bound amino acids were quantified and grouped by class based on functional side group. It was theorized that differences in the amount of bound amino acid class was linked to the mechanism by which the meat-to-film binding occurs. The protein solution was sealed in bags and held in a water bath for 5 s, 20 min, 40 min, and 60 min at 25.8 C for the timed experiment and heated from 25.8 C to 40, 55, 70, and 80 C for the temperature experiment. Protein adhesion occurred due to exposure of the solution to all films at 25.8 C. Greater protein adhesion was found in the two binding films than in the nonbinding film after 60 min of exposure. Heating the protein solution increased adhesion for the Surlyn film and showed a clear delineation in the degree of binding between the film types. Surlyn bound the most protein, followed by the nylon blend and then polyethylene. Bound protein increased in the Surlyn film with heating to 80 C, whereas the polyethylene did not show an increase in the amount of bound protein. Increases in binding observed between 55 and 80 C for Surlyn may be associated with transitional and conformational changes in muscle proteins that affect the adhesion of meat to the film surface. PMID:9603364

  1. Adhesion mechanisms of the mussel foot proteins mfp-1 and mfp-3

    PubMed Central

    Lin, Qi; Gourdon, Delphine; Sun, Chengjun; Holten-Andersen, Niels; Anderson, Travers H.; Waite, J. Herbert; Israelachvili, Jacob N.

    2007-01-01

    Mussels adhere to a variety of surfaces by depositing a highly specific ensemble of 3,4-dihydroxyphenyl-l-alanine (DOPA) containing proteins. The adhesive properties of Mytilus edulis foot proteins mfp-1 and mfp-3 were directly measured at the nano-scale by using a surface forces apparatus (SFA). An adhesion energy of order W ?3 10?4 J/m2 was achieved when separating two smooth and chemically inert surfaces of mica (a common alumino-silicate clay mineral) bridged or glued by mfp-3. This energy corresponds to an approximate force per plaque of ?100 gm, more than enough to hold a mussel in place if no peeling occurs. In contrast, no adhesion was detected between mica surfaces bridged by mfp-1. AFM imaging and SFA experiments showed that mfp-1 can adhere well to one mica surface, but is unable to then link to another (unless sheared), even after prolonged contact time or increased load (pressure). Although mechanistic explanations for the different behaviors are not yet possible, the results are consistent with the apparent function of the proteins, i.e., mfp-1 is disposed as a protective coating, and mfp-3 as the adhesive or glue that binds mussels to surfaces. The results suggest that the adhesion on mica is due to weak physical interactions rather than chemical bonding, and that the strong adhesion forces of plaques arise as a consequence of their geometry (e.g., their inability to be peeled off) rather than a high intrinsic surface or adhesion energy, W. PMID:17360430

  2. 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

  3. Corneal cell adhesion to contact lens hydrogel materials enhanced via tear film protein deposition.

    PubMed

    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

  4. 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

  5. Proteins involved in focal adhesion signaling pathways are differentially regulated in experimental branch retinal vein occlusion.

    PubMed

    Cehofski, Lasse Jørgensen; Kruse, Anders; Kjærgaard, Benedict; Stensballe, Allan; Honoré, Bent; Vorum, Henrik

    2015-09-01

    Branch retinal vein occlusion (BRVO) is a common retinal vascular disease, but global protein changes following the condition remain largely unelucidated. To bring new insights into pathological processes and identify potential therapeutic targets, large-scale retinal protein changes following BRVO were studied by combining a porcine model of experimental BRVO with proteomic analysis by label-free liquid chromatography mass spectrometry. Among a total set of 1974 proteins, 52 significantly upregulated proteins and 10 significantly downregulated proteins were identified in retinas with BRVO after 15 days. Significantly upregulated proteins were involved in signaling pathways of focal adhesion via integrin and blood coagulation. Proteins involved in focal adhesion signaling included collagen α-2 chain, laminin subunit β-2, laminin subunit γ-1, lipocalin-7, nidogen-2, osteopontin, integrin-β, α-actinin-1, isoform 2 of α-actinin-1, talin-2 and filamin C. The identified proteins indicate that BRVO was associated with extracellular matrix remodeling processes. The present study identified focal adhesion signaling and ECM remodeling as important biological mechanisms to evaluate in the search for signaling pathways that promote neovascularisation and macular edema following BRVO. PMID:26086079

  6. Diversity of bone matrix adhesion proteins modulates osteoblast attachment and organization of actin cytoskeleton.

    PubMed

    Demais, V; Audrain, C; Mabilleau, G; Chappard, D; Basl, M F

    2014-06-01

    Interaction of cells with extracellular matrix is an essential event for differentiation, proliferation and activity of osteoblasts. In bone, binding of osteoblasts to bone matrix is required to determine specific activities of the cells and to synthesize matrix bone proteins. Integrins are the major cell receptors involved in the cell linkage to matrix proteins such as fibronectin, type I collagen and vitronectin, via the RGD-sequences. In this study, cultures of osteoblast-like cells (Saos-2) were done on coated glass coverslips in various culture conditions: DMEM alone or DMEM supplemented with poly-L-lysine (PL), fetal calf serum (FCS), fibronectin (FN), vitronectin (VN) and type I collagen (Col-I). The aim of the study was to determine the specific effect of these bone matrix proteins on cell adherence and morphology and on the cytoskeleton status. Morphological characteristics of cultured cells were studied using scanning electron microscopy and image analysis. The heterogeneity of cytoskeleton was studied using fractal analysis (skyscrapers and blanket algorithms) after specific preparation of cells to expose the cytoskeleton. FAK and MAPK signaling pathways were studied by western blotting in these various culture conditions. Results demonstrated that cell adhesion was reduced with PL and VN after 240 min. After 60 min of adhesion, cytoskeleton organization was enhanced with FN, VN and Col-I. No difference in FAK phosphorylation was observed but MAPK phosphorylation was modulated by specific adhesion on extracellular proteins. These results indicate that culture conditions modulate cell adhesion, cytoskeleton organization and intracellular protein pathways according to extracellular proteins present for adhesion. PMID:24735942

  7. 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 ?-casein, ?-casein, ?-casein, and ?-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

  8. 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...

  9. 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...

  10. Substrate, focal adhesions, and actin filaments: a mechanical unit with a weak spot for mechanosensitive proteins.

    PubMed

    Kirchenbchler, David; Born, Simone; Kirchgessner, Norbert; Houben, Sebastian; Hoffmann, Bernd; Merkel, Rudolf

    2010-05-19

    Mechanosensing is a vital prerequisite for dynamic remodeling of focal adhesions and cytoskeletal structures upon substrate deformation. For example, tissue formation, directed cell orientation or cell differentiation are regulated by such mechanosensing processes. Focal adhesions and the actin cytoskeleton are believed to be involved in these processes, but where mechanosensing molecules are located and how elastic substrate, focal adhesions and the cytoskeleton couple with each other upon substrate deformation still remains obscure. To approach these questions we have developed a sensitive method to apply defined spatially decaying deformation fields to cells cultivated on ultrasoft elastic substrates and to accurately quantify the resulting displacements of the actin cytoskeleton, focal adhesions, as well as the substrate. Displacement fields were recorded in live cell microscopy by tracking either signals from fluorescent proteins or marker particles in the substrate. As model cell type we used myofibroblasts. These cells are characterized by highly stable adhesion and force generating structures but are still able to detect mechanical signals with high sensitivity. We found a rigid connection between substrate and focal adhesions. Furthermore, stress fibers were found to be barely extendable almost over their whole lengths. Plastic deformation took place only at the very ends of actin filaments close to focal adhesions. As a result, this area became elongated without extension of existing actin filaments by polymerization. Both ends of the stress fibers were mechanically coupled with detectable plastic deformations on either site. Interestingly, traction force dependent substrate deformation fields remained mostly unaffected even when stress fiber elongations were released. These data argue for a location of mechanosensing proteins at the ends of actin stress fibers and describe, except for these domains, the whole system to be relatively rigid for tensile strain with a mechanical coupling between the front and rear end of a cell. PMID:21386436

  11. Substrate, focal adhesions, and actin filaments: a mechanical unit with a weak spot for mechanosensitive proteins

    NASA Astrophysics Data System (ADS)

    Kirchenbchler, David; Born, Simone; Kirchgener, Norbert; Houben, Sebastian; Hoffmann, Bernd; Merkel, Rudolf

    2010-05-01

    Mechanosensing is a vital prerequisite for dynamic remodeling of focal adhesions and cytoskeletal structures upon substrate deformation. For example, tissue formation, directed cell orientation or cell differentiation are regulated by such mechanosensing processes. Focal adhesions and the actin cytoskeleton are believed to be involved in these processes, but where mechanosensing molecules are located and how elastic substrate, focal adhesions and the cytoskeleton couple with each other upon substrate deformation still remains obscure. To approach these questions we have developed a sensitive method to apply defined spatially decaying deformation fields to cells cultivated on ultrasoft elastic substrates and to accurately quantify the resulting displacements of the actin cytoskeleton, focal adhesions, as well as the substrate. Displacement fields were recorded in live cell microscopy by tracking either signals from fluorescent proteins or marker particles in the substrate. As model cell type we used myofibroblasts. These cells are characterized by highly stable adhesion and force generating structures but are still able to detect mechanical signals with high sensitivity. We found a rigid connection between substrate and focal adhesions. Furthermore, stress fibers were found to be barely extendable almost over their whole lengths. Plastic deformation took place only at the very ends of actin filaments close to focal adhesions. As a result, this area became elongated without extension of existing actin filaments by polymerization. Both ends of the stress fibers were mechanically coupled with detectable plastic deformations on either site. Interestingly, traction force dependent substrate deformation fields remained mostly unaffected even when stress fiber elongations were released. These data argue for a location of mechanosensing proteins at the ends of actin stress fibers and describe, except for these domains, the whole system to be relatively rigid for tensile strain with a mechanical coupling between the front and rear end of a cell.

  12. 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

  13. Retrograde fluxes of focal adhesion proteins in response to cell migration and mechanical signals.

    PubMed

    Guo, Wei-hui; Wang, Yu-li

    2007-11-01

    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

  14. Antibody binding, platelet adhesion, and protein adsorption on various polymer surfaces.

    PubMed

    Nowak, Joanna; Watala, Cezary; Boncler, Magdalena

    2014-01-01

    Commercially available polymer surfaces for in vitro applications are characterized to different extents in terms of hydrophobicity, binding preferences, and immunoglobulin capacity. We compared five, well or poorly defined polystyrene plates, used as standard hydrophobic surfaces for studying biological interactions. Antibody binding (ELISA) and platelet adhesion (release of alkaline phosphatase from adhered platelets) were contrasted with total protein adsorption (alkaline phosphatase assay and bicinchoninic acid assay). In the assays, we utilized four plasma proteins: human serum albumin (HSA), C-reactive protein (CRP), fibronectin, and fibrinogen. At 0.5??g antigen/well, all antibodies bound to their antigens most effectively on Nunc (MaxiSorp and MediSorp, Waltham, Massachusetts, USA) microplates, as compared to Sarstedt (Nmbrecht, Germany) and Corning microplates (Tewksbury, Massachusetts, USA). The significant differences between Nunc and Corning were seen in the binding of anti-HSA (P???0.01) and anti-fibronectin (P???0.0002). Similar patterns were shown in experiments of ADP-induced platelet adhesion to fibrinogen immobilized at 200??g/well. Platelet adhesion noted on Corning microplates was roughly three times lower compared to those observed on MaxiSorp (P?adhesion, was positively correlated with total protein adsorption. Our findings indicate that of five polystyrene surfaces, Nunc microplates are optimal for studies of protein adsorption, as they had the highest binding capacity and relatively the least affected protein structure, pointing to the role of surface chemistry in protein adsorption and adsorption-induced conformational changes in a protein structure. PMID:23945059

  15. Different Roles for Lactococcal Aggregation Factor and Mucin Binding Protein in Adhesion to Gastrointestinal Mucosa

    PubMed Central

    Luki?, Jovanka; Strahini?, Ivana; Jov?i?, Branko; Filipi?, Brankica; Topisirovi?, Ljubia; Koji?, Milan

    2012-01-01

    Adhesion of bacteria to mucosal surfaces and epithelial cells is one of the key features for the selection of probiotics. In this study, we assessed the adhesion property of Lactococcus lactis subsp. lactis BGKP1 based on its strong autoaggregation phenotype and the presence of the mucin binding protein (MbpL). Genes involved in aggregation (aggL) and possible interaction with mucin (mbpL), present on the same plasmid pKP1, were previously separately cloned in the plasmid pAZIL. In vivo and in vitro experiments revealed potentially different physiological roles of these two proteins in the process of adherence to the intestine during the passage of the strain through the gastrointestinal tract. We correlated the in vitro and in vivo aggregation of the BGKP1-20 carrying plasmid with aggL to binding to the colonic mucus through nonspecific hydrophobic interactions. The expression of AggL on the bacterial cell surface significantly increased the hydrophobicity of the strain. On the other hand, the presence of AggL in the strain reduced its ability to adhere to the ileum. Moreover, MbpL protein showed an affinity to bind gastric type mucin proteins such as MUC5AC. This protein did not contribute to the binding of the strain to the ileal or colonic part of the intestine. Different potential functions of lactococcal AggL and MbpL proteins in the process of adhesion to the gastrointestinal tract are proposed. PMID:22961901

  16. A tumor suppressing function in the epithelial adhesion protein Trask

    PubMed Central

    Spassov, Danislav S.; Wong, Ching Hang; Harris, Geoffrey; McDonough, Stephen; Phojanakong, Paul; Wang, Donghui; Hann, Byron; Bazarov, Alexey V.; Yaswen, Paul; Khanafshar, Elham; Moasser, Mark M.

    2011-01-01

    Trask/CDCP1 is a transmembrane glycoprotein widely expressed in epithelial tissues whose functions are just beginning to be understood, but include a role as an anti-adhesive effector of Src kinases. Early studies looking at RNA transcript levels seemed to suggest overexpression in some cancers, but immunostaining studies are now providing more accurate analyses of its expression. In an immunohistochemical survey of human cancer specimens we find that Trask expression is retained, reduced, or sometimes lost in some tumors compared with their normal epithelial tissue counterparts. A survey of human cancer cell lines also show a similar wide variation in the expression of Trask, including some cell types with the loss of Trask expression, and additional cell types that have lost the physiological detachment-induced phosphorylation of Trask. Three experimental models were established to interrogate the role of Trask in tumor progression including two gain-of-function models with tet-inducible expression of Trask in tumor cells lacking Trask expression, and one loss-of-function model to suppress Trask expression in tumor cells with abundant Trask expression. The induction of Trask expression and phosphorylation in MCF-7 cells and in 3T3v-src cells was associated with a reduction in tumor metastases while the shRNA induced knockdown of Trask in L3.6pl cancer cells was associated with increased tumor metastases. The results from these three models are consistent with a tumor suppressing role for Trask. These data identify Trask as one of several potential candidates for functionally relevant tumor suppressors on the 3p21.3 region of the genome frequently lost in human cancers. PMID:21706059

  17. Heat Shock Protein 60 Acts as a Receptor for the Listeria Adhesion Protein in Caco-2 Cells

    PubMed Central

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

    2004-01-01

    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

  18. An integrated transcriptomic and proteomic analysis of sea star epidermal secretions identifies proteins involved in defense and adhesion.

    PubMed

    Hennebert, Elise; Leroy, Baptiste; Wattiez, Ruddy; Ladurner, Peter

    2015-10-14

    Sea stars rely on epidermal secretions to cope with their benthic life. Their integument produces a mucus, which represents the first barrier against invaders; and their tube feet produce adhesive secretions to pry open mussels and attach strongly but temporarily to rocks. In this study, we combined high-throughput sequencing of expressed mRNA and mass-spectrometry-based identification of proteins to establish the first proteome of mucous and adhesive secretions from the sea star Asterias rubens. We show that the two secretions differ significantly, the major adhesive proteins being only present in trace amounts in the mucus secretion. Except for 41 proteins which were present in both secretions, a total of 34 and 244 proteins were identified as specific of adhesive secretions and mucus, respectively. We discuss the role of some of these proteins in the adhesion of sea stars as well as in their protection against oxygen reactive species and microorganisms. In addition, 58% of the proteins identified in adhesive secretions did not present significant similarity to other known proteins, revealing a list of potential novel sea star adhesive proteins uncharacterized so far. The panel of proteins identified in this study offers unprecedented opportunities for the development of sea star-inspired biomimetic materials. PMID:26171724

  19. 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 invitro and invivo 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

  20. The impact of grafted modification of silicone surfaces with quantum-sized materials on protein adsorption and bacterial adhesion.

    PubMed

    Nune, C; Xu, W; Misra, R D K

    2012-12-01

    The majority of the infections associated with the biomedical devices including cardiovascular implants and catheters are instigated by the adhesion of bacteria including staphylococcus aureus, which is subsequently followed by biofilm formation. Keeping in mind the detrimental effect of bacterial adhesion, the objective of the study is to probe the impact of grafted modification of silicone surfaces with quantum-sized carbon on biofilm formation. Also, explored is the effect of protein adsorption on modified surface and its subsequent influence on bacterial adhesion. We compare and contrast the architecture and foot print of protein adsorption on unmodified and modified model silicone surfaces on bacterial adhesion. The study underscores that protein adsorption on quantum-sized carbon-grafted surface acts as a repellant for bacterial adhesion because of steric repulsion between the negatively charged protein and bacteria. Thus, we establish here the efficacy of modified surfaces in preventing biofilm formation. PMID:22707363

  1. The molecular mechanism of mediation of adsorbed serum proteins to endothelial cells adhesion and growth on biomaterials.

    PubMed

    Yang, Dayun; L, Xiaoying; Hong, Ying; Xi, Tingfei; Zhang, Deyuan

    2013-07-01

    To explore molecular mechanism of mediation of adsorbed proteins to cell adhesion and growth on biomaterials, this study examined endothelial cell adhesion, morphology and viability on bare and titanium nitride (TiN) coated nickel titanium (NiTi) alloys and chitosan film firstly, and then identified the type and amount of serum proteins adsorbed on the three surfaces by proteomic technology. Subsequently, the mediation role of the identified proteins to cell adhesion and growth was investigated with bioinformatics analyses, and further confirmed by a series of cellular and molecular biological experiments. Results showed that the type and amount of adsorbed serum proteins associated with cell adhesion and growth was obviously higher on the alloys than on the chitosan film, and these proteins mediated endothelial cell adhesion and growth on the alloys via four ways. First, proteins such as adiponectin in the adsorbed protein layer bound with cell surface receptors to generate signal transduction, which activated cell surface integrins through increasing intracellular calcium level. Another way, thrombospondin 1 in the adsorbed protein layer promoted TGF-? signaling pathway activation and enhanced integrins expression. The third, RGD sequence containing proteins such as fibronectin 1, vitronectin and thrombospondin 1 in the adsorbed protein layer bound with activated integrins to activate focal adhesion pathway, increased focal adhesion formation and actin cytoskeleton organization and mediated cell adhesion and spreading. In addition, the activated focal adhesion pathway promoted the expression of cell growth related genes and resulted in cell proliferation. The fourth route, coagulation factor II (F2) and fibronectin 1 in the adsorbed protein layer bound with cell surface F2 receptor and integrin, activated regulation of actin cytoskeleton pathway and regulated actin cytoskeleton organization. PMID:23660250

  2. The oxidase activity of vascular adhesion protein-1 (VAP-1) is essential for function

    PubMed Central

    Noonan, Thomas; Lukas, Susan; Peet, Gregory W; Pelletier, Josephine; Panzenbeck, Mark; Hanidu, Adedayo; Mazurek, Suzanne; Wasti, Ruby; Rybina, Irina; Roma, Teresa; Kronkaitis, Anthony; Shoultz, Alycia; Souza, Donald; Jiang, Huiping; Nabozny, Gerald; Modis, Louise Kelly

    2013-01-01

    Vascular adhesion protein-1 (VAP-1) has been implicated in the pathogenesis of inflammatory diseases and is suggested to play a role in immune cell trafficking. It is not clear whether this effect is mediated by the oxidase activity or by other features of the protein such as direct adhesion. In order to study the role of VAP-1 oxidase activity in vivo, we have generated mice carrying an oxidase activity-null VAP-1 protein. We demonstrate that the VAP-1 oxidase null mutant mice have a phenotype similar to the VAP-1 null mice in animal models of sterile peritonitis and antibody induced arthritis suggesting that the oxidase activity is responsible for the inflammatory function of VAP-1. PMID:23885334

  3. Study of the adhesion of neurodegenerative proteins on plasma-modified and coated polypropylene surfaces.

    PubMed

    Poncin-Epaillard, F; Mille, C; Debarnot, D; Zorzi, W; El Moualij, B; Coudreuse, A; Legeay, G; Quadrio, I; Perret-Liaudet, A

    2012-01-01

    The inner polymeric surface of an ELISA titration well is plasma-modified and coated with different surfactant molecules. The titration of neurodegenerative proteins markers (prion, Tau and β-synuclein), previously demonstrated as more efficient with such modified tubes, is related to the adhesion behaviour of these proteins and their corresponding capture antibodies. The adhesion process is studied in terms of anchoring and specific mechanisms. The proteins and antibodies binding onto such modified surfaces is related to the substrate hydrophilic character calculated from the angle contact measure, to the polymer surface charge measured through the streaming potential determination at different pH and the inner surface roughness determined from AFM images. Furthermore, the influence of the blocking agent used during the ELISA titration is also studied. PMID:21944054

  4. Applications of traction force microscopy in measuring adhesion molecule dependent cell contractility

    NASA Astrophysics Data System (ADS)

    Mann, Cynthia Marie

    This work describes the use of polyacrylamide hydrogels as controlled elastic modulus substrates for single cell traction force microscopy studies. The first section describes the use of EDC/NHS chemistry to convalently link microbeads to the hydrogel matrix for the purpose of performing long-term traction force studies (7 days). The final study uses the C2C12 cell line to demonstrate that integrin-mediated adhesion to soft substrates causes different cell behavior than N-cahderin-mediated adhesion to soft substrates. Cells plated on laminin-coated hydrogels exhibited stiffness dependent increases in cell spreading, whereas cells plated on N-cadherin-coated substrates. Similarly, cells plated on laminin-coated substrates exhibited substrate stiffness dependent increases in normalized net contractile moment, however the same cells plated on N-cadherin-coated substrates were unable to deform any but the softest hydrogels.

  5. Adhesion of endothelial cells and adsorption of serum proteins on gas plasma-treated polytetrafluoroethylene.

    PubMed

    Dekker, A; Reitsma, K; Beugeling, T; Bantjes, A; Feijen, J; van Aken, W G

    1991-03-01

    From in vitro experiments it is known that human endothelial cells show poor adhesion to hydrophobic polymers. The hydrophobicity of vascular prostheses manufactured from Teflon or Dacron may be the reason why endothelialization of these grafts does not occur after implantation in humans. We modified films of polytetrafluoroethylene (Teflon) by nitrogen plasma and oxygen plasma treatments to make the surfaces more hydrophilic. Depending on the plasma exposure time, modified polytetrafluoroethylene surfaces showed water-contact angles of 15-58 degrees, versus 96 degrees for unmodified polytetrafluoroethylene. ESCA measurements revealed incorporation of both nitrogen- and oxygen-containing groups into the polytetrafluoroethylene surfaces, dependent on the plasma composition and exposure time. The thickness of the modified surface layer was approximately 1 nm. The adhesion of cultured human endothelial cells from 20% human serum-containing culture medium to modified polytetrafluoroethylene surfaces with contact angles of 20-45 degrees led to the formation of a monolayer of cells, which was similar to the one formed on tissue culture polystyrene, the reference surface. This was not the case when endothelial cells were seeded upon unmodified polytetrafluoroethylene. Surface-modified expanded polytetrafluoroethylene prosthesis material (GORE TEX soft tissue) also showed adhesion of endothelial cells comparable to cell adhesion to the reference surface. The amounts of serum proteins, including fibronectin, adsorbed from serum-containing medium to modified polytetrafluoroethylene surfaces were larger than those adsorbed to unmodified polytetrafluoroethylene. Moreover, the modified surfaces probably allow the exchange of adsorbed serum proteins with cellular fibronectin. PMID:1878448

  6. STRUCTURAL ORGANIZATION AND FUNCTION OF MOUSE PHOTORECEPTOR RIBBON SYNAPSES INVOLVE THE IMMUNOGLOBULIN ADHESION PROTEIN SYNCAM 1

    PubMed Central

    Ribic, Adema; Liu, Xinran; Crair, Michael C.; Biederer, Thomas

    2013-01-01

    Adhesive interactions in the retina instruct the developmental specification of inner retinal layers. However, potential roles of adhesion in the development and function of photoreceptor synapses remain incompletely understood. This contrasts with our understanding of synapse development in the central nervous system (CNS), which can be guided by select adhesion molecules such as the Synaptic Cell Adhesion Molecule 1 (SynCAM 1/CADM1/Nectin-like 2 protein). This immunoglobulin superfamily protein modulates the development and plasticity of classical excitatory synapses. We now show by immuno-electron microscopy and immunoblotting that SynCAM 1 is expressed on mouse rod photoreceptors and their terminals in the outer nuclear and plexiform layers (ONL and OPL) in a developmentally regulated manner. Expression of SynCAM 1 on rods is low in early postnatal stages (P3-P7), but increases after eye opening (P14). In support of functional roles in the photoreceptors, electroretinogram recordings demonstrate impaired responses to light stimulation in SynCAM 1 knockout (KO) mice. In addition, the structural integrity of synapses in the OPL requires SynCAM 1. Quantitative ultrastructural analysis of SynCAM 1 KO retina measured fewer fully assembled, triadic rod ribbon synapses. Further, rod synapse ribbons are shortened in KO mice and protein levels of Ribeye, a major structural component of ribbons, are reduced in SynCAM 1 KO retina. Together, our results implicate SynCAM 1 in the synaptic organization of the rod visual pathway and provide evidence for novel roles of synaptic adhesion in the structural and functional integrity of ribbon synapses. PMID:23982969

  7. 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

  8. 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

  9. 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.

  10. 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

  11. 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

  12. Targeting Protein Kinase C Downstream of Growth Factor and Adhesion Signalling

    PubMed Central

    Dowling, Catrona M.; Kiely, Patrick A.

    2015-01-01

    The signaling outputs of Receptor Tyrosine Kinases, G-protein coupled receptors and integrins converge to mediate key cell process such as cell adhesion, cell migration, cell invasion and cell proliferation. Once activated by their ligands, these cell surface proteins recruit and direct a diverse range of proteins to disseminate the appropriate response downstream of the specific environmental cues. One of the key groups of proteins required to regulate these activities is the family of serine/threonine intracellular kinases called Protein Kinase Cs. The activity and subcellular location of PKCs are mediated by a series of tightly regulated events and is dependent on several posttranslational modifications and the availability of second messengers. Protein Kinase Cs exhibit both pro- and anti-tumorigenic effects making them an interesting target for anti-cancer treatment. PMID:26184315

  13. Vascular Adhesion Protein-1 Regulates Leukocyte Transmigration Rate in the Retina During Diabetes

    PubMed Central

    Noda, Kousuke; Nakao, Shintaro; Zandi, Souska; Engelstdter, Verena; Mashima, Yukihiko; Hafezi-Moghadam, Ali

    2009-01-01

    Vascular adhesion protein-1 (VAP-1) is an endothelial adhesion molecule that possesses semicarbazide-sensitive amine oxidase (SSAO) activity and is involved in leukocyte recruitment. Leukocyte adhesion to retinal vessels is a predominant feature of experimentally induced diabetic retinopathy (DR). However, the role of VAP-1 in this process is unknown. Diabetes was induced by i.p. injection of Streptozotocin in LongEvans rats. The specific inhibitor of VAP-1, UV-002, was administered by daily i.p. injections. The expression of VAP-1 mRNA in the retinal extracts of normal and diabetic animals was measured by real time quantitative polymerase chain reaction (PCR). Firm leukocyte adhesion was quantified in retinal flatmounts after intravascular staining with concanavalin A (ConA). Leukocyte transmigration rate was quantified by in vivo acridine orange leukocyte staining (AOLS). In diabetic rats, the rate of leukocyte transmigration into the retinal tissues of live animals was significantly increased, as determined by AOLS. When diabetic animals were treated with daily injections of the VAP-1 inhibitor (0.3mg/kg), leukocyte transmigration rate was significantly reduced (P<0.05). However, firm adhesion of leukocytes in diabetic animals treated with the inhibitor did not differ significantly from vehicle-treated diabetic controls. This work provides evidence for an important role of VAP-1 in the recruitment of leukocyte to the retina in experimental DR. Our results reveal the critical contribution of VAP-1 to leukocyte transmigration, with little impact on firm leukocyte adhesion in the retinas of diabetic animals. VAP-1 inhibition might be beneficial in the treatment of DR. PMID:19635478

  14. Vascular adhesion protein-1 regulates leukocyte transmigration rate in the retina during diabetes.

    PubMed

    Noda, Kousuke; Nakao, Shintaro; Zandi, Souska; Engelstdter, Verena; Mashima, Yukihiko; Hafezi-Moghadam, Ali

    2009-11-01

    Vascular adhesion protein-1 (VAP-1) is an endothelial adhesion molecule that possesses semicarbazide-sensitive amine oxidase (SSAO) activity and is involved in leukocyte recruitment. Leukocyte adhesion to retinal vessels is a predominant feature of experimentally induced diabetic retinopathy (DR). However, the role of VAP-1 in this process is unknown. Diabetes was induced by i.p. injection of Streptozotocin in Long-Evans rats. The specific inhibitor of VAP-1, UV-002, was administered by daily i.p. injections. The expression of VAP-1 mRNA in the retinal extracts of normal and diabetic animals was measured by real-time quantitative polymerase chain reaction (PCR). Firm leukocyte adhesion was quantified in retinal flatmounts after intravascular staining with concanavalin A (ConA). Leukocyte transmigration rate was quantified by in vivo acridine orange leukocyte staining (AOLS). In diabetic rats, the rate of leukocyte transmigration into the retinal tissues of live animals was significantly increased, as determined by AOLS. When diabetic animals were treated with daily injections of the VAP-1 inhibitor (0.3 mg/kg), leukocyte transmigration rate was significantly reduced (P < 0.05). However, firm adhesion of leukocytes in diabetic animals treated with the inhibitor did not differ significantly from vehicle-treated diabetic controls. This work provides evidence for an important role of VAP-1 in the recruitment of leukocyte to the retina in experimental DR. Our results reveal the critical contribution of VAP-1 to leukocyte transmigration, with little impact on firm leukocyte adhesion in the retinas of diabetic animals. VAP-1 inhibition might be beneficial in the treatment of DR. PMID:19635478

  15. Endocytosis Regulates Cell Soma Translocation and the Distribution of Adhesion Proteins in Migrating Neurons

    PubMed Central

    Shieh, Jennifer C.; Schaar, Bruce T.; Srinivasan, Karpagam; Brodsky, Frances M.; McConnell, Susan K.

    2011-01-01

    Newborn neurons migrate from their birthplace to their final location to form a properly functioning nervous system. During these movements, young neurons must attach and subsequently detach from their substrate to facilitate migration, but little is known about the mechanisms cells use to release their attachments. We show that the machinery for clathrin-mediated endocytosis is positioned to regulate the distribution of adhesion proteins in a subcellular region just proximal to the neuronal cell body. Inhibiting clathrin or dynamin function impedes the movement of migrating neurons both in vitro and in vivo. Inhibiting dynamin function in vitro shifts the distribution of adhesion proteins to the rear of the cell. These results suggest that endocytosis may play a critical role in regulating substrate detachment to enable cell body translocation in migrating neurons. PMID:21445347

  16. 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...

  17. 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

  18. Chick neural retina adhesion and survival molecule is a retinol-binding protein

    SciTech Connect

    Schubert, D.; LaCorbiere, M.; Esch, F.

    1986-01-01

    A 20,000-D protein called purpurin has recently been isolated from the growth-conditioned medium of cultured embryonic chick neural retina cells. Purpurin is a constituent of adherons and promotes cell-adheron adhesion by interacting with a cell surface heparan sulfate proteoglycan. It also prolongs the survival of cultured neural retina cells. This paper shows that purpurin is a secretory protein that has sequence homology with a human protein synthesized in the liver that transports retinol in the blood, the serum retinol-binding protein (RBP). Purpurin binds (/sup 3/H)retinol, and both purpurin and chick serum RBP stimulate the adhesion of neural retina cells, although the serum protein is less active than purpurin. Purpurin and the serum RBP are, however, different molecules, for the serum protein is approx.3.000 D larger than purpurin and has different silver-staining characteristics. Finally, purpurin supports the survival of dissociated ciliary ganglion cells, indicating that RBPs can act as ciliary neurotrophic factors.

  19. A genome-wide screen identifies conserved protein hubs required for cadherin-mediated cellcell adhesion

    PubMed Central

    Toret, Christopher P.; DAmbrosio, Michael V.; Vale, Ronald D.; Simon, Michael A.

    2014-01-01

    Cadherins and associated catenins provide an important structural interface between neighboring cells, the actin cytoskeleton, and intracellular signaling pathways in a variety of cell types throughout the Metazoa. However, the full inventory of the proteins and pathways required for cadherin-mediated adhesion has not been established. To this end, we completed a genome-wide (?14,000 genes) ribonucleic acid interference (RNAi) screen that targeted Ca2+-dependent adhesion in DE-cadherinexpressing Drosophila melanogaster S2 cells in suspension culture. This novel screen eliminated Ca2+-independent cellcell adhesion, integrin-based adhesion, cell spreading, and cell migration. We identified 17 interconnected regulatory hubs, based on protein functions and proteinprotein interactions that regulate the levels of the core cadherincatenin complex and coordinate cadherin-mediated cellcell adhesion. Representative proteins from these hubs were analyzed further in Drosophila oogenesis, using targeted germline RNAi, and adhesion was analyzed in MadinDarby canine kidney mammalian epithelial cellcell adhesion. These experiments reveal roles for a diversity of cellular pathways that are required for cadherin function in Metazoa, including cytoskeleton organization, cellsubstrate interactions, and nuclear and cytoplasmic signaling. PMID:24446484

  20. Human Siglec-10 can bind to vascular adhesion protein-1 and serves as its substrate

    PubMed Central

    Kivi, Elina; Elima, Kati; Aalto, Kristiina; Nymalm, Yvonne; Auvinen, Kaisa; Koivunen, Erkki; Otto, Diana M.; Crocker, Paul R.; Salminen, Tiina A.; Salmi, Marko

    2009-01-01

    Leukocytes migrate from the blood into areas of inflammation by interacting with various adhesion molecules on endothelial cells. Vascular adhesion protein-1 (VAP-1) is a glycoprotein expressed on inflamed endothelium where it plays a dual role: it is both an enzyme that oxidizes primary amines and an adhesin that is involved in leukocyte trafficking to sites of inflammation. Although VAP-1 was identified more than 15 years ago, the counterreceptor(s) for VAP-1 on leukocytes has remained unknown. Here we have identified Siglec-10 as a leukocyte ligand for VAP-1 using phage display screenings. The binding between Siglec-10 and VAP-1 was verified by different adhesion assays, and this interaction was also consistent with molecular modeling. Moreover, the interaction between Siglec-10 and VAP-1 led to increased hydrogen peroxide production, indicating that Siglec-10 serves as a substrate for VAP-1. Thus, the Siglec-10VAP-1 interaction seems to mediate lymphocyte adhesion to endothelium and has the potential to modify the inflammatory microenvironment via the enzymatic end products. PMID:19861682

  1. Protein O-mannosylation is crucial for E-cadherin-mediated cell adhesion.

    PubMed

    Lommel, Mark; Winterhalter, Patrick R; Willer, Tobias; Dahlhoff, Maik; Schneider, Marlon R; Bartels, Markus F; Renner-Müller, Ingrid; Ruppert, Thomas; Wolf, Eckhard; Strahl, Sabine

    2013-12-24

    In recent years protein O-mannosylation has become a focus of attention as a pathomechanism underlying severe congenital muscular dystrophies associated with neuronal migration defects. A key feature of these disorders is the lack of O-mannosyl glycans on α-dystroglycan, resulting in abnormal basement membrane formation. Additional functions of O-mannosylation are still largely unknown. Here, we identify the essential cell-cell adhesion glycoprotein epithelial (E)-cadherin as an O-mannosylated protein and establish a functional link between O-mannosyl glycans and cadherin-mediated cell-cell adhesion. By genetically and pharmacologically blocking protein O-mannosyltransferases, we found that this posttranslational modification is essential for preimplantation development of the mouse embryo. O-mannosylation-deficient embryos failed to proceed from the morula to the blastocyst stage because of defects in the molecular architecture of cell-cell contact sites, including the adherens and tight junctions. Using mass spectrometry, we demonstrate that O-mannosyl glycans are present on E-cadherin, the major cell-adhesion molecule of blastomeres, and present evidence that this modification is generally conserved in cadherins. Further, the use of newly raised antibodies specific for an O-mannosyl-conjugated epitope revealed that these glycans are present on early mouse embryos. Finally, our cell-aggregation assays demonstrated that O-mannosyl glycans are crucial for cadherin-based cell adhesion. Our results redefine the significance of O-mannosylation in humans and other mammals, showing the immense impact of cadherins on normal as well as pathogenic cell behavior. PMID:24297939

  2. Activation of focal adhesion kinase enhances the adhesion of Fusarium solani to human corneal epithelial cells via the tyrosine-specific protein kinase signaling pathway

    PubMed Central

    Pan, Xiaojing; Wang, Ye; Zhou, Qingjun; Chen, Peng; Xu, Yuanyuan; Chen, Hao

    2011-01-01

    Purpose To determine the role of the integrin-FAK signaling pathway triggered by the adherence of F. solani to human corneal epithelial cells (HCECs). Methods After pretreatment with/without genistein, HCECs were incubated with F. solani spores at different times (024 h). Cell adhesion assays were performed by optical microscopy. Changes of the ultrastructure were observed using scanning electron microscopy (SEM) and transmission electron microscopy (TEM). The expression of F-actin and Paxillin (PAX) were detected by immunofluorescence and western blotting to detect the expression of these key proteins with/without genistein treatment. Results Cell adhesion assays showed that the number of adhered spores began to rise at 6 h after incubation and peaked at 8 h. SEM and TEM showed that the HCECs exhibited a marked morphological alteration induced by the attachment and entry of the spores. The expression of PAX increased, while the expression of F-actin decreased by stimulation with F. solani. The interaction of F. solani with HCECs causes actin rearrangement in HCECs. Genistein strongly inhibited FAK phosphorylation and the activation of the downstream protein (PAX). F. solani-induced enhancement of cell adhesion ability was inhibited along with the inhibition of FAK phosphorylation. Conclusions Our results suggest that the integrin-FAK signaling pathway is involved in the control of F. solani adhesion to HCECs and that the activation of focal adhesion kinase enhances the adhesion of human corneal epithelial cells to F. solani via the tyrosine-specific protein kinase signaling pathway. PMID:21403855

  3. 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.

  4. Bacterial adhesion to protein-coated surfaces: An AFM and QCM-D study

    NASA Astrophysics Data System (ADS)

    Strauss, Joshua; Liu, Yatao; Camesano, Terri A.

    2009-09-01

    Bacterial adhesion to biomaterials, mineral surfaces, or other industrial surfaces is strongly controlled by the way bacteria interact with protein layers or organic matter and other biomolecules that coat the materials. Despite this knowledge, many studies of bacterial adhesion are performed under clean conditions, instead of in the presence of proteins or organic molecules. We chose fetal bovine serum (FBS) as a model protein, and prepared FBS films on quartz crystals. The thickness of the FBS layer was characterized using atomic force microscopy (AFM) imaging under liquid and quartz crystal microbalance with dissipation (QCM-D). Next, we characterized how the model biomaterial surface would interact with the nocosomial pathogen Staphylococcus epidermidis. An AFM probe was coated with S. epidermidis cells and used to probe a gold slide that had been coated with FBS or another protein, fibronectin (FN). These experiments show that AFM and QCM-D can be used in complementary ways to study the complex interactions between bacteria, proteins, and surfaces.

  5. 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

  6. How to control the recombinant prion protein adhesion for successful storage through modification of surface properties.

    PubMed

    Vrlinic, T; Debarnot, D; Legeay, G; Coudreuse, A; El Moualij, B; Zorzi, W; Perret-Liaudet, A; Quadrio, I; Mozetic, M; Poncin-Epaillard, F

    2012-12-01

    Depletion of neuroproteins on the inner walls of storage tubes influences the accuracy of tests used for identification of various neurodegenerative disorders. In this paper, a strategy is described for surface modification of Eppendorf tubes leading to non-adhesive properties towards the recombinant human prion proteins (PrPrec(hum)). Tubes were pre-activated by helium plasma and grafted with three diverse coatings: pure poly(N-isopropylacrylamide) (PNIPAM), PNIPAM admixed with either neutral PEG(20)sorbitan monolaurate (PEG(20)) or positively charged cetyl trimethylammonium bromide (CTAB) at varying plasma activation times and polymer to surfactant ratios. New functionalized surfaces were analyzed by goniometry, streaming potential measurement and X-ray photoelectron spectroscopy, whereas the protein adhesion was monitored by enzyme linked immunosorbent assays and confocal microscopy. The mapping of PrPrec(hum) adhesion associated with surface analyses enabled us to determine that no or negligible depletion of PrPrec(hum) can be obtained by surfaces possessing basic component in the range between 50 and 60 mJ m(-2) and streaming potential ζ(7.4) - -50 mV. PMID:23109107

  7. Reduction of Adhesion between Steel and Grilled Fish Protein with Ultra-Hydrophobic DLC

    NASA Astrophysics Data System (ADS)

    Honda, Naoko; Kajiya, Makoto; Jang, Young-Jun; Kousaka, Hiroyuki; Umehara, Noritsugu; Tokoroyama, Takayuki

    There is an issue of the fish adhering to the metal grids of grill strongly after grilling fish. Recently, the temperature in a grill equipment is over 500 C, Fluorine resin coating which has low adhesion property can not endurant at 500 C. In order to overcome this issue, we proposed a new grill not to adhere a fish with Ultra-Hydrophobic Diamond-like Carbon coating named as UH-DLC coating. UH-DLC coating has high hydrophobicity. We expected this coating to decrease wettability of water including fish protein and contact area, because fish protein includes about 90% water. Contact angle of water on UH-DLC was over 120 at room temperature. After heating at 500 C for 54 minutes, UH-DLC kept ultra-hydrophobic property. On the other hand, Fluorine resin coating desappeared hydrophobic properties after heating at 500 C for 54 minutes. It was conducted adhesion test for UH-DLC coating, Fluorine resin coating and no coating grilling rods after the grilling of real fish in a grill equipment by gas. The adhesion force between UH-DLC coating grill rods and real fish was as half as that of no coating grill rods and as much as that of Fluorine resin grill rods. So, we believe that Ultra Hydrophobic Diamond-like Carbon coating has a possibility for the usage as new grilling grids at high temperature.

  8. Optical regulation of protein adsorption and cell adhesion by photoresponsive GaN nanowires.

    PubMed

    Li, Jingying; Han, Qiusen; Zhang, Ying; Zhang, Wei; Dong, Mingdong; Besenbacher, Flemming; Yang, Rong; Wang, Chen

    2013-10-01

    Interfacing nanowires with living cells is attracting more and more interest due to the potential applications, such as cell culture engineering and drug delivery. We report on the feasibility of using photoresponsive semiconductor gallium nitride (GaN) nanowires (NWs) for regulating the behaviors of biomolecules and cells at the nano/biointerface. The GaN NWs have been fabricated by a facile chemical vapor deposition method. The superhydrophobicity to superhydrophilicity transition of the NWs is achieved by UV illumination. Bovine serum albumin adsorption could be modulated by photoresponsive GaN NWs. Tunable cell detachment and adhesion are also observed. The mechanism of the NW surface responsible for modulating both of protein adsorption and cell adhesion is discussed. These observations of the modulation effects on protein adsorption and cell adhesion by GaN NWs could provide a novel approach toward the regulation of the behaviors of biomolecules and cells at the nano/biointerface, which may be of considerable importance in the development of high-performance semiconductor nanowire-based biomedical devices for cell culture engineering, bioseparation, and diagnostics. PMID:24073887

  9. Recombinant Probiotic Expressing Listeria Adhesion Protein Attenuates Listeria monocytogenes Virulence In Vitro

    PubMed Central

    Koo, Ok Kyung; Amalaradjou, Mary Anne Roshni; Bhunia, Arun K.

    2012-01-01

    Background Listeria monocytogenes, an intracellular foodborne pathogen, infects immunocompromised hosts. The primary route of transmission is through contaminated food. In the gastrointestinal tract, it traverses the epithelial barrier through intracellular or paracellular routes. Strategies to prevent L. monocytogenes entry can potentially minimize infection in high-risk populations. Listeria adhesion protein (LAP) aids L. monocytogenes in crossing epithelial barriers via the paracellular route. The use of recombinant probiotic bacteria expressing LAP would aid targeted clearance of Listeria from the gut and protect high-risk populations from infection. Methodology/Principal Findings The objective was to investigate the ability of probiotic bacteria or LAP-expressing recombinant probiotic Lactobacillus paracasei (LbpLAP) to prevent L. monocytogenes adhesion, invasion, and transwell-based transepithelial translocation in a Caco-2 cell culture model. Several wild type probiotic bacteria showed strong adhesion to Caco-2 cells but none effectively prevented L. monocytogenes infection. Pre-exposure to LbpLAP for 1, 4, 15, or 24 h significantly (P<0.05) reduced adhesion, invasion, and transepithelial translocation of L. monocytogenes in Caco-2 cells, whereas pre-exposure to parental Lb. paracasei had no significant effect. Similarly, LbpLAP pre-exposure reduced L. monocytogenes translocation by as much as 46% after 24 h. LbpLAP also prevented L. monocytogenes-mediated cell damage and compromise of tight junction integrity. Furthermore, LbpLAP cells reduced L. monocytogenes-mediated cell cytotoxicity by 99.8% after 1 h and 79% after 24 h. Conclusions/Significance Wild type probiotic bacteria were unable to prevent L. monocytogenes infection in vitro. In contrast, LbpLAP blocked adhesion, invasion, and translocation of L. monocytogenes by interacting with host cell receptor Hsp60, thereby protecting cells from infection. These data show promise for the use of recombinant probiotics in preventing L. monocytogenes infection in high-risk populations. PMID:22235279

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

    PubMed

    Hamann, Jrg; Aust, Gabriela; Ara, 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; Prmel, Simone; Schneberg, Torsten; Schwartz, Thue W; Singer, Kathleen; Stacey, Martin; Ushkaryov, Yuri A; Vallon, Mario; Wolfrum, Uwe; Wright, Mathew W; Xu, Lei; Langenhan, Tobias; Schith, 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. Force Activation of a Multimeric Adhesive Protein through Domain Conformational Change

    NASA Astrophysics Data System (ADS)

    Wijeratne Sithara S

    The force-induced activation of adhesive proteins such as von Willebrand factor (VWF), which experience high hydrodynamic forces, is essential in initiating platelet adhesion. The importance of the mechanical force induced functional change is manifested in the multimeric VWF's crucial role in blood coagulation, when high fluid shear stress activates pVWF multimers to bind platelets. Here we showed that a pathological level of high shear flow exposure of pVWF multimers results in domain conformational changes, and the subsequent shifts in the unfolding force allow us to use force as a marker to track the dynamic states of multimeric VWF. We found that shear-activated pVWF multimers (spVWF) are more resistant to mechanical unfolding than non-sheared pVWF multimers, as indicated in the higher peak unfolding force. These results provide insight into the mechanism of shear-induced activation of pVWF multimers.

  12. Mechanical Activation of a Multimeric Adhesive Protein Through Domain Conformational Change

    NASA Astrophysics Data System (ADS)

    Wijeratne, Sithara S.; Botello, Eric; Yeh, Hui-Chun; Zhou, Zhou; Bergeron, Angela L.; Frey, Eric W.; Patel, Jay M.; Nolasco, Leticia; Turner, Nancy A.; Moake, Joel L.; Dong, Jing-fei; Kiang, Ching-Hwa

    2013-03-01

    The mechanical force-induced activation of the adhesive protein von Willebrand factor (VWF), which experiences high hydrodynamic forces, is essential in initiating platelet adhesion. The importance of the mechanical force-induced functional change is manifested in the multimeric VWFs crucial role in blood coagulation, when high fluid shear stress activates plasma VWF (PVWF) multimers to bind platelets. Here, we showed that a pathological level of high shear stress exposure of PVWF multimers results in domain conformational changes, and the subsequent shifts in the unfolding force allow us to use force as a marker to track the dynamic states of the multimeric VWF. We found that shear-activated PVWF multimers are more resistant to mechanical unfolding than nonsheared PVWF multimers, as indicated in the higher peak unfolding force. These results provide insight into the mechanism of shear-induced activation of PVWF multimers.

  13. [The role of adhesive proteins and membrane interactions in the processes of thrombocyte aggregation].

    PubMed

    Belitser, N V; Anishchuk, M G; Pozdniakova, T M; Gorkun, O V; Veklich, V I; Smekhova, T R

    1991-01-01

    The reported data concerning the role of fibrinogen in platelet aggregation are reviewed and compared to the authors' experimental data obtained by electron microscopy and cytochemical techniques. Using fibrinogen and fibrinogen antibodies bound to colloidal gold, it has been shown that the presence of fibrinogen bridged between the adjoining cells is not necessary for primary platelet microaggregation stimulated by ADP or thrombin. The formation of direct interplatelet contacts resembling "pentalaminar membranes" has been shown to participate in that process. Some mechanisms are proposed to explain the enhanced adhesiveness of the activated platelet surfaces. Redistribution of phospholipids in the membrane bilayer leading to the exposure of negatively charged phospholipids may underlie that phenomenon. The clustering and internalization of "occupied" membrane receptors may also contribute to the formation of close contacts between platelets stimulated by primary agonists in the presence of exogenous fibrinogen and other adhesive proteins. PMID:1801463

  14. Fetal bovine serum xenoproteins modulate human monocyte adhesion and protein release on biomaterials in vitro

    PubMed Central

    Schmidt, David; Joyce, Evan James; Kao, Weiyuan John

    2010-01-01

    Monocyte-derived macrophages are critical in the host foreign body response to biomaterials and have been studied extensively in various culture conditions in vitro such as medium supplemented with fetal bovine serum (FBS) or autologous human serum (AHS). Since monocyte maturation into macrophages is highly plastic and may vary considerably depending on the surface, isolation procedures, and in vitro culture conditions, we hypothesize that variations in protein adsorption and serum type will greatly impact monocyte behavior in a surface-dependent manner. The impact of xenoproteins on monocyte-surface interaction is not well studied methodically and the use of AHS rather than FBS for macrophage-biomaterials studies in vitro is far from universal. The commonly used reference materials: tissue culture polystyrene (TCPS), polyethylene glycol (PEG), and poly-dimethylsiloxane (PDMS) were employed in this study and we found a 3-fold higher adherent monocyte density on TCPS when AHS was used versus FBS-supplemented medium. On PEG hydrogels, an 8-10 fold higher adhesion density was observed when AHS was employed versus FBS, while on PDMS no difference in adhesion density was observed between the two sera conditions. Additionally, the presence of lipopolysaccharide abrogated the serum-dependent effect on cell adhesion on TCPS. Significant differential variations in protein release were observed between the serum conditions on these surfaces, in particular there was a 100-fold higher concentration of growth-related oncogene for the AHS condition on PDMS even though the adhesion levels were comparable between the two serum conditions. These results emphasize the combined impact of the surface type and FBS xenoproteins in mediating the observed monocyte response to biomaterials in vitro. PMID:20837169

  15. Onion yellow phytoplasma P38 protein plays a role in adhesion to the hosts.

    PubMed

    Neriya, Yutaro; Maejima, Kensaku; Nijo, Takamichi; Tomomitsu, Tatsuya; Yusa, Akira; Himeno, Misako; Netsu, Osamu; Hamamoto, Hiroshi; Oshima, Kenro; Namba, Shigetou

    2014-12-01

    Adhesins are microbial surface proteins that mediate the adherence of microbial pathogens to host cell surfaces. In Mollicutes, several adhesins have been reported in mycoplasmas and spiroplasmas. Adhesins P40 of Mycoplasma agalactiae and P89 of Spiroplasma citri contain a conserved amino acid sequence known as the Mollicutes adhesin motif (MAM), whose function in the host cell adhesion remains unclear. Here, we show that phytoplasmas, which are plant-pathogenic mollicutes transmitted by insect vectors, possess an adhesion-containing MAM that was identified in a putative membrane protein, PAM289 (P38), of the 'Candidatus Phytoplasma asteris,' OY strain. P38 homologs and their MAMs were highly conserved in related phytoplasma strains. While P38 protein was expressed in OY-infected insect and plant hosts, binding assays showed that P38 interacts with insect extract, and weakly with plant extract. Interestingly, the interaction of P38 with the insect extract depended on MAM. These results suggest that P38 is a phytoplasma adhesin that interacts with the hosts. In addition, the MAM of adhesins is important for the interaction between P38 protein and hosts. PMID:25302654

  16. Developmentally dynamic colocalization patterns of DSCAM with adhesion and synaptic proteins in the mouse retina

    PubMed Central

    Belem de Andrade, Gabriel; Kunzelman, Landon; Merrill, Morgan M.

    2014-01-01

    Purpose The Down syndrome cell adhesion molecule (Dscam) gene is required for normal dendrite arborization and lamination in the mouse retina. In this study, we characterized the developmental localization of the DSCAM protein to better understand the postnatal stages of retinal development during which laminar disorganization occur in the absence of the protein. Methods Immunohistochemistry and colocalization analysis software were used to assay the localization of the DSCAM protein during development of the retina. Results We found that DSCAM was initially localized diffusely throughout mouse retinal neurites but then adopted a punctate distribution. DSCAM colocalized with catenins in the adult retina but was not detected at the active zone of chemical synapses, electrical synapses, and tight junctions. Further analysis identified a wave of colocalization between DSCAM and numerous synaptic and junction proteins coinciding with synaptogenesis between bipolar and retinal ganglion cells. Conclusions Research presented in this study expands our understanding of DSCAM function by characterizing its location during the development of the retina and identifies temporally regulated localization patterns as an important consideration in understanding the function of adhesion molecules in neural development. PMID:25352748

  17. Nanometric protein-patch arrays on glass and polydimethylsiloxane for cell adhesion studies.

    PubMed

    Pi, Fuwei; Dillard, Pierre; Limozin, Laurent; Charrier, Anne; Sengupta, Kheya

    2013-07-10

    We present a simple cost-effective benchtop protocol to functionalize glass and polydimethylsiloxane (PDMS) with nanometric protein patches for cell adhesion studies. Evaporation masks, covering macroscopic areas on glass, were made using improved strategies for self-assembly of colloidal microbeads which then served as templates for creating the protein patch arrays via the intermediate steps of organo-aminosilane deposition and polyethylene-glycol grafting. The diameter of the patches could be varied down to about 80 nm. The glass substrates were used for advanced optical imaging of T-lymphocytes to explore adhesion by reflection interference contrast microscopy and the possible colocalization of T-cell receptor microclusters and the activating protein patches by total internal reflection fluorescence microscopy. The selectively functionalized glass could also serve as template for transferring the protein nanopatches to the surface of a soft elastomer. We demonstrated successful reverse contact printing onto the surface of thin layers of PDMS with stiffness ranging from 30 KPa to 3 MPa. PMID:23808889

  18. Vascular adhesion protein-1: Role in human pathology and application as a biomarker.

    PubMed

    Pannecoeck, Roos; Serruys, Daphne; Benmeridja, Lara; Delanghe, Joris R; Geel, Nanja van; Speeckaert, Reinhart; Speeckaert, Marijn M

    2015-12-01

    Vascular adhesion protein-1 (VAP-1) is a member of the copper-containing amine oxidase/semicarbazide-sensitive amine oxidase (AOC/SSAO) enzyme family. SSAO enzymes catalyze oxidative deamination of primary amines, which results in the production of the corresponding aldehyde, hydrogen peroxide and ammonium. VAP-1 is continuously expressed as a transmembrane glycoprotein in the vascular wall during development and facilitates the accumulation of inflammatory cells into the inflamed environment in concert with other leukocyte adhesion molecules. The soluble form of VAP-1 is released into the circulation mainly from vascular endothelial cells. Over- and under-expression of sVAP-1 result in alterations of the reported reaction product levels, which are involved in the pathogenesis of multiple human diseases. The combination of enzymatic and adhesion capacities as well as its strong association with inflammatory pathologies makes VAP-1 an interesting therapeutic target for drug discovery. In this article, we will review the general characteristics and biological functions of VAP-1, focusing on its important role as a prognostic biomarker in human pathologies. In addition, the potential therapeutic application of VAP-1 inhibitors will be discussed. PMID:26287391

  19. Differential Effect of Actomyosin Relaxation on the Dynamic Properties of Focal Adhesion Proteins

    PubMed Central

    Lavelin, Irena; Wolfenson, Haguy; Patla, Israel; Henis, Yoav I.; Medalia, Ohad; Volberg, Tova; Livne, Ariel; Kam, Zvi; Geiger, Benjamin

    2013-01-01

    Treatment of cultured cells with inhibitors of actomyosin contractility induces rapid deterioration of stress fibers, and disassembly of the associated focal adhesions (FAs). In this study, we show that treatment with the Rho kinase inhibitor Y-27632, which blocks actomyosin contractility, induces disarray in the FA-associated actin bundles, followed by the differential dissociation of eight FA components from the adhesion sites. Live-cell microscopy indicated that the drug triggers rapid dissociation of VASP and zyxin from FAs (? values of 7-8 min), followed by talin, paxillin and ILK (? ~16 min), and then by FAK, vinculin and kindlin-2 (? = 25-28 min). Examination of the molecular kinetics of the various FA constituents, using Fluorescence Recovery After Photobleaching (FRAP), in the absence of or following short-term treatment with the drug, revealed major changes in the kon and koff values of the different proteins tested, which are in close agreement with their differential dissociation rates from the adhesion sites. These findings indicate that mechanical, actomyosin-generated forces differentially regulate the molecular kinetics of individual FA-associated molecules, and thereby modulate FA composition and stability. PMID:24039980

  20. Influence of preadsorbed milk proteins on adhesion of Listeria monocytogenes to hydrophobic and hydrophilic silica surfaces.

    PubMed Central

    al-Makhlafi, H; McGuire, J; Daeschel, M

    1994-01-01

    The adsorption of beta-lactoglobulin, bovine serum albumin, alpha-lactalbumin, and beta-casein for 8 h and beta-lactoglobulin and bovine serum albumin for 1 h at silanized silica surfaces of low and high hydrophobicity, followed by incubation in buffer and contact with Listeria monocytogenes, resulted in different numbers of cells adhered per unit of surface area. Adhesion to both surfaces was greatest when beta-lactoglobulin was present and was lowest when bovine serum albumin was present. Preadsorption of alpha-lactalbumin and beta-casein showed an intermediate effect on cell adhesion. Adsorption of beta-lactoglobulin for 1 h resulted in a generally lower number of cells adhered compared with the 8-h adsorption time, while the opposite result was observed with respect to bovine serum albumin. The adhesion data were explainable in terms of the relative rates of arrival to the surface and postadsorptive conformational change among the proteins, in addition to the extent of surface coverage in each case. PMID:7986033

  1. Plakophilin 2 Affects Cell Migration by Modulating Focal Adhesion Dynamics and Integrin Protein Expression

    PubMed Central

    Koetsier, Jennifer L.; Amargo, Evangeline V.; Todorovi?, Viktor; Green, Kathleen J.; Godsel, Lisa M.

    2014-01-01

    Plakophilin 2 (PKP2), a desmosome component, modulates the activity and localization of the small GTPase RhoA at sites of cellcell contact. PKP2 regulates cortical actin rearrangement during junction formation, and its loss is accompanied by an increase in actin stress fibers. We hypothesized that PKP2 may regulate focal adhesion dynamics and cell migration. Here we show that PKP2-deficient cells bind efficiently to the extracellular matrix, but upon spreading display total cell areas ~30% smaller than control cells. Focal adhesions in PKP2-deficient cells are ~2 larger and more stable than in control cells, and vinculin displays an increased time for fluorescence recovery after photobleaching. Furthermore, ?4 and ?1 integrin protein and mRNA expression is elevated in PKP2-silenced cells. Normal focal adhesion phenotypes can be restored in PKP2-null cells by dampening the RhoA pathway or silencing ?1 integrin. However, integrin expression levels are not restored by RhoA signaling inhibition. These data uncover a potential role for PKP2 upstream of ?1 integrin and RhoA in integrating cellcell and cellsubstrate contact signaling in basal keratinocytes necessary for the morphogenesis, homeostasis, and reepithelialization of the stratified epidermis. PMID:23884246

  2. Serum protein layers on parylene-C and silicon oxide: effect on cell adhesion.

    PubMed

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

    2015-02-01

    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

  3. 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

  4. Platelet adhesion and plasma protein adsorption control of collagen surfaces by He + ion implantation

    NASA Astrophysics Data System (ADS)

    Kurotobi, K.; Suzuki, Y.; Nakajima, H.; Suzuki, H.; Iwaki, M.

    2003-05-01

    He + ion implanted collagen-coated tubes with a fluence of 1 10 14 ions/cm 2 were exhibited antithrombogenicity. To investigate the mechanisms of antithrombogenicity of these samples, plasma protein adsorption assay and platelet adhesion experiments were performed. The adsorption of fibrinogen (Fg) and von Willebrand factor (vWf) was minimum on the He + ion implanted collagen with a fluence of 1 10 14 ions/cm 2. Platelet adhesion (using platelet rich plasma) was inhibited on the He + ion implanted collagen with a fluence of 1 10 14 ions/cm 2 and was accelerated on the untreated collagen and ion implanted collagen with fluences of 1 10 13, 1 10 15 and 1 10 16 ions/cm 2. Platelet activation with washed platelets was observed on untreated collagen and He + ion implanted collagen with a fluence of 1 10 14 ions/cm 2 and was inhibited with fluences of 1 10 13, 1 10 15 and 1 10 16 ions/cm 2. Generally, platelets can react with a specific ligand inside the collagen (GFOGER sequence). The results of platelets adhesion experiments using washed platelets indicated that there were no ligands such as GFOGER on the He + ion implanted collagen over a fluence of 1 10 13 ions/cm 2. On the 1 10 14 ions/cm 2 implanted collagen, no platelet activation was observed due to the influence of plasma proteins. From the above, it is concluded that the decrease of adsorbed Fg and vWf caused the antithrombogenicity of He + ion implanted collagen with a fluence of 1 10 14 ions/cm 2 and that plasma protein adsorption took an important role repairing the graft surface.

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

    PubMed Central

    Leech, Astrid O.; Cruz, Rodrigo G.B.; Hill, Arnold D.K.

    2015-01-01

    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

  6. CLCA Splicing Isoform Associated with Adhesion through ?1-Integrin and Its Scaffolding Protein

    PubMed Central

    Yamazaki, Jun; Okamura, Kazuhiko; Uehara, Kiyoko; Hatta, Mitsutoki

    2013-01-01

    We previously found that a rat CLCA homologue (rCLCA-f) modulates Ca2+-dependent Cl? transport in the ductal cells of the rat submandibular gland. CLCA proteins have been shown to be multifunctional, with roles in, for example, cell adhesion. Here, we describe the mRNA and protein expressions of a splicing isoform of rat rCLCA (rCLCA-t). This isoform is a 514-amino acid protein containing a C-terminal 59-amino acid that is distinct from the rCLCA-f sequence. Immunohistochemistry revealed rCLCA-t to be located in the basal cells of the rat submandibular gland excretory duct and the stratum basale of rat epidermis, whereas rCLCA-f was detected in cells during the process of differentiation. In a heterologous expression system, rCLCA-t was found to be a membrane protein present predominantly in the perinuclear region, and not to be either present on the cell surface or secreted. rCLCA-t failed to enhance ionomycin-induced Cl? conductance (unlike rCLCA-f). When compared with rCLCA-f, it weakened cell attachment to a greater extent and in a manner that was evidently modulated by intracellular Ca2+, protein kinase C, and ?1-integrin. rCLCA-t was found to associate with RACK1 (receptor for activated C kinase) and to reduce expression of mature ?1-integrin. Treatment of rat skin with rCLCA-t siRNA increased the expression of ?1-integrin in the stratum basale of the epidermis. These results are consistent with cell-specific splicing of rCLCA mRNA playing a role in the modulation of the adhesive potential of undifferentiated epithelial cells. PMID:23297403

  7. Signalling 'SELF' to phagocytes with adhesion proteins that turn off default engulfment

    NASA Astrophysics Data System (ADS)

    Discher, Dennis; Photos, Peter; Subramanian, Shyam; Parthasarathy, Ranganath

    2004-03-01

    'Marker of self' proteins are being discovered that reportedly have the important role in multicellular organisms of signalling self to the many phagocytes (macrophages, neutrophils, etc) in the body. These cells generally succeed in engulfing, degrading, or at least isolating (even by cell fusion into giant cells) foreign objects ranging from the microbes that have existed for eons to more modern implant biomaterials and injectibles. We have conducted wide-ranging studies aimed at clarifying the function of one 'marker of self' surface protein, CD47, found on all cells and its counter-receptor, SIRPa, found on phagocytes. The Ig domains of these proteins, from human and mouse, are being recombinantly expressed and used to not only verify binding to the suitable receptors in solution and on cells, but the proteins are also being used to begin evaluating the signaling dynamics that underlie phagocytosis inhibition through adhesion. Conceptually, activators of engulfment on the surfaces of targeted cells (complement , antibodies, etc.) compete with CD47-SIRPa signalling through phosphorylation cascades that couple to cytoskeleton activation in the phagocytes. Collectively the results suggest new protein-protein means of understanding and perhaps augmenting biocompatibility.

  8. Effect of interfacial serum proteins on melanoma cell adhesion to biodegradable poly(l-lactic acid) microspheres coated with hydroxyapatite.

    PubMed

    Shinto, Hiroyuki; Hirata, Takuya; Fukasawa, Tomonori; Fujii, Syuji; Maeda, Hayata; Okada, Masahiro; Nakamura, Yoshinobu; Furuzono, Tsutomu

    2013-08-01

    We have measured the interaction forces between a murine melanoma cell and a poly(l-lactic acid) (PLLA) microsphere coated with/without hydroxyapatite (HAp) nanoparticles (i.e., an HAp/PLLA or a bare PLLA microsphere) in a serum-free culture medium, using atomic force microscopy (AFM) with colloid probe technique, in order to investigate how the HAp-nanoparticle coating as well as interfacial serum proteins influence the cell-microsphere adhesion. The cell adhesion force of the HAp/PLLA microspheres was 1.4-fold stronger than that of the bare PLLA microspheres. When the microspheres were pretreated with a culture medium supplemented with 10% fetal bovine serum, the cell adhesion force of the HAp/PLLA microspheres was increased by a factor of 2.1; in contrast, no change was observed in the cell adhesion force of the bare PLLA microspheres before/after the pretreatment. Indeed, the cell adhesion force of the HAp/PLLA was 2.8-fold larger than that of the bare PLLA after the pretreatment. Additionally, we have investigated the effect of interfacial serum proteins on the zeta potentials of these microspheres. On the basis of the obtained results, possible mechanism of cell adhesion to the HAp/PLLA and bare PLLA microspheres in the presence/absence of the interfacial serum proteins is discussed. PMID:23524077

  9. RNA-Binding Protein Musashi1 Is a Central Regulator of Adhesion Pathways in Glioblastoma

    PubMed Central

    Uren, Philip J.; Vo, Dat T.; de Araujo, Patricia Rosa; Pötschke, Rebecca; Burns, Suzanne C.; Bahrami-Samani, Emad; Qiao, Mei; de Sousa Abreu, Raquel; Nakaya, Helder I.; Correa, Bruna R.; Kühnöl, Caspar; Ule, Jernej; Martindale, Jennifer L.; Abdelmohsen, Kotb; Gorospe, Myriam

    2015-01-01

    The conserved RNA-binding protein Musashi1 (MSI1) has emerged as a key oncogenic factor in numerous solid tumors, including glioblastoma. However, its mechanism of action has not yet been established comprehensively. To identify its target genes comprehensively and determine the main routes by which it influences glioblastoma phenotypes, we conducted individual-nucleotide resolution cross-linking and immunoprecipitation (iCLIP) experiments. We confirmed that MSI1 has a preference for UAG sequences contained in a particular structural context, especially in 3′ untranslated regions. Although numerous binding sites were also identified in intronic sequences, our RNA transcriptome sequencing analysis does not favor the idea that MSI1 is a major regulator of splicing in glioblastoma cells. MSI1 target mRNAs encode proteins that function in multiple pathways of cell proliferation and cell adhesion. Since these associations indicate potentially new roles for MSI1, we investigated its impact on glioblastoma cell adhesion, morphology, migration, and invasion. These processes are known to underpin the spread and relapse of glioblastoma, in contrast to other tumors where metastasis is the main driver of recurrence and progression. PMID:26100017

  10. Dishevelled proteins regulate cell adhesion in mouse blastocyst and serve to monitor changes in Wnt signaling.

    PubMed

    Na, Jie; Lykke-Andersen, Karin; Torres Padilla, Maria Elena; Zernicka-Goetz, Magdalena

    2007-02-01

    Wnt signaling is essential for the regulation of cell polarity and cell fate in the early embryogenesis of many animal species. Multiple Wnt genes and its pathway members are expressed in the mouse early embryo, raising the question whether they play any roles in preimplantation development. Dishevelled is an important transducer of divergent Wnt pathways. Here we show that three of the mouse Dishevelled proteins are not only expressed in oocytes and during preimplantation development, but also display distinct spatio-temporal localization. Interestingly, as embryos reach blastocyst stage, Dishevelled 2 becomes increasingly associated with cell membrane in trophectoderm cells, while at E4.5, Dishevelled 3 is highly enriched in the cytoplasm of ICM cells. These changes are coincident with an increase in the active form of beta-catenin, p120catenin transcription and decrease of Kaiso expression, indicating an upregulation of Wnt signaling activity before implantation. When Dishevelled-GFP fusion proteins are overexpressed in single blastomeres of the 4-cell stage embryo, the progeny of this cell show reduction in cell adhesiveness and a rounded shape at the blastocyst stage. This suggests that perturbing Dvl function interferes with cell-cell adhesion through the non-canonical Wnt pathway in blastocysts. PMID:17005174

  11. Protein adsorption and cell adhesion on polyurethane/Pluronic surface with lotus leaf-like topography.

    PubMed

    Zheng, Jun; Song, Wei; Huang, He; Chen, Hong

    2010-06-01

    Lotus leaf-like polyurethane/Pluronic F-127 surface was fabricated via replica molding using a natural lotus leaf as the template. Water contact angle measurements showed that both the hydrophobicity of the unmodified polyurethane (PU) surface and the hydrophilicity of the PU/Pluronic surface were enhanced by the construction of lotus leaf-like topography. Protein adsorption on the PU/Pluronic surface without topographic modification was significantly lower than on the PU surface. Adsorption was further reduced when lotus leaf-like topography was constructed on the PU/Pluronic surface. Cell culture experiments with L929 cells showed that adhesion on the PU/Pluronic surface with lotus leaf-like topography was low and adherent cells were spherical and of low viability. The PU/Pluronic surface with lotus leaf-like topography thus appears to be resistant to nonspecific protein adsorption and to cell adhesion, and these effects derive from the both chemical composition and topography. The results suggest a new strategy based on surface topography for the design of antifouling materials. PMID:20172699

  12. NELIN, a new F-actin associated protein, stimulates HeLa cell migration and adhesion.

    PubMed

    Wang, Wei; Zhang, Weili; Han, Yu; Chen, Jingzhou; Wang, Yibo; Zhang, Zhen; Hui, Rutai

    2005-05-20

    A new gene (GenBank Accession No. AF114264) was cloned from umbilical vein wall tissue by using RT-PCR. The gene shares high similarity to the gene encoding F-actin binding protein nexilin, so named as NELIN. A clone of 2737bp contains open reading frame of 1344bp extending from 412 to 1755. NELIN was expressed primarily in the heart and skeletal muscle among eight tested normal tissues. Immunofluorescence and immunoprecipitation demonstrated that NELIN product was associated with F-actin. Stable transfection of NELIN into HeLa cells increased the cell migration by 2.17-fold and the adhesion by 1.67-fold, respectively, compared to cells with the empty vector (P<0.05). The results support that NELIN product is an F-actin associated protein and mediates cell motility. PMID:15823560

  13. Characterization and binding analysis of a microneme adhesive repeat domain-containing protein from Toxoplasma gondii.

    PubMed

    Gong, Haiyan; Kobayashi, Kyousuke; Sugi, Tatsuki; Takemae, Hitoshi; Ishiwa, Akiko; Recuenco, Frances C; Murakoshi, Fumi; Xuan, Xuenan; Horimoto, Taisuke; Akashi, Hiroomi; Kato, Kentaro

    2014-04-01

    The intracellular parasite Toxoplasma gondii invades almost all nucleated cells, and has infected approximately 34% of the world's population to date. In order to develop effective vaccines against T. gondii infection, understanding of the role of the molecules that are involved in the invasion process is important. For this purpose, we characterized T. gondii proteins that contain microneme adhesive repeats (MARs), which are common in moving junction proteins. T. gondii MAR domain-containing protein 4a (TgMCP4a), which contains repeats of 17-22 amino acid segments at the N-terminus and three putative MAR domains at the C-terminus, is localized near the rhoptry of extracellular parasites. Following infection, TgMCP4a was detected in the parasitophorous vacuole. The recombinant Fc-TgMCP4a N-terminus protein (rTgMCP4a-1/Fc) showed binding activity to the surface proteins of Vero, 293T, and CHO cells. The recombinant GST-TgMCP4a N-terminus protein (rTgMCP4a-1/GST), which exhibited binding activity, was used to pull down the interacting factors from 293T cell lysate, and subsequent mass spectrometry analysis revealed that three types of heat shock proteins (HSPs) interacted with TgMCP4a. Transfection of a FLAG fusion protein of TgMCP4a-1 (rTgMCP4a-1/FLAG) into 293T cell and the following immunoprecipitation with anti-FLAG antibody confirmed the interactions of HSC70 with TgMCP4a. The addition of rTgMCP4a-1/GST into the culture medium significantly affected the growth of the parasite. This study hints that T. gondii may employ HSP proteins of host cell to facilitate their growth. PMID:24361285

  14. Barnacle larvae exploring surfaces with variable hydrophilicity: influence of morphology and adhesion of "footprint" proteins by AFM.

    PubMed

    Guo, Shifeng; Puniredd, Sreenivasa Reddy; Ja?czewski, Dominik; Lee, Serina Siew Chen; Teo, Serena Lay Ming; He, Tao; Zhu, Xiaoying; Vancso, G Julius

    2014-08-27

    Interaction forces of adhesive proteins employed by cyprid larvae of Amphibalanus amphitrite for temporary attachment during surface exploration in marine fouling were studied by AFM force spectroscopy using chemically modified, reactive colloidal probes. The proteins were covalently attached to the surfaces of the probes by incubation in the protein deposits (footprints) left behind at the surface by the cyprids. This covalent coupling enabled robust and reproducible probing of adhesion of the attachment proteins to model surfaces with variable hydrophilicity. Three model monolayer surfaces were designed and prepared that exhibited different wettabilities derived from variations in the monolayer chemical composition. The morphology and size of cyprid protein deposits was imaged by AFM. The deposits showed larger area of spreading on more hydrophobic surfaces, whereas the overall volume of the secreted proteins exhibited no significant variation. Notable difference in adhesion forces was found among the surfaces by force spectroscopy, with substantially higher values measured on the hydrophobic surface (21 2 nN) than that measured on the more hydrophilic surface (7.2 1 nN). The same surfaces were also tested in laboratory essays. Rather surprisingly, no significant differences were found in values of fractional cyprid settlement among the surfaces studied, indicating that variations of surface wettability and adhesion strength of settlement proteins may be insufficient to explain settlement trends. PMID:25055115

  15. 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-?)-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 g/ml to 1 mg/ml. Treatment with 0.1 mg/ml BLE caused 63% inhibition of monocyte adhesion in TNF-?-activated HUVECs, which was associated with 38.4% suppression of vascular cell adhesion molecule-1 expression. Furthermore, TNF-?-induced reactive oxygen species generation was decreased to 47.9% in BLE treated TNF-?-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

  16. 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

  17. 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.

  18. High affinity neurexin binding to cell adhesion G-protein-coupled receptor CIRL1/latrophilin-1 produces an intercellular adhesion complex.

    PubMed

    Boucard, Antony A; Ko, Jaewon; Sdhof, Thomas C

    2012-03-16

    The G-protein-coupled receptor CIRL1/latrophilin-1 (CL1) and the type-1 membrane proteins neurexins represent distinct neuronal cell adhesion molecules that exhibit no similarities except for one common function: both proteins are receptors for ?-latrotoxin, a component of black widow spider venom that induces massive neurotransmitter release at synapses. Unexpectedly, we have now identified a direct binding interaction between the extracellular domains of CL1 and neurexins that is regulated by alternative splicing of neurexins at splice site 4 (SS4). Using saturation binding assays, we showed that neurexins lacking an insert at SS4 bind to CL1 with nanomolar affinity, whereas neurexins containing an insert at SS4 are unable to bind. CL1 competed for neurexin binding with neuroligin-1, a well characterized neurexin ligand. The extracellular sequences of CL1 contain five domains (lectin, olfactomedin-like, serine/threonine-rich, hormone-binding, and G-protein-coupled receptor autoproteolysis-inducing (GAIN) domains). Of these domains, the olfactomedin-like domain mediates neurexin binding as shown by deletion mapping. Cell adhesion assays using cells expressing neurexins and CL1 revealed that their interaction produces a stable intercellular adhesion complex, indicating that their interaction can be trans-cellular. Thus, our data suggest that CL1 constitutes a novel ligand for neurexins that may be localized postsynaptically based on its well characterized interaction with intracellular SH3 and multiple ankyrin repeats adaptor proteins (SHANK) and could form a trans-synaptic complex with presynaptic neurexins. PMID:22262843

  19. Regulation of Focal Adhesion Dynamics and Cell Motility by the EB2 and Hax1 Protein Complex.

    PubMed

    Liu, Han; Yue, Jiping; Huang, He; Gou, Xuewen; Chen, Shao-Yu; Zhao, Yingming; Wu, Xiaoyang

    2015-12-25

    Cell migration is a fundamental cellular process requiring integrated activities of the cytoskeleton, membrane, and cell/extracellular matrix adhesions. Many cytoskeletal activities rely on microtubule filaments. It has been speculated that microtubules can serve as tracks to deliver proteins essential for focal adhesion turnover. Three microtubule end-binding proteins (EB1, EB2, and EB3) in mammalian cells can track the plus ends of growing microtubules. EB1 and EB3 together can regulate microtubule dynamics by promoting microtubule growth and suppressing catastrophe, whereas, in contrast, EB2 does not play a direct role in microtubule dynamic instability, and little is known about the cellular function of EB2. By quantitative proteomics, we identified mammalian HCLS1-associated protein X-1 (HAX1) as an EB2-specific interacting protein. Knockdown of HAX1 and EB2 in skin epidermal cells stabilizes focal adhesions and impairs epidermal migration in vitro and in vivo. Our results further demonstrate that cell motility and focal adhesion turnover require interaction between Hax1 and EB2. Together, our findings provide new insights for this critical cellular process, suggesting that EB2 association with Hax1 plays a significant role in focal adhesion turnover and epidermal migration. PMID:26527684

  20. Adhesion of Staphylococcus aureus to the vessel wall under flow is mediated by von Willebrand factor-binding protein.

    PubMed

    Claes, Jorien; Vanassche, Thomas; Peetermans, Marijke; Liesenborghs, Laurens; Vandenbriele, Christophe; Vanhoorelbeke, Karen; Missiakas, Dominique; Schneewind, Olaf; Hoylaerts, Marc F; Heying, Ruth; Verhamme, Peter

    2014-09-01

    Adhesion of Staphylococcus aureus to blood vessels under shear stress requires von Willebrand factor (VWF). Several bacterial factors have been proposed to interact with VWF, including VWF-binding protein (vWbp), a secreted coagulase that activates the host's prothrombin to generate fibrin. We measured the adhesion of S aureus Newman and a vWbp-deficient mutant (vwb) to VWF, collagen, and activated endothelial cells in a microparallel flow chamber. In vivo adhesion of S aureus was evaluated in the mesenteric circulation of wild-type (WT) and VWF-deficient mice. We found a shear-dependent increase in adhesion of S aureus to the (sub)endothelium that was dependent on interactions between vWbp and the A1-domain of VWF. Adhesion was further enhanced by coagulase-mediated fibrin formation that clustered bacteria and recruited platelets into bacterial microthrombi. In vivo, deficiency of vWbp or VWF as well as inhibition of coagulase activity reduced S aureus adhesion. We conclude that vWbp contributes to vascular adhesion of S aureus through 2 independent mechanisms: shear-mediated binding to VWF and activation of prothrombin to form S aureus-fibrin-platelet aggregates. PMID:24951431

  1. The involvement of an integrin-like protein and protein kinase C in amoebic adhesion to fibronectin and amoebic cytotoxicity.

    PubMed

    Han, Kyu-Lee; Lee, Hyun-Ju; Shin, Myeong Heon; Shin, Ho-Joon; Im, Kyung-Il; Park, Soon-Jung

    2004-09-01

    Adherence of a pathogen to the host cell is one of the critical steps in microbial infections. Naegleria fowleri, a causative agent of primary amoebic meningoencephalitis in humans, is expected to interact with extracellular components of the host, such as fibronectin, in a receptor-mediated mode. In this study, we investigated the interaction between N. fowleri and fibronectin to understand its cytopathology. In binding assays using immobilized fibronectin, the number of amoebae bound to fibronectin was increased compared to the controls, and was dependent on the amount of coated fibronectin present. A fibronectin binding protein of 60 kDa was found in extracts of N. fowleri. Western blot and immunolocalization assays using integrin alpha(5)/FnR antibodies showed that a 60 kDa protein reacted with the antibodies in extracts of N. fowleri, which was localized on the surface of N. fowleri. Preincubation of N. fowleri with the integrin antibodies significantly inhibited amoebic binding to fibronectin and cytotoxicity to the CHO cells. Additionally, protein kinase C activity was detected in the extract of N. fowleri. When N. fowleri was pretreated with protein kinase C activator or inhibitor, the abilities of amoebic adhesion to fibronectin and cytotoxicity to the host cells were markedly affected compared to untreated amoebae. These results suggest that an amoebic integrin-like receptor and protein kinase C play important roles in amoebic cellular processes in response to fibronectin. PMID:15338291

  2. Hematopoietic PBX-interacting protein (HPIP) is over expressed in breast infiltrative ductal carcinoma and regulates cell adhesion and migration through modulation of focal adhesion dynamics.

    PubMed

    Bugide, S; David, D; Nair, A; Kannan, N; Samanthapudi, V S K; Prabhakar, J; Manavathi, B

    2015-08-27

    The scaffolding protein, hematopoietic PBX-interacting protein (HPIP/PBXIP1), regulates cell migration necessary for cancer cell dissemination. However, the mechanism that governs this process remains unknown. We show here that HPIP expression is associated with stages of breast cancer where cell dissemination results in poor patient outcome. Our investigation finds a novel association of HPIP with focal adhesion kinase (FAK) regulating FA dynamics. Interestingly, this interaction that led to activation of FAK protein was mediated by the C-terminal domain of HPIP and not the typical integrin-binding motif. Further, short hairpin RNA-mediated knockdown of FAK expression significantly reduced HPIP-induced cell migration indicating participation of FAK pathway. Live-cell time-lapse imaging and biochemical analysis further established the role of HPIP in microtubule-induced FA disassembly. We also found that HPIP-mediated MAPK activation led to phosphorylation and subsequent activation of calpain2, and the activated calpain2 in turn proteolyses FA protein, talin. Interestingly, HPIP is also proteolysed by calpain2 in breast cancer cells. The proteolysis of HPIP and talin by calpain2, and the activation of calapin2 by HPIP-mediated MAPK phosphorylation, is a novel regulatory axis to modulate the cell migration signal. Together, we have determined HPIP as a novel activator of FAK and a new substrate of calpain2. These molecular interactions between HPIP and FAK, and HPIP and calpain2 regulate cell adhesion and migration through modulation of FA dynamics. PMID:25486428

  3. Secreted Listeria adhesion protein (Lap) influences Lap-mediated Listeria monocytogenes paracellular translocation through epithelial barrier

    PubMed Central

    2013-01-01

    Background Listeria adhesion protein (Lap), an alcohol acetaldehyde dehydrogenase (lmo1634) promotes bacterial paracellular translocation through epithelial cell junctions during gastrointestinal phase of infection. Secreted Lap is critical for pathogenesis and is mediated by SecA2 system; however, if strain dependent variation in Lap secretion would affect L. monocytogenes paracellular translocation through epithelial barrier is unknown. Methods Amounts of Lap secretion were examined in clinical isolates of L. monocytogenes by cell fractionation analysis using Western blot. Quantitative reverse transcriptase PCR (qRT-PCR) was used to verify protein expression profiles. Adhesion and invasion of isolates were analyzed by in vitro Caco-2 cell culture model and paracellular translocation was determined using a trans-well model pre-seeded with Caco-2 cells. Results Western blot revealed that expression of Lap in whole cell preparation of isolates was very similar; however, cell fractionation analysis indicated variable Lap secretion among isolates. The strains showing high Lap secretion in supernatant exhibited significantly higher adhesion (3.4 - 4.8% vs 1.5 - 2.3%, P < 0.05), invasion and paracellular translocation in Caco-2 cells than the low secreting isolates. In cell wall fraction, Lap level was mostly uniform for both groups, while Lap accumulated in cytosol in low secreting strains indicating that Lap distribution in cellular compartments is a strain-dependent phenomenon, which may be controlled by the protein transport system, SecA2. ΔsecA2 mutants showed significantly reduced paracellular translocation through epithelial barrier (0.48 ± 0.01 vs 0.24 ± 0.02, P < 0.05). qRT-PCR did not show any discernible variation in lap transcript levels in either high or low secreting isolates. Conclusion This study revealed that secreted Lap is an important determinant in Lap-mediated L. monocytogenes translocation through paracellular route and may serve as an indicator for pathogenic potential of an isolate. PMID:23799938

  4. 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

  5. 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.

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

    PubMed

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

    2015-02-01

    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

  7. Unraveling the role of surface mucus-binding protein and pili in muco-adhesion of Lactococcus lactis.

    PubMed

    Le, Doan Thanh Lam; Tran, Thi-Ly; Duviau, Marie-Pierre; Meyrand, Mickael; Gurardel, Yann; Castelain, Mickal; Loubire, Pascal; Chapot-Chartier, Marie-Pierre; Dague, Etienne; Mercier-Bonin, Muriel

    2013-01-01

    Adhesion of bacteria to mucus may favor their persistence within the gut and their beneficial effects to the host. Interactions between pig gastric mucin (PGM) and a natural isolate of Lactococcus lactis (TIL448) were measured at the single-cell scale and under static conditions, using atomic force microscopy (AFM). In parallel, these interactions were monitored at the bacterial population level and under shear flow. AFM experiments with a L. lactis cell-probe and a PGM-coated surface revealed a high proportion of specific adhesive events (60%) and a low level of non-adhesive ones (2%). The strain muco-adhesive properties were confirmed by the weak detachment of bacteria from the PGM-coated surface under shear flow. In AFM, rupture events were detected at short (100-200 nm) and long distances (up to 600-800 nm). AFM measurements on pili and mucus-binding protein defective mutants demonstrated the comparable role played by these two surface proteinaceous components in adhesion to PGM under static conditions. Under shear flow, a more important contribution of the mucus-binding protein than the pili one was observed. Both methods differ by the way of probing the adhesion force, i.e. negative force contact vs. sedimentation and normal-to-substratum retraction vs. tangential detachment conditions, using AFM and flow chamber, respectively. AFM blocking assays with free PGM or O-glycan fractions purified from PGM demonstrated that neutral oligosaccharides played a major role in adhesion of L. lactis TIL448 to PGM. This study dissects L. lactis muco-adhesive phenotype, in relation with the nature of the bacterial surface determinants. PMID:24260308

  8. Unraveling the Role of Surface Mucus-Binding Protein and Pili in Muco-Adhesion of Lactococcus lactis

    PubMed Central

    Duviau, Marie-Pierre; Meyrand, Mickael; Guérardel, Yann; Castelain, Mickaël; Loubière, Pascal; Chapot-Chartier, Marie-Pierre; Dague, Etienne; Mercier-Bonin, Muriel

    2013-01-01

    Adhesion of bacteria to mucus may favor their persistence within the gut and their beneficial effects to the host. Interactions between pig gastric mucin (PGM) and a natural isolate of Lactococcus lactis (TIL448) were measured at the single-cell scale and under static conditions, using atomic force microscopy (AFM). In parallel, these interactions were monitored at the bacterial population level and under shear flow. AFM experiments with a L. lactis cell-probe and a PGM-coated surface revealed a high proportion of specific adhesive events (60%) and a low level of non-adhesive ones (2%). The strain muco-adhesive properties were confirmed by the weak detachment of bacteria from the PGM-coated surface under shear flow. In AFM, rupture events were detected at short (100−200 nm) and long distances (up to 600−800 nm). AFM measurements on pili and mucus-binding protein defective mutants demonstrated the comparable role played by these two surface proteinaceous components in adhesion to PGM under static conditions. Under shear flow, a more important contribution of the mucus-binding protein than the pili one was observed. Both methods differ by the way of probing the adhesion force, i.e. negative force contact vs. sedimentation and normal-to-substratum retraction vs. tangential detachment conditions, using AFM and flow chamber, respectively. AFM blocking assays with free PGM or O-glycan fractions purified from PGM demonstrated that neutral oligosaccharides played a major role in adhesion of L. lactis TIL448 to PGM. This study dissects L. lactis muco-adhesive phenotype, in relation with the nature of the bacterial surface determinants. PMID:24260308

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

    PubMed

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

    2012-10-01

    Acanthamoeba castellanii is a single-celled protozoan that is widely distributed in the environment and is a well-known of causing human keratitis, a vision-threatening infection. In this study, an ethyl methane sulfonate (EMS) and a selection of saccharide were applied to A. castellanii by chemical mutagenesis. To understand the functional roles of a mannose-binding protein (MBP). A. castellanii were treated with methyl-alpha-D-mannopyranoside abbreviated Man, with and without the EMS pre-treatment, and their adhesion and cytotoxicity were analyzed, using a human brain microvascular endothelial cell (HBMEC) as the target cell. Both EMS and Man mutants exhibited significantly decreased levels of MBP expression and cytotoxicity to HBMEC, but showed similar levels of binding to HBMEC, as compared with the wild type. Of interest was that the exogenous mannose inhibited amoebae (i.e., Man mutant) binding to the HBMEC by <20%. Only the mutant Man exhibited a significant decrease in bacterial uptake, as compared to the wild type, 0.020 vs 0.032 (p<0.05) and proteolytic activity. The results showed that MBP should be clearly provided as the pathogenic target candidate, to further target-based therapy, but EMS mutation should not be associated with initial adhesion and phagocytosis of A. castellanii. PMID:22940016

  10. 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

  11. Vascular adhesion protein-1 promotes liver inflammation and drives hepatic fibrosis

    PubMed Central

    Weston, Chris J.; Shepherd, Emma L.; Claridge, Lee C.; Rantakari, Pia; Curbishley, Stuart M.; Tomlinson, Jeremy W.; Hubscher, Stefan G.; Reynolds, Gary M.; Aalto, Kristiina; Anstee, Quentin M.; Jalkanen, Sirpa; Salmi, Marko; Smith, David J.; Day, Christopher P.; Adams, David H.

    2014-01-01

    Nonalcoholic fatty liver disease (NAFLD) encompasses a range of manifestations, including steatosis and cirrhosis. Progressive disease is characterized by hepatic leukocyte accumulation in the form of steatohepatitis. The adhesion molecule vascular adhesion protein-1 (VAP-1) is a membrane-bound amine oxidase that promotes leukocyte recruitment to the liver, and the soluble form (sVAP-1) accounts for most circulating monoamine oxidase activity, has insulin-like effects, and can initiate oxidative stress. Here, we determined that hepatic VAP-1 expression is increased in patients with chronic liver disease and that serum sVAP-1 levels are elevated in patients with NAFLD compared with those in control individuals. In 4 murine hepatic injury models, an absence or blockade of functional VAP-1 reduced inflammatory cell recruitment to the liver and attenuated fibrosis. Moreover, disease was reduced in animals expressing a catalytically inactive form of VAP-1, implicating enzyme activity in the disease pathogenesis. Within the liver, hepatic stromal cells expressed functional VAP-1, and evaluation of cultured cells revealed that sVAP-1 promotes leukocyte migration through catalytic generation of ROS, which depended on VAP-1 enzyme activity. VAP-1 enhanced stromal cell spreading and wound closure and modulated expression of profibrotic genes. Together, these results link the amine oxidase activity of VAP-1 with hepatic inflammation and fibrosis and suggest that targeting VAP-1 has therapeutic potential for NAFLD and other chronic fibrotic liver diseases. PMID:25562318

  12. 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

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

    PubMed Central

    Bligt-Lindn, Eva; Pihlavisto, Marjo; Szatmri, Istvn; Otwinowski, Zbyszek; Smith, David J.; Lzr, Lszl; Flp, 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

  14. 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.

  15. Adhesive properties of YapV and paralogous autotransporter proteins of Yersinia pestis.

    PubMed

    Nair, Manoj K M; De Masi, Leon; Yue, Min; Galvn, Estela M; Chen, Huaiqing; Wang, Fang; Schifferli, Dieter M

    2015-05-01

    Yersinia pestis is the causative agent of plague. This bacterium evolved from an ancestral enteroinvasive Yersinia pseudotuberculosis strain by gene loss and acquisition of new genes, allowing it to use fleas as transmission vectors. Infection frequently leads to a rapidly lethal outcome in humans, a variety of rodents, and cats. This study focuses on the Y. pestis KIM yapV gene and its product, recognized as an autotransporter protein by its typical sequence, outer membrane localization, and amino-terminal surface exposure. Comparison of Yersinia genomes revealed that DNA encoding YapV or each of three individual paralogous proteins (YapK, YapJ, and YapX) was present as a gene or pseudogene in a strain-specific manner and only in Y. pestis and Y. pseudotuberculosis. YapV acted as an adhesin for alveolar epithelial cells and specific extracellular matrix (ECM) proteins, as shown with recombinant Escherichia coli, Y. pestis, or purified passenger domains. Like YapV, YapK and YapJ demonstrated adhesive properties, suggesting that their previously related in vivo activity is due to their capacity to modulate binding properties of Y. pestis in its hosts, in conjunction with other adhesins. A differential host-specific type of binding to ECM proteins by YapV, YapK, and YapJ suggested that these proteins participate in broadening the host range of Y. pestis. A phylogenic tree including 36 Y. pestis strains highlighted an association between the gene profile for the four paralogous proteins and the geographic location of the corresponding isolated strains, suggesting an evolutionary adaption of Y. pestis to specific local animal hosts or reservoirs. PMID:25690102

  16. Adhesive Properties of YapV and Paralogous Autotransporter Proteins of Yersinia pestis

    PubMed Central

    Nair, Manoj K. M.; De Masi, Leon; Yue, Min; Galván, Estela M.; Chen, Huaiqing; Wang, Fang

    2015-01-01

    Yersinia pestis is the causative agent of plague. This bacterium evolved from an ancestral enteroinvasive Yersinia pseudotuberculosis strain by gene loss and acquisition of new genes, allowing it to use fleas as transmission vectors. Infection frequently leads to a rapidly lethal outcome in humans, a variety of rodents, and cats. This study focuses on the Y. pestis KIM yapV gene and its product, recognized as an autotransporter protein by its typical sequence, outer membrane localization, and amino-terminal surface exposure. Comparison of Yersinia genomes revealed that DNA encoding YapV or each of three individual paralogous proteins (YapK, YapJ, and YapX) was present as a gene or pseudogene in a strain-specific manner and only in Y. pestis and Y. pseudotuberculosis. YapV acted as an adhesin for alveolar epithelial cells and specific extracellular matrix (ECM) proteins, as shown with recombinant Escherichia coli, Y. pestis, or purified passenger domains. Like YapV, YapK and YapJ demonstrated adhesive properties, suggesting that their previously related in vivo activity is due to their capacity to modulate binding properties of Y. pestis in its hosts, in conjunction with other adhesins. A differential host-specific type of binding to ECM proteins by YapV, YapK, and YapJ suggested that these proteins participate in broadening the host range of Y. pestis. A phylogenic tree including 36 Y. pestis strains highlighted an association between the gene profile for the four paralogous proteins and the geographic location of the corresponding isolated strains, suggesting an evolutionary adaption of Y. pestis to specific local animal hosts or reservoirs. PMID:25690102

  17. Photorhabdus adhesion modification protein (Pam) binds extracellular polysaccharide and alters bacterial attachment

    PubMed Central

    2010-01-01

    Background Photorhabdus are Gram-negative nematode-symbiotic and insect-pathogenic bacteria. The species Photorhabdus asymbiotica is able to infect humans as well as insects. We investigated the secreted proteome of a clinical isolate of P. asymbiotica at different temperatures in order to identify proteins relevant to the infection of the two different hosts. Results A comparison of the proteins secreted by a clinical isolate of P. asymbiotica at simulated insect (28C) and human (37C) temperatures led to the identification of a small and highly abundant protein, designated Pam, that is only secreted at the lower temperature. The pam gene is present in all Photorhabdus strains tested and shows a high level of conservation across the whole genus, suggesting it is both ancestral to the genus and probably important to the biology of the bacterium. The Pam protein shows limited sequence similarity to the 13.6 kDa component of a binary toxin of Bacillus thuringiensis. Nevertheless, injection or feeding of heterologously produced Pam showed no insecticidal activity to either Galleria mellonella or Manduca sexta larvae. In bacterial colonies, Pam is associated with an extracellular polysaccharide (EPS)-like matrix, and modifies the ability of wild-type cells to attach to an artificial surface. Interestingly, Surface Plasmon Resonance (SPR) binding studies revealed that the Pam protein itself has adhesive properties. Although Pam is produced throughout insect infection, genetic knockout does not affect either insect virulence or the ability of P. luminescens to form a symbiotic association with its host nematode, Heterorhabditis bacteriophora. Conclusions We studied a highly abundant protein, Pam, which is secreted in a temperature-dependent manner in P. asymbiotica. Our findings indicate that Pam plays an important role in enhancing surface attachment in insect blood. Its association with exopolysaccharide suggests it may exert its effect through mediation of EPS properties. Despite its abundance and conservation in the genus, we find no evidence for a role of Pam in either virulence or symbiosis. PMID:20462430

  18. The role of plasma proteins in cell adhesion to PEG surface-density-gradient-modified titanium oxide.

    PubMed

    Pei, Jia; Hall, Heike; Spencer, Nicholas D

    2011-12-01

    Surface-density gradients of poly(ethylene glycol) (PEG) were fabricated, in order to carry out a systematic study of the influence of PEG chain density on protein adsorption and cell-adhesion behavior, as well as the correlation between them. Gradients with a linear change in coverage of the polycationic polymer Poly(L-lysine)-g-poly(ethylene glycol) (PLL-g-PEG) were prepared on titanium dioxide surfaces by a controlled dipping process and characterized by variable-angle spectroscopic ellipsometry and fluorescence microscopy. The adsorption behavior of single proteins (fibrinogen and albumin) generally correlated with semiempirical geometric models, illustrating the effect of the PEG-chain surface distribution on the inhibition of protein adsorption. Distinct differences could be observed between individual adsorbing proteins, attributable to their mode of surface attachment. The single and competitive adsorption of protein solutions containing albumin and fibrinogen was then investigated by fluorescence microscopy, indicating a larger amount of fibrinogen adsorption compared with albumin adsorption (in minutes to hours) along the entire PLL-g-PEG gradient samples. To further elucidate the underlying mechanism of cell adhesion and spreading as a function of PEG coverage and the potential involvement of integrins, cell-adhesion assays were carried out with human foreskin fibroblasts (hFF). The use of surface-gradient samples demonstrated the importance for protein adsorption of PEG conformation, the amount of exposed titanium dioxide surface area (and its distribution), and the structure and chemistry of the proteins involved. Correspondingly the influence of these factors on cell adhesion could be directly observed, and insights gained into the roles of both nonspecific binding and specific integrin binding in cell adhesion. PMID:21872325

  19. Adhesion G protein-coupled receptors are activated by exposure of a cryptic tethered agonist

    PubMed Central

    Stoveken, Hannah M.; Hajduczok, Alexander G.; Xu, Lei; Tall, Gregory G.

    2015-01-01

    The large class of adhesion G protein-coupled receptors (aGPCRs) bind extracellular matrix or neighboring cell-surface ligands to regulate organ and tissue development through an unknown activation mechanism. We examined aGPCR activation using two prototypical aGPCRs, GPR56 and GPR110. Active dissociation of the noncovalently bound GPR56 or GPR110 extracellular domains (ECDs) from the respective seven-transmembrane (7TM) domains relieved an inhibitory influence and permitted both receptors to activate defined G protein subtypes. After ECD displacement, the newly revealed short N-terminal stalk regions of the 7TM domains were found to be essential for G protein activation. Synthetic peptides comprising these stalks potently activated GPR56 or GPR110 in vitro or in cells, demonstrating that the stalks comprise a tethered agonist that was encrypted within the ECD. Establishment of an aGPCR activation mechanism provides a rational platform for the development of aGPCR synthetic modulators that could find clinical utility toward aGPCR-directed disease. PMID:25918380

  20. 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

  1. Complete sequence and transcript regulation of a cell adhesion protein from aggregating Dictyostelium cells

    PubMed Central

    Noegel, A.; Gerisch, G.; Stadler, J.; Westphal, M.

    1986-01-01

    Three cDNA clones coding for the contact site A (csA) protein, a cell adhesion molecule of Dictyostelium discoideum, were isolated by screening a cDNA library with monoclonal antibodies. Two of these clones contained the complete coding region for the csA protein of 1542 bp including a sequence of 57 bp coding for the leader. The N terminus of the mature protein, as it was published previously, was identified in the amino acid sequence derived from both full-length cDNA clones. Southern blot analysis suggests the presence of only one csA gene in the haploid genome. Accumulation of the csA-specific message of 1.9 kb begins during development on nitrocellulose filters at 9 h of starvation, and reaches a maximum at 12 h, the time of cell aggregation. Expression of the csA glycoprotein follows closely accumulation of the transcripts. In the multicellular slug stage following cell aggregation, the amount of csA transcripts rapidly declines to low levels. ImagesFig. 2.Fig. 3. PMID:16453689

  2. 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

  3. [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 ? (TNF?)-activated human aortic endothelial cells (HAECs) were treated with different concentrations of AMPK agonist 5-Aminoimidazole-4-carboxamide-1-?-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 TNF?. 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-?B p65 at lysine 221 site was assessed by Western blot. NF-?B 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 TNF?. NF-?B 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 TNF?-activated HAECs. Moreover, transfection of constitutive active AMPK? but not dominant negative AMPK? strongly diminished TNF?-induced upregulation of ICAM-1 and VCAM-1 mRNA expressions and secretions, as well as monocyte-endothelial adhesion. Furthermore, AMPK activation decreased TNF?-mediated acetylation of NF-?B p65 at Lys221 site and reduced NF-?B p65 DNA binding activity. Silencing p300 by siRNA significantly abolished the effect of TNF?- induced adhesion molecules expression and monocyte-endothelial adhesion. Blocking AMPK activation by compound C almost completely reversed the effect of AICAR exerted on HAECs. These results suggest AMPK activation suppresses monocyte-endothelial adhesion, and the underlying mechanism is relevant to the inhibition of p300 activity and NF-?B p65 transcriptional activity. PMID:26915321

  4. Lateral assembly of the immunoglobulin protein SynCAM 1 controls its adhesive function and instructs synapse formation

    PubMed Central

    Fogel, Adam I; Stagi, Massimiliano; Perez de Arce, Karen; Biederer, Thomas

    2011-01-01

    Synapses are specialized adhesion sites between neurons that are connected by protein complexes spanning the synaptic cleft. These trans-synaptic interactions can organize synapse formation, but their macromolecular properties and effects on synaptic morphology remain incompletely understood. Here, we demonstrate that the synaptic cell adhesion molecule SynCAM 1 self-assembles laterally via its extracellular, membrane-proximal immunoglobulin (Ig) domains 2 and 3. This cis oligomerization generates SynCAM oligomers with increased adhesive capacity and instructs the interactions of this molecule across the nascent and mature synaptic cleft. In immature neurons, cis assembly promotes the adhesive clustering of SynCAM 1 at new axo-dendritic contacts. Interfering with the lateral self-assembly of SynCAM 1 in differentiating neurons strongly impairs its synaptogenic activity. At later stages, the lateral oligomerization of SynCAM 1 restricts synaptic size, indicating that this adhesion molecule contributes to the structural organization of synapses. These results support that lateral interactions assemble SynCAM complexes within the synaptic cleft to promote synapse induction and modulate their structure. These findings provide novel insights into synapse development and the adhesive mechanisms of Ig superfamily members. PMID:21926970

  5. Cytoplasmic Domain of Zebrafish Myelin Protein Zero: Adhesive Role Depends on ?-Conformation

    PubMed Central

    Luo, XiaoYang; Inouye, Hideyo; Gross, Abby A. R.; Hidalgo, Marla M.; Sharma, Deepak; Lee, Daniel; Avila, Robin L.; Salmona, Mario; Kirschner, Daniel A.

    2007-01-01

    Solution spectroscopy studies on the cytoplasmic domain of human myelin protein zero (P0) (hP0-cyt) suggest that H-bonding between ?-strands from apposed molecules is likely responsible for the tight cytoplasmic apposition in compact myelin. As a follow-up to these findings, in the current study we used circular dichroism and x-ray diffraction to analyze the same type of model membranes previously used for hP0-cyt to investigate the molecular mechanism underlying the zebrafish cytoplasmic apposition. This space is significantly narrower in teleosts compared with that in higher vertebrates, and can be accounted for in part by the much shorter cytoplasmic domain in the zebrafish protein (zP0-cyt). Circular dichroism measurements on zP0-cyt showed similar structural characteristics to those of hP0-cyt, i.e., the protein underwent a ??? structural transition at lipid/protein (L/P) molar ratios >50, and adopted a ?-conformation at lower L/P molar ratios. X-ray diffraction was carried out on lipid vesicle solutions with zP0-cyt before and after dehydration to study the effect of protein on membrane lipid packing. Solution diffraction revealed the electron-density profile of a single membrane bilayer. Diffraction patterns of dried samples suggested a multilamellar structure with the ?-folded P0-cyt located at the intermembrane space. Our findings support the idea that the adhesive role of P0 at the cytoplasmic apposition in compact myelin depends on the cytoplasmic domain of P0 being in the ?-conformation. PMID:17693467

  6. 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 90fmol/cm(2) and 700fmol/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

  7. 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

    As the broader effort towards device and material miniaturization progresses in all fields, it becomes increasingly important to understand the implications of working with functional structures that approach the size scale of molecules, 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.As the broader effort towards device and material miniaturization progresses in all fields, it becomes increasingly important to understand the implications of working with functional structures that approach the size scale of molecules, 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

  8. 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

  9. 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

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

    PubMed

    Leo-Macias, Alejandra; Agullo-Pascual, Esperanza; Sanchez-Alonso, Jose L; Keegan, Sarah; Lin, Xianming; Arcos, Tatiana; Feng-Xia-Liang; Korchev, Yuri E; Gorelik, Julia; Feny, 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. Immunolocalization of specific keratin associated beta-proteins (beta-keratins) in the adhesive setae of Gekko gecko.

    PubMed

    Alibardi, Lorenzo

    2013-08-01

    The previous identification of 21 proteins in the digital setae transcriptome of Gekko gecko, 2 alpha-keratins of 52-53kDa and 19 beta-proteins (beta-keratins) of 10-21kDa, has indicated that most of setal corneous proteins are cysteine-rich. The production of specific antibodies for two of the main beta-protein subfamilies expressed in gecko setae has allowed the ultrastructural localization of two beta-proteins indicated as Ge-cprp-9 (cysteine-rich) and Ge-gprp-6 (glycine-rich). Only Ge-cprp-9, representing most of the 16 cysteine-rich beta-proteins, is present in the oberhautchen, setae and in the terminal spatula where adhesion takes place, supporting the previous expression study. Instead, the glycine-rich beta-proteins (Ge-gprp-6), representing the 3 glycine-rich beta-proteins of digital epidermis is only present in the stiff beta-layer of the digital scales and in the thin beta layer of the pad lamella sustaining the setae. Ge-cprp-9 is representative for most of the remaining 15 cys-rich proteins (Ge-cprp 1-16) and may have a structural and functional role in the process of adhesion. Most of the cysteine-rich setal proteins have a net positive charge and it is here hypothesized that these proteins may induce the formation of dipoles at the surface interface between the spatula and the substrate, enhancing the van der Waals forces and therefore adhesion to the substrate. The selection and improvement of these proteins during the evolution of geckos may have represented a successful factor for the survival and ecological adaptations of these climbing lizards. PMID:23639767

  12. Pathogenic Naegleria fowleri and non-pathogenic Naegleria lovaniensis exhibit differential adhesion to, and invasion of, extracellular matrix proteins.

    PubMed

    Jamerson, Melissa; da Rocha-Azevedo, Bruno; Cabral, Guy A; Marciano-Cabral, Francine

    2012-03-01

    Naegleria fowleri and Naegleria lovaniensis are closely related free-living amoebae found in the environment. N. fowleri causes primary amoebic meningoencephalitis (PAM), a rapidly fatal disease of the central nervous system, while N. lovaniensis is non-pathogenic. N. fowleri infection occurs when the amoebae access the nasal passages, attach to the nasal mucosa and its epithelial lining, and migrate to the brain. This process involves interaction with components of the host extracellular matrix (ECM). Since the ability to invade tissues can be a characteristic that distinguishes pathogenic from non-pathogenic amoebae, the objective of this study was to assess adhesion to, and invasion of, the ECM by these two related but distinct Naegleria species. N. fowleri exhibited a higher level of adhesion to the ECM components laminin-1, fibronectin and collagen I. Scanning electron microscopy revealed that N. fowleri attached on ECM substrata exhibited a spread-out appearance that included the presence of focal adhesion-like structures. Western immunoblotting revealed two integrin-like proteins for both species, but one of these, with a molecular mass of approximately 70 kDa, was detected at a higher level in N. fowleri. Confocal microscopy indicated that the integrin-like proteins co-localized to the focal adhesion-like structures. Furthermore, anti-integrin antibody decreased adhesion of N. fowleri to ECM components. Finally, N. fowleri disrupted 3D ECM scaffolds, while N. lovaniensis had a minimal effect. Collectively, these results indicate a distinction in adhesion to, and invasion of, ECM proteins between N. fowleri and N. lovaniensis. PMID:22222499

  13. Pathogenic Naegleria fowleri and non-pathogenic Naegleria lovaniensis exhibit differential adhesion to, and invasion of, extracellular matrix proteins

    PubMed Central

    Jamerson, Melissa; da Rocha-Azevedo, Bruno; Cabral, Guy A.

    2012-01-01

    Naegleria fowleri and Naegleria lovaniensis are closely related free-living amoebae found in the environment. N. fowleri causes primary amoebic meningoencephalitis (PAM), a rapidly fatal disease of the central nervous system, while N. lovaniensis is non-pathogenic. N. fowleri infection occurs when the amoebae access the nasal passages, attach to the nasal mucosa and its epithelial lining, and migrate to the brain. This process involves interaction with components of the host extracellular matrix (ECM). Since the ability to invade tissues can be a characteristic that distinguishes pathogenic from non-pathogenic amoebae, the objective of this study was to assess adhesion to, and invasion of, the ECM by these two related but distinct Naegleria species. N. fowleri exhibited a higher level of adhesion to the ECM components laminin-1, fibronectin and collagen I. Scanning electron microscopy revealed that N. fowleri attached on ECM substrata exhibited a spread-out appearance that included the presence of focal adhesion-like structures. Western immunoblotting revealed two integrin-like proteins for both species, but one of these, with a molecular mass of approximately 70 kDa, was detected at a higher level in N. fowleri. Confocal microscopy indicated that the integrin-like proteins co-localized to the focal adhesion-like structures. Furthermore, anti-integrin antibody decreased adhesion of N. fowleri to ECM components. Finally, N. fowleri disrupted 3D ECM scaffolds, while N. lovaniensis had a minimal effect. Collectively, these results indicate a distinction in adhesion to, and invasion of, ECM proteins between N. fowleri and N. lovaniensis. PMID:22222499

  14. Functional Modulation of Vascular Adhesion Protein-1 by a Novel Splice Variant

    PubMed Central

    Kaitaniemi, Sam; Grn, Kirsi; Elovaara, Heli; Salmi, Marko; Jalkanen, Sirpa; Elima, Kati

    2013-01-01

    Vascular Adhesion Protein-1 (VAP-1) is an endothelial adhesion molecule belonging to the primary amine oxidases. Upon inflammation it takes part in the leukocyte extravasation cascade facilitating transmigration of leukocytes into the inflamed tissue. Screening of a human lung cDNA library revealed the presence of an alternatively spliced shorter transcript of VAP-1, VAP-1?3. Here, we have studied the functional and structural characteristics of VAP-1?3, and show that the mRNA for this splice variant is expressed in most human tissues studied. In comparison to the parent molecule this carboxy-terminally truncated isoform lacks several of the amino acids important in the formation of the enzymatic groove of VAP-1. In addition, the conserved His684, which takes part in coordinating the active site copper, is missing from VAP-1?3. Assays using the prototypic amine substrates methylamine and benzylamine demonstrated that VAP-1?3 is indeed devoid of the semicarbazide-sensitive amine oxidase (SSAO) activity characteristic to VAP-1. When VAP-1?3-cDNA is transfected into cells stably expressing VAP-1, the surface expression of the full-length molecule is reduced. Furthermore, the SSAO activity of the co-transfectants is diminished in comparison to transfectants expressing only VAP-1. The observed down-regulation of both the expression and enzymatic activity of VAP-1 may result from a dominant-negative effect caused by heterodimerization between VAP-1 and VAP-1?3, which was detected in co-immunoprecipitation studies. This alternatively spliced transcript adds thus to the repertoire of potential regulatory mechanisms through which the cell-surface expression and enzymatic activity of VAP-1 can be modulated. PMID:23349812

  15. Functional modulation of vascular adhesion protein-1 by a novel splice variant.

    PubMed

    Kaitaniemi, Sam; Grn, Kirsi; Elovaara, Heli; Salmi, Marko; Jalkanen, Sirpa; Elima, Kati

    2013-01-01

    Vascular Adhesion Protein-1 (VAP-1) is an endothelial adhesion molecule belonging to the primary amine oxidases. Upon inflammation it takes part in the leukocyte extravasation cascade facilitating transmigration of leukocytes into the inflamed tissue. Screening of a human lung cDNA library revealed the presence of an alternatively spliced shorter transcript of VAP-1, VAP-1?3. Here, we have studied the functional and structural characteristics of VAP-1?3, and show that the mRNA for this splice variant is expressed in most human tissues studied. In comparison to the parent molecule this carboxy-terminally truncated isoform lacks several of the amino acids important in the formation of the enzymatic groove of VAP-1. In addition, the conserved His684, which takes part in coordinating the active site copper, is missing from VAP-1?3. Assays using the prototypic amine substrates methylamine and benzylamine demonstrated that VAP-1?3 is indeed devoid of the semicarbazide-sensitive amine oxidase (SSAO) activity characteristic to VAP-1. When VAP-1?3-cDNA is transfected into cells stably expressing VAP-1, the surface expression of the full-length molecule is reduced. Furthermore, the SSAO activity of the co-transfectants is diminished in comparison to transfectants expressing only VAP-1. The observed down-regulation of both the expression and enzymatic activity of VAP-1 may result from a dominant-negative effect caused by heterodimerization between VAP-1 and VAP-1?3, which was detected in co-immunoprecipitation studies. This alternatively spliced transcript adds thus to the repertoire of potential regulatory mechanisms through which the cell-surface expression and enzymatic activity of VAP-1 can be modulated. PMID:23349812

  16. Binding of CD157 Protein to Fibronectin Regulates Cell Adhesion and Spreading*

    PubMed Central

    Morone, Simona; Augeri, Stefania; Cuccioloni, Massimiliano; Mozzicafreddo, Matteo; Angeletti, Mauro; Lo Buono, Nicola; Giacomino, Alice; Ortolan, Erika; Funaro, Ada

    2014-01-01

    CD157/BST-1 behaves both as an ectoenzyme and signaling receptor and is an important regulator of leukocyte trafficking and ovarian cancer progression. However, the molecular interactions underpinning the role of CD157 in these processes remain obscure. The biological functions of CD157 and its partnership with members of the integrin family prompted us to assume the existence of a direct interaction between CD157 and an unknown component of the extracellular matrix. Using solid-phase binding assays and surface plasmon resonance analysis, we demonstrated that CD157 binds fibronectin with high affinity within its heparin-binding domains 1 and 2. Furthermore, we found that CD157 binds to other extracellular matrix proteins containing heparin-binding domains. Finally, we proved that the CD157-fibronectin interaction occurs with living cells, where it elicits CD157-mediated cell responses. Indeed, knockdown of CD157 in Met-5A mesothelial cells changed their morphology and cytoskeleton organization and attenuated the activation of intracellular signaling pathways triggered by fibronectin. This led to impaired cell spreading and adhesion to selected extracellular matrix proteins. Collectively, these findings indicate a central role of CD157 in cell-extracellular matrix interactions and make CD157 an attractive therapeutic target in inflammation and cancer. PMID:24753259

  17. The adhesive protein invasin of Yersinia pseudotuberculosis induces neutrophil extracellular traps via ?1 integrins.

    PubMed

    Gillenius, Erik; Urban, Constantin F

    2015-05-01

    Yersinia pseudotuberculosis adhesive protein invasin is crucial for the bacteria to cross the intestine epithelium by binding to ?1 integrins on M-cells and gaining access to the underlying tissues. After the crossing invasin can bind to ?1 integrins on other cell surfaces, however effector proteins delivered by the type III secretion system Y. pseudotuberculosis efficiently inhibit potential immune responses induced by this interaction. Here, we use mutant Y. pseudotuberculosis strains lacking the type III secretion system and additionally invasin-expressing Escherichia coli to analyze neutrophil responses towards invasin. Our data reveals that invasin induces production of reactive oxygen species and release of chromatin into the extracellular milieu, which we confirmed to be neutrophil extracellular traps by immunofluorescence microscopy. This was mediated through ?1 integrins and was dependent on both the production of reactive oxygen species and signaling through phosphoinositide 3-kinase. We therefore have gained insight into a potential role of integrins in inflammation and infection clearance that has not previously been described, suggesting that targeting of ?1 integrins could be utilized as an adjunctive therapy against yersiniosis. PMID:25576025

  18. 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

  19. 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. Cancer Res; 76(6); 1603-14. ©2016 AACR. PMID:26795349

  20. 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

  1. The Bro1-domain-containing protein Myopic/HDPTP coordinates with Rab4 to regulate cell adhesion and migration.

    PubMed

    Chen, Dong-Yuan; Li, Meng-Yen; Wu, Shih-Yun; Lin, Yu-Ling; Tsai, Sung-Po; Lai, Pei-Lun; Lin, Yu-Tsen; Kuo, Jean-Cheng; Meng, Tzu-Ching; Chen, Guang-Chao

    2012-10-15

    Protein tyrosine phosphatases (PTPs) are a group of tightly regulated enzymes that coordinate with protein tyrosine kinases to control protein phosphorylation during various cellular processes. Using genetic analysis in Drosophila non-transmembrane PTPs, we identified one role that Myopic (Mop), the Drosophila homolog of the human His domain phosphotyrosine phosphatase (HDPTP), plays in cell adhesion. Depletion of Mop results in aberrant integrin distribution and border cell dissociation during Drosophila oogenesis. Interestingly, Mop phosphatase activity is not required for its role in maintaining border cell cluster integrity. We further identified Rab4 GTPase as a Mop interactor in a yeast two-hybrid screen. Expression of the Rab4 dominant-negative mutant leads to border cell dissociation and suppression of Mop-induced wing-blade adhesion defects, suggesting a critical role of Rab4 in Mop-mediated signaling. In mammals, it has been shown that Rab4-dependent recycling of integrins is necessary for cell adhesion and migration. We found that human HDPTP regulates the spatial distribution of Rab4 and integrin trafficking. Depletion of HDPTP resulted in actin reorganization and increased cell motility. Together, our findings suggest an evolutionarily conserved function of HDPTP-Rab4 in the regulation of endocytic trafficking, cell adhesion and migration. PMID:22825871

  2. Multiscale approaches to protein-mediated interactions between membranesrelating microscopic and macroscopic dynamics in radially growing adhesions

    NASA Astrophysics Data System (ADS)

    Bihr, Timo; Seifert, Udo; Smith, Ana-Sun?ana

    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.

  3. 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 by wounds following suture removal. This hypothesis is supported by the data of this study, as well as, the acute tensile strength data of Part I of this study.

  4. Interleukin-1 receptor accessory protein organizes neuronal synaptogenesis as a cell adhesion molecule.

    PubMed

    Yoshida, Tomoyuki; Shiroshima, Tomoko; Lee, Sung-Jin; Yasumura, Misato; Uemura, Takeshi; Chen, Xigui; Iwakura, Yoichiro; Mishina, Masayoshi

    2012-02-22

    Interleukin-1 receptor accessory protein (IL-1RAcP) is the essential component of receptor complexes mediating immune responses to interleukin-1 family cytokines. IL-1RAcP in the brain exists in two isoforms, IL-1RAcP and IL-1RAcPb, differing only in the C-terminal region. Here, we found robust synaptogenic activities of IL-1RAcP in cultured cortical neurons. Knockdown of IL-1RAcP isoforms in cultured cortical neurons suppressed synapse formation as indicated by decreases of active zone protein Bassoon puncta and dendritic protrusions. IL-1RAcP recovered the accumulation of presynaptic Bassoon puncta, while IL-1RAcPb rescued both Bassoon puncta and dendritic protrusions. Consistently, the expression of IL-1RAcP in cortical neurons enhances the accumulation of Bassoon puncta and that of IL-1RAcPb stimulated both Bassoon puncta accumulation and spinogenesis. IL-1RAcP interacted with protein tyrosine phosphatase (PTP) ? through the extracellular domain. Mini-exon peptides in the Ig-like domains of PTP? splice variants were critical for their efficient binding to IL-1RAcP. The synaptogenic activities of IL-1RAcP isoforms were diminished in cortical neurons from PTP? knock-out mice. Correspondingly, PTP? required IL-1RAcPb to induce postsynaptic differentiation. Thus, IL-1RAcPb bidirectionally regulated synapse formation of cortical neurons. Furthermore, the spine densities of cortical and hippocampal pyramidal neurons were reduced in IL-1RAcP knock-out mice lacking both isoforms. These results suggest that IL-1RAcP isoforms function as trans-synaptic cell adhesion molecules in the brain and organize synapse formation. Thus, IL-1RAcP represents an interesting molecular link between immune systems and synapse formation in the brain. PMID:22357843

  5. Vascular adhesion protein-1 and renalase in regard to diabetes in hemodialysis patients

    PubMed Central

    Koc-Zorawska, Ewa; Zbroch, Edyta; Malyszko, Jacek; Mysliwiec, Michal

    2012-01-01

    Introduction Vascular adhesion protein-1 (VAP-1) is a copper-containing semicarbazide-sensitive amine oxidase (SSAO) secreted by vascular smooth muscle cells, adipocytes, and endothelial cells with functional monoamine oxidase activity. Renalase, with possible monoamine oxidase activity, which breaks down catecholamines like SSAO, is also expressed in the endothelium as well as in the kidney. The aim of the study was to assess VAP-1 level and its correlation with renalase level in 60 hemodialyzed (HD) patients. Material and methods Complete blood count, urea, serum lipids, fasting glucose and creatinine were studied by the standard laboratory method in the hospital central laboratory. We assessed VAP-1 and renalase with commercially available assays. Results The mean level of VAP-1 as well as renalase was significantly higher in HD patients when compared to the control group (291.01 94.91 ng/ml vs. 158.34 56.89 ng/ml, p < 0.01; 27.53 9.394.91 g/ml vs. 4.00 1.37 g/ml, p < 0.001, respectively). In hemodialysis patients VAP-1 correlated with presence of diabetes (r = 0.27, p < 0.05), presence of hypertension (r = 0.32, p < 0.05), use of calcium channel blockers (r = 0.30, p < 0.05), use of ?-blockers (r = 0.25, p < 0.05), ejection fraction (r = 0.38, p < 0.01), systolic blood pressure before (r = 0.52, p < 0.001) and after hemodialysis (r = 0.30, p < 0.01), and weight gain (r = 0.41, p < 0.01). Renalase was not significantly different in diabetic and non-diabetic patients or between hypertensive and normotensive patients. In multiple regression analysis VAP-1 was predicted 77% by serum ejection fraction and fibrinogen. Conclusions Vascular adhesion protein-1, elevated in patients on hemodialysis, was predominantly dependent on blood pressure and diabetes, both factors associated with endothelial damage and promoting cardiovascular complications. Renalase appeared to be unrelated to VAP, at least in the HD population. PMID:23319980

  6. Low density lipoprotein receptor-related protein 1 mediated endocytosis of ?1-integrin influences cell adhesion and cell migration.

    PubMed

    Rabiej, Verena K; Pflanzner, Thorsten; Wagner, Timo; Goetze, Kristina; Storck, Steffen E; Eble, Johannes A; Weggen, Sascha; Mueller-Klieser, Wolfgang; Pietrzik, Claus U

    2016-01-01

    The low density lipoprotein receptor-related protein 1 (LRP1) has been shown to interact with ?1-integrin and regulate its surface expression. LRP1 knock-out cells exhibit altered cytoskeleton organization and decreased cell migration. Here we demonstrate coupled endocytosis of LRP1 and ?1-integrin and the involvement of the intracellular NPxY2 motif of LRP1 in this process. Mouse embryonic fibroblasts harboring a knock in replacement of the NPxY2 motif of LRP1 by a multiple alanine cassette (AAxA) showed elevated surface expression of ?1-integrin and decreased ?1-integrin internalization rates. As a consequence, cell spreading was altered and adhesion rates were increased in our cell model. Cells formed more focal adhesion complexes, whereby in vitro cell migration rates were decreased. Similar results could be observed in a corresponding mouse model, the C57Bl6 LRP1 NPxYxxL knock in mice, therefore, the biochemistry of cellular adhesion was altered in primary cortical neurons. In vivo cell migration experiments demonstrated a disturbance of neuroblast cell migration along the rostral migratory stream. In summary, our results indicate that LRP1 interacts with ?1-integrin mediating integrin internalization and thus correlates with downstream signaling of ?1-integrin such as focal adhesion dynamics. Consequently, the disturbance of this interaction resulted in a dysfunction in in vivo and in vitro cell adhesion and cell migration. PMID:26610862

  7. Cytomegalovirus Destruction of Focal Adhesions Revealed in a High-Throughput Western Blot Analysis of Cellular Protein Expression ?

    PubMed Central

    Stanton, R. J.; McSharry, B. P.; Rickards, C. R.; Wang, E. C. Y.; Tomasec, P.; Wilkinson, G. W. G.

    2007-01-01

    Human cytomegalovirus (HCMV) systematically manages the expression of cellular functions, rather than exerting the global shutoff of host cell protein synthesis commonly observed with other herpesviruses during the lytic cycle. While microarray technology has provided remarkable insights into viral control of the cellular transcriptome, HCMV is known to encode multiple mechanisms for posttranscriptional and posttranslation regulation of cellular gene expression. High-throughput Western blotting (BD Biosciences Powerblot technology) with 1,009 characterized antibodies was therefore used to analyze and compare the effects of infection with attenuated high-passage strain AD169 and virulent low-passage strain Toledo at 72 hpi across gels run in triplicate for each sample. Six hundred ninety-four proteins gave a positive signal in the screen, of which 68 from strain AD169 and 71 from strain Toledo were defined as being either positively or negatively regulated by infection with the highest level of confidence (BD parameters). In follow-up analyses, a subset of proteins was selected on the basis of the magnitude of the observed effect or their potential to contribute to defense against immune recognition. In analyses performed at 24, 72, and 144 hpi, connexin 43 was efficiently downregulated during HCMV infection, implying a breakdown of intercellular communication. Mitosis-associated protein Eg-5 was found to be differentially upregulated in the AD169 and Toledo strains of HCMV. Focal adhesions link the actin cytoskeleton to the extracellular matrix and have key roles in initiating signaling pathways and substrate adhesion and regulating cell migration. HCMV suppressed expression of the focal-adhesion-associated proteins Hic-5, paxillin, and ?-actinin. Focal adhesions were clearly disrupted in HCMV-infected fibroblasts, with their associated intracellular and extracellular proteins being dispersed. Powerblot shows potential for rapid screening of the cellular proteome during HCMV infection. PMID:17522202

  8. Exchange of adsorbed serum proteins during adhesion of Staphylococcus aureus to an abiotic surface and Candida albicans hyphae--an AFM study.

    PubMed

    Ovchinnikova, Ekaterina S; van der Mei, Henny C; Krom, Bastiaan P; Busscher, Henk J

    2013-10-01

    Staphylococcus aureus and Candida albicans are the second and third most commonly isolated microorganisms in hospital-related-infections, that are often multi-species in nature causing high morbidity and mortality. Here, adhesion forces between a S. aureus strain and abiotic (tissue-culture-polystyrene, TCPS) or partly biotic (TCPS with adhering hyphae of C. albicans) surfaces were investigated in presence of fetal-bovine-serum or individual serum proteins and related with staphylococcal adhesion. Atomic-force-microscopy was used to measure adhesion forces between S. aureus and the abiotic and biotic surfaces. Adsorption of individual serum proteins like albumin and apo-transferrin to abiotic TCPS surfaces during 60min, impeded development of strong adhesion forces as compared to fibronectin, while 60min adsorption of proteins from fetal-bovine-serum yielded a decrease in adhesion force from -5.7nN in phosphate-buffered-saline to -0.6nN. Adsorption of albumin and apo-transferrin also decreased staphylococcal adhesion forces to hyphae as compared with fibronectin. During 60min exposure to fetal-bovine-serum however, initial (5min protein adsorption) staphylococcal adhesion forces were low (-1.6nN), but strong adhesion forces of around -5.5nN were restored within 60min. This suggests for the first time that in whole fetal-bovine-serum exchange of non-adhesive proteins by fibronectin occurs on biotic C. albicans hyphal surfaces. No evidence was found for such protein exchange on abiotic TCPS surfaces. Staphylococcal adhesion of abiotic and biotic surfaces varied in line with the adhesion forces and was low on TCPS in presence of fetal-bovine-serum. On partly biotic TCPS, staphylococci aggregated in presence of fetal-bovine-serum around adhering C. albicans hyphae. PMID:23707849

  9. Non-adhesive behavior of new nanostructured PNIPAM surfaces towards specific neurodegenerative proteins: application to storage and titration of Tau proteins.

    PubMed

    Vrlinic, Tjasa; Debarnot, Dominique; Legeay, Gilbert; Coudreuse, Arnaud; El Moualij, Benaissa; Zorzi, Willy; Perret-Liaudet, Armand; Quadrio, Isabelle; Mozetic, Miran; Poncin-Epaillard, Fabienne

    2012-10-01

    New nonfouling tubes are developed and their influence on the adhesion of neuroproteins is studied. The biomarkers are considered as single components (recombinant prion and Tau proteins) or in a solution of native and pathological forms. The samples are stored for 24 h at 4 °C in virgin and treated tubes layered with two different nanostructured coatings based on poly(N-isopropylacrylamide) with either a positive or a neutral charge, and the protein adhesion is monitored. The recombinant protein with a high pI is repelled from the nanostructured surface that has a negative ζ potential, whereas the recombinant protein with the lower pI is attracted. Furthermore, in the case of complex solutions, neutral nanostructured surfaces are able to retain all amyloid biomarkers. PMID:22927330

  10. Non-adhesive behavior of new nanostructured PNIPAM surfaces towards specific neurodegenerative proteins: application to storage and titration of Tau proteins.

    TOXLINE Toxicology Bibliographic Information

    Vrlinic T; Debarnot D; Legeay G; Coudreuse A; El Moualij B; Zorzi W; Perret-Liaudet A; Quadrio I; Mozetic M; Poncin-Epaillard F

    2012-10-01

    New nonfouling tubes are developed and their influence on the adhesion of neuroproteins is studied. The biomarkers are considered as single components (recombinant prion and Tau proteins) or in a solution of native and pathological forms. The samples are stored for 24 h at 4 °C in virgin and treated tubes layered with two different nanostructured coatings based on poly(N-isopropylacrylamide) with either a positive or a neutral charge, and the protein adhesion is monitored. The recombinant protein with a high pI is repelled from the nanostructured surface that has a negative ζ potential, whereas the recombinant protein with the lower pI is attracted. Furthermore, in the case of complex solutions, neutral nanostructured surfaces are able to retain all amyloid biomarkers.

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

    PubMed

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

    2015-12-01

    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. The species-specific egg receptor for sea urchin sperm adhesion is EBR1,a novel ADAMTS protein.

    PubMed

    Kamei, Noriko; Glabe, Charles G

    2003-10-15

    Species-specific adhesion of sperm to the egg during sea urchin fertilization involves the interaction of the sperm adhesive protein,bindin, and a complementary receptor on the egg surface,and serves to restrict the gene pool to individuals of the same species. We used PCR representation difference analysis to clone the species-specific egg receptor for bindin, EBR1, from Strongylocentrotus franciscanus (Sf) and S. purpuratus (Sp). Sf-EBR1 contains a novel ADAMTS-like N-terminal domain followed by approximately 19 tandem EBR repeats consisting of alternating CUB and thrombospondin type 1 (TSP-1) domains where the last 10 EBR repeats are species-specific and highly conserved. Recombinant protein corresponding to the species-specific EBR repeat displays species-specific sperm adhesion and bindin-binding activity. The Sp-EBR1 ortholog has the same ADAMTS (a disintegrin and metalloprotease with thrombospondin type-1 modules) core region followed by eight and one-half tandem egg bindin receptor (EBR) repeats that share 88% identity with the Sf-EBR1 repeats,but has an entirely different species-specific domain consisting of hyalin-like (HYR) repeats. Thus,the species-specific domains of egg bindin receptor 1 (EBR1) from both species function as the egg surface receptor to mediate species-specific sperm adhesion. PMID:14561772

  13. Mammalian Adenylyl Cyclase-associated Protein 1 (CAP1) Regulates Cofilin Function, the Actin Cytoskeleton, and Cell Adhesion*

    PubMed Central

    Zhang, Haitao; Ghai, Pooja; Wu, Huhehasi; Wang, Changhui; Field, Jeffrey; Zhou, Guo-Lei

    2013-01-01

    CAP (adenylyl cyclase-associated protein) was first identified in yeast as a protein that regulates both the actin cytoskeleton and the Ras/cAMP pathway. Although the role in Ras signaling does not extend beyond yeast, evidence supports that CAP regulates the actin cytoskeleton in all eukaryotes including mammals. In vitro actin polymerization assays show that both mammalian and yeast CAP homologues facilitate cofilin-driven actin filament turnover. We generated HeLa cells with stable CAP1 knockdown using RNA interference. Depletion of CAP1 led to larger cell size and remarkably developed lamellipodia as well as accumulation of filamentous actin (F-actin). Moreover, we found that CAP1 depletion also led to changes in cofilin phosphorylation and localization as well as activation of focal adhesion kinase (FAK) and enhanced cell spreading. CAP1 forms complexes with the adhesion molecules FAK and Talin, which likely underlie the cell adhesion phenotypes through inside-out activation of integrin signaling. CAP1-depleted HeLa cells also had substantially elevated cell motility as well as invasion through Matrigel. In summary, in addition to generating in vitro and in vivo evidence further establishing the role of mammalian CAP1 in actin dynamics, we identified a novel cellular function for CAP1 in regulating cell adhesion. PMID:23737525

  14. Structural organization and function of mouse photoreceptor ribbon synapses involve the immunoglobulin protein synaptic cell adhesion molecule 1.

    PubMed

    Ribic, Adema; Liu, Xinran; Crair, Michael C; Biederer, Thomas

    2014-03-01

    Adhesive interactions in the retina instruct the developmental specification of inner retinal layers. However, potential roles of adhesion in the development and function of photoreceptor synapses remain incompletely understood. This contrasts with our understanding of synapse development in the CNS, which can be guided by select adhesion molecules such as the Synaptic Cell Adhesion Molecule 1 (SynCAM 1/CADM1/nectin-like 2 protein). This immunoglobulin superfamily protein modulates the development and plasticity of classical excitatory synapses. We show here by immunoelectron microscopy and immunoblotting that SynCAM 1 is expressed on mouse rod photoreceptors and their terminals in the outer nuclear and plexiform layers in a developmentally regulated manner. Expression of SynCAM 1 on rods is low in early postnatal stages (P3-P7) but increases after eye opening (P14). In support of functional roles in the photoreceptors, electroretinogram recordings demonstrate impaired responses to light stimulation in SynCAM 1 knockout (KO) mice. In addition, the structural integrity of synapses in the OPL requires SynCAM 1. Quantitative ultrastructural analysis of SynCAM 1 KO retina measured fewer fully assembled, triadic rod ribbon synapses. Furthermore, rod synapse ribbons are shortened in KO mice, and protein levels of Ribeye, a major structural component of ribbons, are reduced in SynCAM 1 KO retina. Together, our results implicate SynCAM 1 in the synaptic organization of the rod visual pathway and provide evidence for novel roles of synaptic adhesion in the structural and functional integrity of ribbon synapses. PMID:23982969

  15. C1q/TNF-related protein-9 inhibits cytokine-induced vascular inflammation and leukocyte adhesiveness via AMP-activated protein kinase activation in endothelial cells.

    PubMed

    Jung, Chang Hee; Lee, Min Jung; Kang, Yu Mi; Lee, Yoo La; Seol, So Mi; Yoon, Hae Kyeong; Kang, Sang-Wook; Lee, Woo Je; Park, Joong-Yeol

    2016-01-01

    Although recent studies have reported cardioprotective effects of C1q/TNF-related protein 9 (CTRP9), the closet adiponectin paralog, its role on cytokine-induced endothelial inflammation is unknown. We investigated whether CTRP9 prevented inflammatory cytokine-induced nuclear factor-kappa B (NF-?B) activation and inhibited the expression of adhesion molecules and a chemokine in the vascular endothelial cell. We used human aortic endothelial cells (HAECs) to examine the effects of CTRP9 on NF-?B activation and the expression of NF-?B-mediated genes, including intercellular adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1), and monocyte chemoattractant protein-1 (MCP-1). Tumor necrosis factor alpha (TNF?) was used as a representative proinflammatory cytokine. In an adhesion assay using THP-1 cells, CTRP9 reduced TNF?-induced adhesion of monocytes to HAECs. Treatment with CTRP9 significantly decreased TNF?-induced activation of NF-?B, as well as the expression of ICAM-1, VCAM-1, and MCP-1. In addition, treatment with CTRP9 significantly increased the phosphorylation of AMP-activated protein kinase (AMPK) and acetyl-CoA carboxylase (ACC), the downstream target of AMPK. The inhibitory effect of CTRP9 on the expression of ICAM-1, VCAM-1, and MCP-1 and monocyte adhesion to HAECs was abolished after transfection with an AMPK?1-specific siRNA. Our study is the first to demonstrate that CTRP9 attenuates cytokine-induced vascular inflammation in endothelial cells mediated by AMPK activation. PMID:26523509

  16. 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

  17. 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 ?-catenin as a transcriptional regulator affected in PrP-deficient cells. Indeed, pharmacological blockade or siRNA-based knockdown of ?-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

  18. Conformational control of inorganic adhesion in a designer protein engineered for cuprous oxide binding.

    PubMed

    Choe, Woo-Seok; Sastry, M S R; Thai, Corrine K; Dai, Haixia; Schwartz, Daniel T; Baneyx, Franois

    2007-11-01

    Combinatorial selection of peptides that bind technological materials has emerged as a valuable tool for room-temperature nucleation and assembly of complex nanostructured materials. At present, the parameters that control peptide-solid binding are poorly understood, but such knowledge is needed to build the next generation of hybrid materials. Here, we use a derivative of the DNA binding protein TraI engineered with a disulfide-bonded cuprous oxide binding sequence called CN225 to probe the influence of sequence composition and conformation on Cu2O binding affinity. We previously reported a statistically significant enrichment in paired arginines (RR) among a family of cuprous oxide binding peptides and hypothesized that this is a key motif for binding. However, systematic alanine (A) substitutions in the CN225 RR motif (creating RA, AR, and AA pairs) do not support the hypothesis that RR is critical for Cu2O binding by CN225. Instead, we find that the presentation of the peptide in a disulfide-constrained loop (i.e., the conformation present during combinatorial selection) is crucial for binding to the metal oxide. Our results suggest that caution should be exerted when extrapolating from statistical data and that, in some cases, conformation is more important than composition in determining peptide-inorganic adhesion. PMID:17918983

  19. 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...

  20. 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

  1. Enhanced Adhesion of Campylobacter jejuni to Abiotic Surfaces Is Mediated by Membrane Proteins in Oxygen-Enriched Conditions

    PubMed Central

    Sulaeman, Sheiam; Hernould, Mathieu; Schaumann, Annick; Coquet, Laurent; Bolla, Jean-Michel; D, Emmanuelle; Tresse, Odile

    2012-01-01

    Campylobacter jejuni is responsible for the major foodborne bacterial enteritis in humans. In contradiction with its fastidious growth requirements, this microaerobic pathogen can survive in aerobic food environments, suggesting that it must employ a variety of protection mechanisms to resist oxidative stress. For the first time, C. jejuni 81176 inner and outer membrane subproteomes were analyzed separately using two-dimensional protein electrophoresis (2-DE) of oxygen-acclimated cells and microaerobically grown cells. LC-MS/MS analyses successfully identified 42 and 25 spots which exhibited a significantly altered abundance in the IMP-enriched fraction and in the OMP-enriched fraction, respectively, in response to oxidative conditions. These spots corresponded to 38 membrane proteins that could be grouped into different functional classes: (i) transporters, (ii) chaperones, (iii) fatty acid metabolism, (iv) adhesion/virulence and (v) other metabolisms. Some of these proteins were up-regulated at the transcriptional level in oxygen-acclimated cells as confirmed by qRT-PCR. Downstream analyses revealed that adhesion of C. jejuni to inert surfaces and swarming motility were enhanced in oxygen-acclimated cells or paraquat-stressed cells, which could be explained by the higher abundance of membrane proteins involved in adhesion and biofilm formation. The virulence factor CadF, over-expressed in the outer membrane of oxygen-acclimated cells, contributes to the complex process of C. jejuni adhesion to inert surfaces as revealed by a reduction in the capability of C. jejuni 81176 ?CadF cells compared to the isogenic strain. Taken together, these data demonstrate that oxygen-enriched conditions promote the over-expression of membrane proteins involved in both the biofilm initiation and virulence of C. jejuni. PMID:23029510

  2. Effect of avidin-like proteins and biotin modification on mesenchymal stem cell adhesion

    PubMed Central

    Schmidt, Ray C.; Healy, Kevin E.

    2013-01-01

    The avidin-biotin system is a highly specific reaction that has been used in a wide range of biomedical applications, including surface modification and cell patterning. We systematically examined a number of avidin derivatives as the basis for a simple and cost effective tissue culture polystyrene substrate surface modification for human stem cell culture. Non-specific adhesion between human mesenchymal stem cells and various avidin derivatives, media conditions, and subsequent biotinylation reactions was quantified. We observed significant non-specific cell adhesion to avidin and strepthavidin, indicating that previous observations using this system may be artifactual. Seeding of cells in serum free media, blocking with bovine-serum albumin, and the use of the avidin derivative Neutravidin were all necessary for elimination of background adhesion. Neutravidin conjugated with biotinylated bsp-RGD(15) peptide provided the most robust cell adhesion, as well as the greatest increase in cell adhesion over background levels. PMID:23452388

  3. Epithelial cell adhesion molecule (EpCAM) complex proteins promote transcription factor-mediated pluripotency reprogramming.

    PubMed

    Huang, Hsiang-Po; Chen, Pin-Hsun; Yu, Chun-Ying; Chuang, Ching-Yu; Stone, Lee; Hsiao, Wen-Chu; Li, Chung-Leung; Tsai, Shih-Chih; Chen, Kai-Yun; Chen, Hsin-Fu; Ho, Hong-Nerng; Kuo, Hung-Chih

    2011-09-23

    Epithelial cell adhesion molecule (EpCAM) is a transmembrane glycoprotein that is highly expressed in embryonic stem cells (ESCs) and its role in maintenance of pluripotency has been suggested previously. In epithelial cancer cells, activation of the EpCAM surface-to-nucleus signaling transduction pathway involves a number of membrane proteins. However, their role in somatic cell reprogramming is still unknown. Here we demonstrate that EpCAM and its associated protein, Cldn7, play a critical role in reprogramming. Quantitative RT-PCR analysis of Oct4, Sox2, Klf4, and c-Myc (OSKM) infected mouse embryonic fibroblasts (MEFs) indicated that EpCAM and Cldn7 were up-regulated during reprogramming. Analysis of numbers of alkaline phosphatase- and Nanog-positive clones, and the expression level of pluripotency-related genes demonstrated that inhibition of either EpCAM or Cldn7 expression resulted in impairment in reprogramming efficiency, whereas overexpression of EpCAM, EpCAM plus Cldn7, or EpCAM intercellular domain (EpICD) significantly enhanced reprogramming efficiency in MEFs. Furthermore, overexpression of EpCAM or EpICD significantly repressed the expression of p53 and p21 in the reprogramming MEFs, and both EpCAM and EpICD activated the promoter activity of Oct4. These observations suggest that EpCAM signaling may enhance reprogramming through up-regulation of Oct4 and possible suppression of the p53-p21 pathway. In vitro and in vivo characterization indicated that the EpCAM-reprogrammed iPSCs exhibited similar molecular and functional features to the mouse ESCs. In summary, our studies provide additional insight into the molecular mechanisms of reprogramming and suggest a more effective means of induced pluripotent stem cell generation. PMID:21799003

  4. Anti-neutrophil cytoplasmic antibody-enriched IgG induces adhesion of human T lymphocytes to extracellular matrix proteins.

    PubMed

    Tomer, Y; Lider, O; Gilburd, B; Hershkoviz, R; Meroni, P L; Wiik, A; Shoenfeld, Y

    1997-06-01

    Recent studies have shown that anti-neutrophil cytoplasmic antibodies (ANCA) can activate neutrophils to adhere to endothelium, degranulate, and cause endothelial cell injury. These data have lead to the hypothesis that the T cell inflammatory response causing the vasculitis in Wegener's granulomatosis (WG) is secondary to stimulation of neutrophils by ANCA. So far there is no evidence for a direct effect of ANCA on lymphocytes. The present study was designed to examine whether lymphocytes can be directly stimulated by ANCA to adhere to endothelial extracellular matrix (ECM) proteins. Human and mouse ANCA-enriched IgG were tested for their ability to increase adhesion of human T lymphocytes to fibronectin, laminin, and intact ECM. Incubation of human T lymphocytes with human ANCA-enriched IgG increased adhesion of the lymphocytes in a dose-dependent manner to fibronectin, laminin, and intact ECM (the percentage adhesion to intact ECM was 55.7 +/- 3.1 and 45.0 +/- 1.0% for lymphocytes incubated with human IgG containing ANCA or control human IgG, respectively; P = 0.0045). The same induction of adhesion to fibronectin, laminin, and intact ECM was observed when the cells were incubated with the F(ab)2 fragment of ANCA-enriched IgG. Similarly, ANCA-enriched IgG produced in mice increased the adhesion of lymphocytes to fibronectin (the percentage adhesion to fibronectin was 29.7 +/- 4.3 and 16.6 +/- 1.9% for lymphocytes incubated with mouse IgG-ANCA or control mouse IgG, respectively; P = 0.0008). These results may suggest that ANCA can directly stimulate lymphocytes to adhere to endothelial ECM and to induce the vasculitic lesions of WG. It remains to be shown by which mechanisms ANCA stimulate lymphocytes to adhere to ECM. PMID:9175913

  5. Protein 4.1G Regulates Cell Adhesion, Spreading, and Migration of Mouse Embryonic Fibroblasts through the ?1 Integrin Pathway.

    PubMed

    Chen, Lixiang; Wang, Ting; Wang, Yaomei; Zhang, Jingxin; Qi, Yuanming; Weng, Haibo; Kang, Qiaozhen; Guo, Xinhua; Baines, Anthony J; Mohandas, Narla; An, Xiuli

    2016-01-29

    Protein 4.1G is a membrane skeletal protein that can serve as an adapter between transmembrane proteins and the underlying membrane skeleton. The function of 4.1G remains largely unexplored. Here, using 4.1G knockout mouse embryonic fibroblasts (MEFs) as a model system, we explored the function of 4.1G in motile cells. We show that the adhesion, spreading, and migration of 4.1G(-/-) MEF cells are impaired significantly. We further show that, although the total cellular expression of ?1 integrin is unchanged, the surface expression of ?1 integrin and its active form are decreased significantly in 4.1G(-/-) MEF cells. Moreover, the phosphorylation of focal adhesion kinase, a downstream component of the integrin-mediated signal transduction pathway, is suppressed in 4.1G(-/-) MEF cells. Co-immunoprecipitation experiments and in vitro binding assays showed that 4.1G binds directly to ?1 integrin via its membrane-binding domain. These findings identified a novel role of 4.1G in cell adhesion, spreading, and migration in MEF cells by modulating the surface expression of ?1 integrin and subsequent downstream signal transduction. PMID:26644476

  6. 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-sensitive fibrils 91 and 72 nm in length, respectively. A third class of only very sparsely distributed short fibrils (63 nm) was observed on mutant HB-V51, which lacks both wall-associated AgB and AgC antigens. The identity of these fibrils and whether they are present on the wild type are not clear. The function of long, protease-resistant fibrils of 178 nm, which are also present on the wild-type strain, remains unknown. Images PMID:2419308

  7. Regulation of cadherin-mediated adhesion by the small GTP-binding protein Rho in small cell lung carcinoma cells.

    PubMed

    Tokman, M G; Porter, R A; Williams, C L

    1997-05-01

    Metastasis is one of the most important factors responsible for the pathogenesis of small cell lung carcinoma (SCLC). SCLC cells express cadherins, which are homophilic cell-cell adhesion molecules that play an important role in the regulation of metastasis. We present the first evidence that altering the activity of the small GTP-binding protein Rho induces cadherin-mediated adhesion. ADP-ribosylation of Rho upon incubation or electroporation with recombinant C3 exoenzyme induces rapid aggregation and compaction of SCLC cells. Aggregation and compaction induced by C3 exoenzyme are diminished by removal of extracellular Ca2+ and by the HECD blocking antibody to E-cadherin but not by antibodies to other adhesion molecules. Altering the activity of Rho by ADP-ribosylation does not alter surface expression of E-cadherin, but it alters G actin content, as indicated by the binding of DNase I. Treatment with cytochalasin D also alters G actin content and increases aggregation and compaction of SCLC cells. These findings implicate Rho in the regulation of cadherin-mediated adhesion and identify Rho as a potential therapeutic target for the control of SCLC metastasis. PMID:9135023

  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. Dok-2 Adaptor Protein Regulates the Shear-dependent Adhesive Function of Platelet Integrin ?IIb?3 in Mice*

    PubMed Central

    Hughan, Sascha C.; Spring, Christopher M.; Schoenwaelder, Simone M.; Sturgeon, Sharelle; Alwis, Imala; Yuan, Yuping; McFadyen, James D.; Westein, Erik; Goddard, Duncan; Ono, Akiko; Yamanashi, Yuji; Nesbitt, Warwick S.; Jackson, Shaun P.

    2014-01-01

    The Dok proteins are a family of adaptor molecules that have a well defined role in regulating cellular migration, immune responses, and tumor progression. Previous studies have demonstrated that Doks-1 to 3 are expressed in platelets and that Dok-2 is tyrosine-phosphorylated downstream of integrin ?IIb?3, raising the possibility that it participates in integrin ?IIb?3 outside-in signaling. We demonstrate that Dok-2 in platelets is primarily phosphorylated by Lyn kinase. Moreover, deficiency of Dok-2 leads to dysregulated integrin ?IIb?3-dependent cytosolic calcium flux and phosphatidylinositol(3,4)P2 accumulation. Although agonist-induced integrin ?IIb?3 affinity regulation was unaltered in Dok-2?/? platelets, Dok-2 deficiency was associated with a shear-dependent increase in integrin ?IIb?3 adhesive function, resulting in enhanced platelet-fibrinogen and platelet-platelet adhesive interactions under flow. This increase in adhesion was restricted to discoid platelets and involved the shear-dependent regulation of membrane tethers. Dok-2 deficiency was associated with an increased rate of platelet aggregate formation on thrombogenic surfaces, leading to accelerated thrombus growth in vivo. Overall, this study defines an important role for Dok-2 in regulating biomechanical adhesive function of discoid platelets. Moreover, they define a previously unrecognized prothrombotic mechanism that is not detected by conventional platelet function assays. PMID:24385425

  10. Reaction of Vascular Adhesion Protein-1 (VAP-1) with Primary Amines

    PubMed Central

    Heuts, Dominic P. H. M.; Gummadova, Jennet O.; Pang, Jiayun; Rigby, Stephen E. J.; Scrutton, Nigel S.

    2011-01-01

    Human vascular adhesion protein-1 (VAP-1) is an endothelial copper-dependent amine oxidase involved in the recruitment and extravasation of leukocytes at sites of inflammation. VAP-1 is an important therapeutic target for several pathological conditions. We expressed soluble VAP-1 in HEK293 EBNA1 cells at levels suitable for detailed mechanistic studies with model substrates. Using the model substrate benzylamine, we analyzed the steady-state kinetic parameters of VAP-1 as a function of solution pH. We found two macroscopic pKa values that defined a bell-shaped plot of turnover number kcat,app as a function of pH, representing ionizable groups in the enzyme-substrate complex. The dependence of (kcat/Km)app on pH revealed a single pKa value (?9) that we assigned to ionization of the amine group in free benzylamine substrate. A kinetic isotope effect (KIE) of 6 to 7.6 on (kcat/Km)app over the pH range of 6 to 10 was observed with d2-benzylamine. Over the same pH range, the KIE on kcat was found to be close to unity. The unusual KIE values on (kcat/Km)app were rationalized using a mechanistic scheme that includes the possibility of multiple isotopically sensitive steps. We also report the analysis of quantitative structure-activity relationships (QSAR) using para-substituted protiated and deuterated phenylethylamines. With phenylethylamines we observed a large KIE on kcat,app (8.01 0.28 with phenylethylamine), indicating that CH bond breakage is limiting for 2,4,5-trihydroxyphenylalanine quinone reduction. Poor correlations were observed between steady-state rate constants and QSAR parameters. We show the importance of combining KIE, QSAR, and structural studies to gain insight into the complexity of the VAP-1 steady-state mechanism. PMID:21737458

  11. Localization of Vascular Adhesion Protein-1 (VAP-1) in the Human Eye

    PubMed Central

    Almulki, Lama; Noda, Kousuke; Nakao, Shintaro; Hisatomi, Toshio; Thomas, Kennard L.; Hafezi-Moghadam, Ali

    2009-01-01

    Recently we showed a critical role for Vascular Adhesion Protein-1 (VAP-1) in rodents during acute ocular inflammation, angiogenesis, and diabetic retinal leukostasis. However, the expression of VAP-1 in the human eye is unknown. VAP-1 localization was investigated by immunohistochemistry. Five ?m thick sections were generated from human ocular tissues embedded in paraffin. Sections were incubated overnight with primary mAbs against VAP-1 (5?g/ml), smooth muscle actin (1?g/ml), CD31 or isotype-matched IgG at 4C. Subsequently, a secondary mAb was used for 30min at room temperature, followed by Dako Envision + HRP (AEC) System for signal detection. The stained sections were examined using light microscopy and the signal intensity was quantified by two masked evaluators and graded into 4 discrete categories. In all examined ocular tissues, VAP-1 staining was confined to the vasculature. VAP-1 labeling showed the highest intensity in both arteries and veins of neuronal tissues; retina, and optic nerve, and the lowest intensity in the iris vasculature (p<0.05). Scleral and choroidal vessels showed moderate staining for VAP-1. VAP-1 intensity was significantly higher in the arteries compared to veins (p<0.05). Furthermore, VAP-1 staining in arteries co-localized with both CD31 and smooth muscle actin (sm-actin) staining, suggesting expression of VAP-1 in endothelial cells, smooth muscle cells or potentially pericytes. In conclusion, Immunohistochemistry reveals constitutive expression of VAP-1 in human ocular tissues. VAP-1 expression is exclusive to the vasculature with arteries showing significantly higher expression than veins. Furthermore, VAP-1 expression in the ocular vasculature is heterogeneous, with the vessels of the optic nerve and the retina showing highest expressions. These results characterize VAP-1 expression in human ocular tissues. PMID:19761765

  12. Calcium Dobesilate Inhibits the Alterations in Tight Junction Proteins and Leukocyte Adhesion to Retinal Endothelial Cells Induced by Diabetes

    PubMed Central

    Leal, Ermelindo C.; Martins, Joo; Voabil, Paula; Liberal, Joana; Chiavaroli, Carlo; Bauer, Jacques; Cunha-Vaz, Jos; Ambrsio, Antnio F.

    2010-01-01

    OBJECTIVE Calcium dobesilate (CaD) has been used in the treatment of diabetic retinopathy in the last decades, but its mechanisms of action are not elucidated. CaD is able to correct the excessive vascular permeability in the retina of diabetic patients and in experimental diabetes. We investigated the molecular and cellular mechanisms underlying the protective effects of CaD against the increase in bloodretinal barrier (BRB) permeability induced by diabetes. RESEARCH DESIGN AND METHODS Wistar rats were divided into three groups: controls, streptozotocin-induced diabetic rats, and diabetic rats treated with CaD. The BRB breakdown was evaluated using Evans blue. The content or distribution of tight junction proteins (occludin, claudin-5, and zonula occluden-1 [ZO-1]), intercellular adhesion molecule-1 (ICAM-1), and p38 mitogen-activated protein kinase (p38 MAPK) was evaluated by Western blotting and immunohistochemistry. Leukocyte adhesion was evaluated in retinal vessels and in vitro. Oxidative stress was evaluated by the detection of oxidized carbonyls and tyrosine nitration. NF-?B activation was measured by enzyme-linked immunosorbent assay. RESULTS Diabetes increased the BRB permeability and retinal thickness. Diabetes also decreased occludin and claudin-5 levels and altered the distribution of ZO-1 and occludin in retinal vessels. These changes were inhibited by CaD treatment. CaD also inhibited the increase in leukocyte adhesion to retinal vessels or endothelial cells and in ICAM-1 levels, induced by diabetes or elevated glucose. Moreover, CaD decreased oxidative stress and p38 MAPK and NF-?B activation caused by diabetes. CONCLUSIONS CaD prevents the BRB breakdown induced by diabetes, by restoring tight junction protein levels and organization and decreasing leukocyte adhesion to retinal vessels. The protective effects of CaD are likely to involve the inhibition of p38 MAPK and NF-?B activation, possibly through the inhibition of oxidative/nitrosative stress. PMID:20627932

  13. Hydroxyalkenals and Oxidized Phospholipids Modulation of Endothelial Cytoskeleton, Focal Adhesion and Adherens Junction Proteins in Regulating Endothelial Barrier Function

    PubMed Central

    Usatyuk, Peter V.; Natarajan, Viswanathan

    2011-01-01

    Lipid peroxidation of polyunsaturated fatty acids generates bioactive aldehydes, which exhibit pro- and anti-inflammatory effects in cells and tissues. Accumulating evidence indicates that 4-hydroxynonenal (4-HNE), a major aldehyde derived from lipid peroxidation of n-6 polyunsaturated fatty acids trigger signals that modulates focal adhesion and adherens junction proteins thereby inducing endothelial barrier dysfunction. Similarly, oxidized phospholipids (Ox-PLs) generated by lipid peroxidation of phospholipids with polyunsaturated fatty acids have been implicated in atherogenesis, inflammation and gene expression. Interestingly, physiological concentration of Ox-PLs are anti-inflammatory and protect against endotoxin- and ventilator-associated acute lung injury. Thus, excess generation of bioactive hydroxyalkenals and Ox-PLs during oxidative stress contributes to pathophysiology of various diseases by modulating signaling pathways that regulate pro- and anti-inflammatory responses and barrier regulation. This review summarizes the role of 4-HNE and Ox-PLs affecting cell signaling pathways and endothelial barrier dysfunction through modulation of the activities of proteins/enzymes by Michael adducts formation, enhancing the level of protein tyrosine phosphorylation of the target proteins, and by reorganization of cytoskeletal, focal adhesion, and adherens junction proteins. A better understanding of molecular mechanisms of hydroxyalkenals- and Ox-PLs-mediated pro-and anti-inflammatory responses and barrier function may lead to development of novel therapies to ameliorate oxidative stress related cardio-pulmonary disorders. PMID:21570987

  14. Amalgam, an axon guidance Drosophila adhesion protein belonging to the immunoglobulin superfamily: over-expression, purification and biophysical characterization.

    PubMed

    Zeev-Ben-Mordehai, Tzviya; Paz, Aviv; Peleg, Yoav; Toker, Lilly; Wolf, Sharon G; Rydberg, Edwin H; Sussman, Joel L; Silman, Israel

    2009-02-01

    Amalgam, a multi-domain member of the immunoglobulin superfamily, possesses homophilic and heterophilic cell adhesion properties. It is required for axon guidance during Drosophila development in which it interacts with the extracellular domain of the transmembrane protein, neurotactin, to promote adhesion. Amalgam was heterologously expressed in Pichia pastoris, and the secreted protein product, bearing an NH(2)-terminal His(6)Tag, was purified from the growth medium by metal affinity chromatography. Size exclusion chromatography separated the purified protein into two fractions: a major, multimeric fraction and a minor, dimeric one. Two protocols to reduce the percentage of multimers were tested. In one, protein induction was performed in the presence of the zwitterionic detergent CHAPS, yielding primarily the dimeric form of amalgam. In a second protocol, agitation was gradually reduced during the course of the induction and antifoam was added daily to reduce the air/liquid interfacial foam area. This latter protocol lowered the percentage of multimer 2-fold, compared to constant agitation. Circular dichroism measurements showed that the dimeric fraction had a high beta-sheet content, as expected for a protein with an immunoglobulin fold. Dynamic light scattering and sedimentation velocity measurements showed that the multimeric fraction displays a monodisperse distribution, with R(H)=16 nm. When co-expressed together with amalgam the ectodomain of neurotactin copurified with it. Furthermore, both purified fractions of amalgam were shown to interact with Torpedo californica acetylcholinesterase, a structural homolog of neurotactin. PMID:18938249

  15. 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

  16. 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 practical applications and can be successfully applied to prepare other Dopa/Dopaquinone-based biomaterials. PMID:23095646

  17. Flagellar adhesion-dependent regulation of Chlamydomonas adenylyl cyclase in vitro: a possible role for protein kinases in sexual signaling

    PubMed Central

    1994-01-01

    Interactions between adhesion molecules, agglutinins, on the surfaces of the flagella of mt+ and mt- gametes in Chlamydomonas rapidly generate a sexual signal, mediated by cAMP, that prepares the cells for fusion to form a zygote. The mechanism that couples agglutinin interactions to increased cellular levels of cAMP is unknown. In previous studies on the adenylyl cyclase in flagella of a single mating type (i.e., non-adhering flagella) we presented evidence that the gametic form of the enzyme, but not the vegetative form, was regulated by phosphorylation and dephosphorylation (Zhang, Y., E. M. Ross, and W. J. Snell. 1991. J. Biol. Chem. 266:22954-22959; Zhang, Y., and W. J. Snell. 1993. J. Biol. Chem. 268:1786-1791). In the present report we describe studies on regulation of flagellar adenylyl cyclase during adhesion in a cell-free system. The results show that the activity of gametic flagellar adenylyl cyclase is regulated by adhesion in vitro between flagella isolated from mt+ and mt- gametes. After mixing mt+ and mt- flagella together for 15 s in vitro, adenylyl cyclase activity was increased two- to threefold compared to that of the non-mixed (non- adhering), control flagella. This indicates that the regulation of gametic flagellar adenylyl cyclase during the early steps of fertilization is not mediated by signals from the cell body, but is a direct and primary response to interactions between mt+ and mt- agglutinins. By use of this in vitro assay, we discovered that 50 nM staurosporine (a protein kinase inhibitor) blocked adhesion-induced activation of adenylyl cyclase in vitro, while it had no effect on adenylyl cyclase activity of non-adhering gametic flagella. This same low concentration of staurosporine also inhibited adhesion-induced increases in vivo in cellular cAMP and blocked subsequent cellular responses to adhesion. Taken together, our results indicate that flagellar adenylyl cyclase in Chlamydomonas gametes is coupled to interactions between mt+ and mt- agglutinins by a staurosporine- sensitive activity, probably a protein kinase. PMID:8175884

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

    PubMed

    Becker, Pierre T; Lambert, Aurlie; Lejeune, Annabelle; Lanterbecq, Dborah; 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

  19. 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

  20. Influence of Temperature and Growth Phase on Expression of a 104-Kilodalton Listeria Adhesion Protein in Listeria monocytogenes

    PubMed Central

    Santiago, Nivia I.; Zipf, Allan; Bhunia, Arun K.

    1999-01-01

    Interaction of Listeria monocytogenes with mammalian intestinal cells is believed to be an important first step in Listeria pathogenesis. Transposon (Tn916) mutagenesis provided strong evidence that a 104-kDa surface protein, designated the Listeria adhesion protein (LAP), was involved in adherence of L. monocytogenes to a human enterocyte-like Caco-2 cell line (V. Pandiripally, D. Westbrook, G. Sunki, and A. Bhunia, J. Med. Microbiol. 48:117–124, 1999). In this study, expression of LAP in L. monocytogenes at various growth temperatures (25, 37, and 42°C) and in various growth phases was determined by performing an enzyme-linked immunoassay (ELISA) and Western blotting with a specific monoclonal antibody (monoclonal antibody H7). The ELISA and Western blot results indicated that there was a significant increase in LAP expression over time only at 37 and 42°C and that the level of LAP expression was low during the exponential phase and high during the stationary phase. In contrast, there were not significant differences in LAP expression between the exponential and stationary phases at 25°C. Examination of the adhesion of L. monocytogenes cells from exponential-phase (12-h) or stationary-phase (24-h) cultures grown at 37°C to Caco-2 cells revealed that there were not significant differences in adhesion. Although expression of L. monocytogenes LAP was different at different growth temperatures and in different growth phases, enhanced expression did not result in increased adhesion, possibly because only a few LAP molecules were sufficient to initiate binding to Caco-2 cells. PMID:10347076

  1. Protein adsorption and platelet adhesion onto polyurethane grafted with methoxy-poly(ethylene glycol) methacrylate by plasma technique.

    PubMed

    Fujimoto, K; Inoue, H; Ikada, Y

    1993-12-01

    Graft polymerization of methoxy-poly(ethylene glycol) methacrylate, an ester of methacrylic acid and monomethoxy-poly(ethylene glycol) (PEO), was performed onto a polyetherurethane (PU) film and tube under different polymerization conditions by a plasma treatment technique. The surface of grafted PU film was characterized by staining with dye, x-ray photoelectron spectroscopy, contact angle, and zeta potential. All these measurements indicated that water-soluble chains were immobilized on the PU surface, their location being restricted to the film surface region. The PU surface showed reduced protein adsorption in vitro and reduced platelet adhesion in vitro and ex vivo. The optimum graft density suppressing the protein adsorption was as low as 5 micrograms cm(-2). When a small amount of dimethacrylate was added to the monomer solution for graft polymerization to introduce crosslinking in the grafted layer, protein adsorption was further slightly reduced. The extent of reduction in serum albumin adsorption was always less than that of gamma-globulin. Although platelet adhesion was largely reduced by the surface graft polymerization, a definite amount of protein was always adsorbed to the grafted surface. PMID:8113244

  2. 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 37C 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

  3. 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

  4. 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

  5. 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

  6. Differential Impact of Plasma Proteins on the Adhesion Efficiency of Vascular-Targeted Carriers (VTCs) in Blood of Common Laboratory Animals.

    PubMed

    Namdee, Katawut; Sobczynski, Daniel J; Onyskiw, Peter J; Eniola-Adefeso, Omolola

    2015-12-16

    Vascular-targeted carrier (VTC) interaction with human plasma is known to reduce targeted adhesion efficiency in vitro. However, the role of plasma proteins on the adhesion efficiency of VTCs in laboratory animals remains unknown. Here, in vitro blood flow assays are used to explore the effects of plasma from mouse, rabbit, and porcine on VTC adhesion. Porcine blood exhibited a strong negative plasma effect on VTC adhesion while no significant plasma effect was found with rabbit and mouse blood. A brush density poly(ethylene glycol) (PEG) on VTCs was effective at improving adhesion of microsized, but not nanosized, VTCs in porcine blood. Overall, the results suggest that porcine models, as opposed to mouse, can serve as better models in preclinical research for predicting the in vivo functionality of VTCs for use in humans. These considerations hold great importance for the design of various pharmaceutical products and development of reliable drug delivery systems. PMID:26505780

  7. Influence of blood proteins in the in vitro adhesion of Staphylococcus epidermidis to teflon, polycarbonate, polyethylene and bovine pericardium.

    PubMed

    Carballo, J; Ferreirs, C M; Criado, M T

    1991-12-01

    The influence of human plasma proteins (fibrinogen, albumin and fibronectin) on the adherence of Staphylococcus epidermis to teflon, polyethylene, polycarbonate and bovine pericardium was studied in an in vitro quantitative assay by scintillation counting. Bacterial adhesion was generally reduced by the presence of protein during the adherence assay except in the case of bovine pericardium, in which adherence remained almost unaffected. The effect of these plasma proteins on bacterial surface properties resulted in strong increases of surface charge as measured by ion-exchange chromatography and with no effect on hydrophobicity, estimated as contact angles. Adherence was not found to be correlated with these two properties, suggesting that bacteria-surface interactions must not be simplified to the influence of interfacial forces. PMID:1812542

  8. Mechanism for Adhesion G Protein-Coupled Receptor GPR56-Mediated RhoA Activation Induced By Collagen III Stimulation

    PubMed Central

    Luo, Rong; Jeong, Sung-Jin; Yang, Annie; Wen, Miaoyun; Saslowsky, David E.; Lencer, Wayne I.; Ara, Demet; Piao, Xianhua

    2014-01-01

    GPR56 is a member of the adhesion G protein-coupled receptor (GPCR) family. Despite the importance of GPR56 in brain development, where mutations cause a devastating human brain malformation called bilateral frontoparietal polymicrogyria (BFPP), the signaling mechanism(s) remain largely unknown. Like many other adhesion GPCRs, GPR56 is cleaved via a GPCR autoproteolysis-inducing (GAIN) domain into N- and C-terminal fragments (GPR56N and GPR56C); however, the biological significance of this cleavage is elusive. Taking advantage of the recent identification of a GPR56 ligand and the presence of BFPP-associated mutations, we investigated the molecular mechanism of GPR56 signaling. We demonstrate that ligand binding releases GPR56N from the membrane-bound GPR56C and triggers the association of GPR56C with lipid rafts and RhoA activation. Furthermore, one of the BFPP-associated mutations, L640R, does not affect collagen III-induced lipid raft association of GPR56. Instead, it specifically abolishes collagen III-mediated RhoA activation. Together, these findings reveal a novel signaling mechanism that may apply to other members of the adhesion GPCR family. PMID:24949629

  9. hE-cadherin-Fc fusion protein coated surface enhances the adhesion and proliferation of human mesenchymal stem cells.

    PubMed

    Xu, Jianbin; Zhu, Chuanshun; Zhang, Yan; Jiang, Nan; Li, Suhua; Su, Zhongyu; Akaike, Toshihiro; Yang, Jun

    2013-09-01

    A fusion protein consisting of human E-cadherin extracellular domain and the immunoglobulin G Fc region (hE-cadherin-Fc) was prepared and used as a cell-cell adhesion biomimicking matrix for the in vitro expansion of human mesenchymal stem cells (hMSCs) for use in regenerative medicine. The hE-cadherin-Fc was stably immobilized onto a polystyrene plate due to the hydrophobicity of the Fc domain, enhancing the surface wettability and topography of the plate. The hE-cadherin-Fc matrix markedly promoted the cell adhesion and proliferation of hMSCs compared with the tissue culture-treated plate (TC-PS) and the gelatin-coated plate. Furthermore, the expanded hMSCs on the hE-cadherin-Fc were positive for CD105, similar to those from the gelatin. Additionally, the expression of E-cadherin and ?-catenin in the hMSCs was improved on the hE-cadherin-Fc matrix, suggesting that the interactions of the hE-cadherin-Fc matrix with the hMSCs were substitutes for the cell-cell adhesion junctions during the initial culture stage in the absence of intercellular interactions. The hE-cadherin-Fc was shown to be a promising artificial ECM for the in vitro expansion of hMSCs. PMID:23624276

  10. 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

  11. 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

  12. 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

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

    SciTech Connect

    Lu, Renquan; Department of Oncology, Shanghai Medical College, Fudan University, Shanghai 200032 ; Sun, Xinghui; Department of Medicine, Harvard Medical School, MA 02115 ; Xiao, Ran; Zhou, Lei; Gao, Xiang; Guo, Lin; Department of Oncology, Shanghai Medical College, Fudan University, Shanghai 200032

    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.

  14. Block Copolymer Arrangement and Composition Effects on Protein Conformation Using AFM-Based Antigen-Antibody Adhesion

    PubMed Central

    Palacio, Manuel L. B.; Schricker, Scott R.; Bhushan, Bharat

    2013-01-01

    The conformational changes of fibronectin deposited on various block copolymers where one block is composed of poly(methyl methacrylate) (PMMA) and the other block is either poly(acrylic acid) (PAA) or poly(2-hydroxyethyl methacrylate) (PHEMA) were investigated using a functionalized atomic force microscope (AFM) tip. The tip was modified with an antibody sensitive to the exposure of the arginine-glycine-aspartic acid (RGD) groups in fibronectin. By studying the adhesive interactions between the antibody and the proteins adsorbed on the block copolymer surface and phase imaging, it was found that the triblock copolymers PAA-b-PMMA-b-PAA and PMMA-b-PHEMA-b-PMMA, which both have large domain sizes, are conducive to the exposure of the fibronectin RGD groups on the surface. Based on these results, it is concluded that the surface chemistry as well as the nanomorphology dictated by the block copolymer arrangement could both tune protein conformation and orientation and optimize cell adhesion to the biomaterial surface. PMID:22278846

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

    PubMed

    Leyva-Leyva, Margarita; Lpez-Daz, Annia; Barrera, Lourdes; Camacho-Morales, Alberto; Hernandez-Aguilar, Felipe; Carrillo-Casas, Erika M; Arriaga-Pizano, Lourdes; Caldern-Prez, Jaime; Garca-lvarez, Jorge; Orozco-Hoyuela, Gabriel; Pia-Barba, Cristina; Rojas-Martnez, Augusto; Romero-Daz, Vktor; Lara-Arias, Jorge; Rivera-Bolaos, Nancy; Lpez-Camarillo, Csar; Moncada-Saucedo, Nidia; Galvn-De los Santos, Alejandra; Meza-Urza, Ftima; Villarreal-Gmez, 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 ?-actinin expression compared with CD105(+) cells at the maturation stage. Interestingly, levels of activated ?1-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 ?-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 orthopedic bioengineering. PMID:26230358

  16. Translocation of a human focal adhesion LIM-only protein, FHL2, during myofibrillogenesis and identification of LIM2 as the principal determinants of FHL2 focal adhesion localization.

    PubMed

    Li, H Y; Kotaka, M; Kostin, S; Lee, S M; Kok, L D; Chan, K K; Tsui, S K; Schaper, J; Zimmermann, R; Lee, C Y; Fung, K P; Waye, M M

    2001-01-01

    LIM domain proteins are found to be important regulators in cell growth, cell fate determination, cell differentiation, and remodeling of the cell cytoskeleton. Human Four-and-a-half LIM-only protein 2 (FHL2) is expressed predominantly in human heart and is only slightly expressed in skeletal muscle. Since FHL2 is an abundant protein in human heart, it may play an important role in the regulation of cell differentiation and myofibrillogenesis of heart at defined subcellular compartment. Therefore, we hypothesized that FHL2 act as a multi-functional protein by the specific arrangement of the LIM domains of FHL2 and that one of the LIM domains of FHL2 can function as an anchor and localizes it into a specific subcellular compartment in a cell type specific manner to regulate myofibrillogenesis. From our results, we observed that FHL2 is localized at the focal adhesions of the C2C12, H9C2 myoblast as well as a nonmyogenic cell line, HepG2 cells. Colocalization of vinculin-CFP and FHL2-GFP at focal adhesions was also observed in cell lines. Site-directed mutagenesis, in turn, suggested that the second LIM domain-LIM2 is essential for its specific localization to focal adhesions. Moreover, FHL2 was observed along with F-actin and focal adhesion of C2C12 and H9C2 myotubes. Finally, we believe that FHL2 moves from focal adhesions and then stays at the Z-discs of terminally differentiated heart muscle. PMID:11124707

  17. Connexon-mediated cell adhesion drives microtissue self-assembly

    PubMed Central

    Bao, Brian; Jiang, Jean; Yanase, Toshihiko; Nishi, Yoshihiro; Morgan, Jeffrey R.

    2011-01-01

    Microtissue self-assembly is thought to be driven primarily by cadherins, while connexons have been examined mainly in intercellular coupling. We investigated whether connexon 43 (Cx43)-mediated cell adhesion modulates self-assembly of human KGN granulosa cells, normal human fibroblasts (NHFs), and MCF-7 breast cancer cells seeded into nonadhesive agarose gels. We found that treatment with anti-Cx43 E2 (112 ?g/ml), which suppresses Cx43 docking, significantly inhibited the kinetics of KGN and NHF self-assembly compared to the preimmune sera control (41.14.5 and 24.510.4% at 8 h, respectively). Likewise, gap junction inhibitor carbenoxolone also inhibited self-assembly of KGN, NHF, and MCF-7 cells in a dose-dependent manner that was specific to cell type. In contrast, Gap26 connexin mimetic peptide, which inhibits channel permeability but not docking, accelerated self-assembly of KGN and NHF microtissues. Experiments using selective enzymatic digestion of cell adhesion molecules and neutralizing N-cadherin antibodies further showed that self-assembly was comparably disrupted by inhibiting connexin- and cadherin-mediated adhesion. These findings demonstrate that connexon-mediated cell adhesion and intercellular communication differentially influence microtissue self-assembly, and that their contributions are comparable to those of cadherins.Bao, B., Jiang, J., Yanase, T., Nishi, Y., Morgan, J. R. Connexon-mediated cell adhesion drives microtissue self-assembly. PMID:20876208

  18. Cross Talk between Cell Cell and Cell Matrix Adhesion Signaling Pathways during Heart Organogenesis: Implications for Cardiac Birth Defects

    NASA Astrophysics Data System (ADS)

    Linask, Kersti K.; Manisastry, Shyam; Han, Mingda

    2005-06-01

    The anterior posterior and dorsal ventral progression of heart organogenesis is well illustrated by the patterning and activity of two members of different families of cell adhesion molecules: the calcium-dependent cadherins, specifically N-cadherin, and the extracellular matrix glycoproteins, fibronectin. N-cadherin by its binding to the intracellular molecule [beta]-catenin and fibronectin by its binding to integrins at focal adhesion sites, are involved in regulation of gene expression by their association with the cytoskeleton and through signal transduction pathways. The ventral precardiac mesoderm cells epithelialize and become stably committed by the activation of these cell matrix and intracellular signaling transduction pathways. Cross talk between the adhesion signaling pathways initiates the characteristic phenotypic changes associated with cardiomyocyte differentiation: electrical activity and organization of myofibrils. The development of both organ form and function occurs within a short interval thereafter. Mutations in any of the interacting molecules, or environmental insults affecting either of these signaling pathways, can result in embryonic lethality or fetuses born with severe heart defects. As an example, we have defined that exposure of the embryo temporally to lithium during an early sensitive developmental period affects a canonical Wnt pathway leading to [beta]-catenin stabilization. Lithium exposure results in an anterior posterior progression of severe cardiac defects.

  19. Discovery of a novel and conserved Plasmodium falciparum exported protein that is important for adhesion of PfEMP1 at the surface of infected erythrocytes

    PubMed Central

    Nacer, Adla; Claes, Aurlie; Roberts, Amy; Scheidig-Benatar, Christine; Sakamoto, Hiroshi; Ghorbal, Mehdi; Lopez-Rubio, Jose-Juan; Mattei, Denise

    2015-01-01

    Plasmodium falciparum virulence is linked to its ability to sequester in post-capillary venules in the human host. Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1) is the main variant surface antigen implicated in this process. Complete loss of parasite adhesion is linked to a large subtelomeric deletion on chromosome 9 in a number of laboratory strains such as D10 and T9-96. Similar to the cytoadherent reference line FCR3, D10 strain expresses PfEMP1 on the surface of parasitized erythrocytes, however without any detectable cytoadhesion. To investigate which of the deleted subtelomeric genes may be implicated in parasite adhesion, we selected 12 genes for D10 complementation studies that are predicted to code for proteins exported to the red blood cell. We identified a novel single copy gene (PF3D7_0936500) restricted to P. falciparum that restores adhesion to CD36, termed here virulence-associated protein 1 (Pfvap1). Protein knockdown and gene knockout experiments confirmed a role of PfVAP1 in the adhesion process in FCR3 parasites. PfVAP1 is co-exported with PfEMP1 into the host cell via vesicle-like structures called Maurer's clefts. This study identifies a novel highly conserved parasite molecule that contributes to parasite virulence possibly by assisting PfEMP1 to establish functional adhesion at the host cell surface. PMID:25703704

  20. Discovery of a novel and conserved Plasmodium falciparum exported protein that is important for adhesion of PfEMP1 at the surface of infected erythrocytes.

    PubMed

    Nacer, Adéla; Claes, Aurélie; Roberts, Amy; Scheidig-Benatar, Christine; Sakamoto, Hiroshi; Ghorbal, Mehdi; Lopez-Rubio, Jose-Juan; Mattei, Denise

    2015-08-01

    Plasmodium falciparum virulence is linked to its ability to sequester in post-capillary venules in the human host. Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1) is the main variant surface antigen implicated in this process. Complete loss of parasite adhesion is linked to a large subtelomeric deletion on chromosome 9 in a number of laboratory strains such as D10 and T9-96. Similar to the cytoadherent reference line FCR3, D10 strain expresses PfEMP1 on the surface of parasitized erythrocytes, however without any detectable cytoadhesion. To investigate which of the deleted subtelomeric genes may be implicated in parasite adhesion, we selected 12 genes for D10 complementation studies that are predicted to code for proteins exported to the red blood cell. We identified a novel single copy gene (PF3D7_0936500) restricted to P. falciparum that restores adhesion to CD36, termed here virulence-associated protein 1 (Pfvap1). Protein knockdown and gene knockout experiments confirmed a role of PfVAP1 in the adhesion process in FCR3 parasites. PfVAP1 is co-exported with PfEMP1 into the host cell via vesicle-like structures called Maurer's clefts. This study identifies a novel highly conserved parasite molecule that contributes to parasite virulence possibly by assisting PfEMP1 to establish functional adhesion at the host cell surface. PMID:25703704

  1. Glutamine Synthetase and Glucose-6-Phosphate Isomerase Are Adhesive Moonlighting Proteins of Lactobacillus crispatus Released by Epithelial Cathelicidin LL-37

    PubMed Central

    Kainulainen, Veera; Loimaranta, Vuokko; Pekkala, Anna; Edelman, Sanna; Antikainen, Jenni; Kylväjä, Riikka; Laaksonen, Maiju; Laakkonen, Liisa; Finne, Jukka

    2012-01-01

    Glutamine synthetase (GS) and glucose-6-phosphate isomerase (GPI) were identified as novel adhesive moonlighting proteins of Lactobacillus crispatus ST1. Both proteins were bound onto the bacterial surface at acidic pHs, whereas a suspension of the cells to pH 8 caused their release into the buffer, a pattern previously observed with surface-bound enolase and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) of L. crispatus. The pH shift was associated with a rapid and transient increase in cell wall permeability, as measured by cell staining with propidium iodide. A gradual increase in the release of the four moonlighting proteins was also observed after the treatment of L. crispatus ST1 cells with increasing concentrations of the antimicrobial cationic peptide LL-37, which kills bacteria by disturbing membrane integrity and was here observed to increase the cell wall permeability of L. crispatus ST1. At pH 4, the fusion proteins His6-GS, His6-GPI, His6-enolase, and His6-GAPDH showed localized binding to cell division septa and poles of L. crispatus ST1 cells, whereas no binding to Lactobacillus rhamnosus GG was detected. Strain ST1 showed a pH-dependent adherence to the basement membrane preparation Matrigel. Purified His6-GS and His6-GPI proteins bound to type I collagen, and His6-GS also bound to laminin, and their level of binding was higher at pH 5.5 than at pH 6.5. His6-GS also expressed a plasminogen receptor function. The results show the strain-dependent surface association of moonlighting proteins in lactobacilli and that these proteins are released from the L. crispatus surface after cell trauma, under conditions of alkaline stress, or in the presence of the antimicrobial peptide LL-37 produced by human cells. PMID:22389474

  2. Inactivation of a fibronectin-binding TonB-dependent protein increases adhesion properties of Bacteroides fragilis.

    PubMed

    Pauer, Heidi; Cavalcanti, Soraia N V; Teixeira, Felipe L; Santos-Filho, Joaquim; Vommaro, Rossiane C; Oliveira, Ana Carolina S C; Ferreira, Eliane O; Domingues, Regina R M C P

    2013-10-01

    Bacteroides fragilis is the Gram-negative strictly anaerobic bacterium most frequently isolated from clinical infections, including intra-abdominal abscess and bacteraemia. A number of factors can contribute to its virulence, including the expression of adhesins. Some of them are already characterized and can recognize and bind to extracellular matrix components, such as fibronectin. One of the molecules responsible for fibronectin-binding is an outer-membrane protein previously described by our group, which belongs to the TonB-dependent family. The aim of the present work was to characterize this protein. Initially, it was confirmed by fluorescence and electron microscopy that the fibronectin-binding molecules were located in the bacterial surface, but the distribution of these molecules on the surface was not uniform. To further evaluate the role of this protein, the gene bf1991, responsible for encoding this protein, was inactivated by a suicide vector and the mutant strains generated were used in several experiments to verify possible phenotypical alterations. In adherence assays with fibronectin immobilized on latex beads an increased adhesion was observed with the mutant strains compared with the wild-type strain. Western blot analysis in the mutant strain revealed the absence of the 120 kDa TonB-dependent outer-membrane protein and an alteration in the expression of an unknown 30 kDa protein. Killing assays using peritoneal macrophages were performed to evaluate the role of this protein as a virulence attribute and it was observed that the mutant strains were more efficiently internalized than the wild-type strains, with more internalization in the samples covered with fibronectin than in the samples not covered with it. PMID:23893918

  3. Investigation of cell adhesion to silica nanoparticle-decorated surfaces and the associated protein-mediated mechanisms

    NASA Astrophysics Data System (ADS)

    Ballard, Jake D.

    Nanostructured materials have shown promise to improve the interface between prosthetic devices and living cells, tissues, and organs through the ability to evoke cell type-specific and size-selective functions from various cell types of in vivo importance. However, the underlying molecular level mechanisms responsible for enhancement of select cell functions on these materials are not fully understood. Silica particles of either 4, 20, or 100 nm diameters were successfully coated onto native-oxide coated silicon substrates in the range of 0 to 100% coverage by particles. The materials formulated and fabricated for the present study provide a controlled and characterized set of substrates needed for investigation of the effects of nanoscale features on the adsorption and conformation of proteins, and subsequent functions of mammalian cells that are critical to the clinical efficacy of biomaterials. The size of nanoscale surface features constituted by silica nanoparticles on native oxide-coated silicon pieces affected the adhesion of rat calvarial osteoblasts and rat skin fibroblasts differently. It was also demonstrated, for the first time, that a threshold density of nanoscale surface features is necessary to elicit size-selective, and cell type-specific, adhesion from osteoblasts or fibroblasts. Adsorption of fibronectin and vitronectin onto native oxide-coated silicon surfaces decorated with either 4, 20, or 100 nm diameter silica particles at either 25, 45, or 80% surface coverage was quantified and examined by scanning electron microscopy. Circular dichroism spectroscopy provided evidence that the secondary structures of fibronectin in the presence of either 4 or 20 nm diameter particles were similar, but fibronectin exhibited decreased beta sheet content and increased unordered structure in the presence of 100 nm particles. The secondary structure of vitronectin in the presence of silica particles exhibited similar levels of structure loss for all particle sizes examined. For the first time, this study offers insight into a molecular mechanism that is linked to nanostructured material surface feature size through quantified changes in protein structure and cell adhesion behavior. These results provide an explanation of the molecular level events occurring on nanostructured material surfaces that contribute to protein-mediated size-selective and cell type-specific responses of various cell types.

  4. A short-term Borrelia burgdorferi infection model identifies tissue tropisms and bloodstream survival conferred by adhesion proteins.

    PubMed

    Caine, Jennifer A; Coburn, Jenifer

    2015-08-01

    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

  5. The GPS Motif Is a Molecular Switch for Bimodal Activities of Adhesion Class G Protein-Coupled Receptors

    PubMed Central

    Prmel, Simone; Frickenhaus, Marie; Hughes, Samantha; Mestek, Lamia; Staunton, David; Woollard, Alison; Vakonakis, Ioannis; Schneberg, Torsten; Schnabel, Ralf; Russ, AndreasP.; 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 invivo 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

  6. 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

  7. Streptavidin-coated TiO2 surfaces are biologically inert: protein adsorption and osteoblast adhesion studies.

    PubMed

    Lehnert, Michael; Gorbahn, Miriam; Klein, Marcus; Al-Nawas, Bilal; Kper, Ingo; Knoll, Wolfgang; Veith, Michael

    2012-02-01

    Non-fouling TiO2 surfaces are attractive for a wide range of applications such as biosensors and medical devices, where biologically inert surfaces are needed. Typically, this is achieved by controlled surface modifications which prevent protein adsorption. For example, polyethylene glycol (PEG) or PEG-derived polymers have been widely applied to render TiO2 surfaces biologically inert. These surfaces have been further modified in order to achieve specific bio-activation. Therefore, there have been efforts to specifically functionalize TiO2 surfaces with polymers with embedded biotin motives, which can be used to couple streptavidin for further functionalization. As an alternative, here a streptavidin layer was immobilized by self-assembly directly on a biotinylated TiO2 surface, thus forming an anti-adhesive matrix, which can be selectively bio-activated. The anti-adhesive properties of these substrates were analyzed by studying the interaction of the surface coating with fibronectin, lysozym, and osteoblast cells using surface plasmon resonance spectroscopy, atomic force microscopy, and light microscopy. In contrast to non-modified TiO2 surfaces, streptavidin-coated TiO2 surfaces led to a very biologically inert substrate, making this type of surface coating a promising alternative to polymer coatings of TiO2 surfaces. PMID:22083833

  8. Post-ischemic vascular adhesion protein-1 inhibition provides neuroprotection in a rat temporary middle cerebral artery occlusion model

    PubMed Central

    Watcharotayangul, Jittiya; Mao, Lizhen; Xu, Haoliang; Vetri, Francesco; Baughman, Verna L.; Paisansathan, Chanannait; Pelligrino, Dale A.

    2012-01-01

    We examined the neuroprotective efficacy associated with post-ischemic vascular adhesion protein-1 (VAP-1) blockade in rats subjected to transient (1h) middle cerebral artery occlusion (MCAo). We compared saline-treated control rats to rats treated with a highly-selective VAP-1 inhibitor, LJP-1586 [Z-3-fluoro-2-(4-methoxybenzyl) allylamine hydrochloride]. Initial intraperitoneal LJP-1586 (or saline control) treatments were delayed until 6h or 12h reperfusion. At 72h reperfusion, LJP-1586-treated rats displayed 51% and 33% smaller infarct volumes, relative to their controls, in the 6- and 12h-treatment groups, respectively. However, only in the 6h-treatment group was the infarct volume reduction significant (p<0.05). On the other hand, we observed significantly improved neurologic functions in both 6- and 12h-treatment groups, versus their matched controls (p<0.05). Also, the effect of 6h LJP-1586 treatment on post-ischemic leukocyte trafficking in pial venules overlying the ischemic cortex was evaluated using intravital microscopy. These experiments revealed that: 1) LJP-1586 did not affect intravascular leukocyte (largely neutrophil) adhesion, at least out to 12h reperfusion; and 2) the onset of neutrophil extravasation, which occurred between 6-8h reperfusion in control rats, was prevented by LJP-1586-treatment. In conclusion, in rats subjected to transient MCAo, selective VAP-1 pharmacologic blockade provided neuroprotection, with a prolonged therapeutic window of 6 to 12h reperfusion. PMID:23050649

  9. Interaction of Zyxin, a Focal Adhesion Protein, with the E6 Protein from Human Papillomavirus Type 6 Results in Its Nuclear Translocation

    PubMed Central

    Degenhardt, Yan Yan; Silverstein, Saul

    2001-01-01

    Zyxin, a focal adhesion molecule, interacts specifically with the E6 protein from human papillomavirus (HPV) type 6 in a yeast two-hybrid screen of a cDNA library prepared from human keratinocytes. Zyxin does not interact significantly with E6 proteins from HPV types 11, 16, or 18. The interaction was confirmed by in vitro and in vivo analyses and it requires the LIM domains (Lin-11, Isl-1, and Mec-3 [G. Freyd, S. K. Kim, and H. R. Horvitz, Nature 344:876879, 1990]) found at the carboxyl terminus of zyxin. Cotransfection of E6 from HPV (6E6) and zyxin results in the accumulation of zyxin in the nucleus where it can function as a transcriptional activator. 6E6 can also mobilize endogenous zyxin to the nucleus. PMID:11689660

  10. 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 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 proteinprotein 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 under preliminary cryoconditions, using an in-house generator. This work leads the way to the determination of the first structure of an OLF domain.

  11. Interaction of Paxillin with Poly(A)-Binding Protein 1 and Its Role in Focal Adhesion Turnover and Cell Migration

    PubMed Central

    Woods, Alison J.; Kantidakis, Theodoros; Sabe, Hisataka; Critchley, David R.; Norman, Jim C.

    2005-01-01

    We have previously identified poly(A)-binding protein 1 (PABP1) as a ligand for paxillin and shown that the paxillin-PABP1 complex undergoes nucleocytoplasmic shuttling. By targeting the paxillin-binding subdomain sequences in PABP1, we have generated mutants of PABP1 that do not bind to cellular paxillin. Here we report that paxillin association is necessary for efficient nuclear export of PABP1 and that RNA interference of paxillin drives the nuclear accumulation of PABP1. Furthermore, ablation of paxillin-PABP1 association impeded a number of indices of cell motility including spreading on fibronectin, cell migration on two-dimensional matrices, and transmigration in Boyden chambers. These data indicate that PABP1 must associate with paxillin in order to be efficiently transported from the nucleus to the cytoplasm and that this event is necessary for cells to remodel their focal adhesions during cell migration. PMID:15831480

  12. The tight-adhesion proteins TadGEF of Bradyrhizobium diazoefficiens USDA 110 are involved in cell adhesion and infectivity on soybean roots.

    PubMed

    Mongiardini, Elas J; Parisi, Gustavo D; Quelas, Juan I; Lodeiro, Anbal 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

  13. Biotin ligase tagging identifies proteins proximal to E-cadherin, including lipoma preferred partner, a regulator of epithelial cellcell and cellsubstrate adhesion

    PubMed Central

    Van Itallie, Christina M.; Tietgens, Amber Jean; Aponte, Angel; Fredriksson, Karin; Fanning, Alan S.; Gucek, Marjan; Anderson, James M.

    2014-01-01

    ABSTRACT Known proteins associated with the cell-adhesion protein E-cadherin include catenins and proteins involved in signaling, trafficking and actin organization. However, the list of identified adherens junction proteins is likely to be incomplete, limiting investigation into this essential cell structure. To expand the inventory of potentially relevant proteins, we expressed E-cadherin fused to biotin ligase in MDCK epithelial cells, and identified by mass spectrometry neighboring proteins that were biotinylated. The most abundant of the 303 proteins identified were catenins and nearly 40 others that had been previously reported to influence cadherin function. Many others could be rationalized as novel candidates for regulating the adherens junction, cytoskeleton, trafficking or signaling. We further characterized lipoma preferred partner (LPP), which is present at both cell contacts and focal adhesions. Knockdown of LPP demonstrated its requirement for E-cadherin-dependent adhesion and suggested that it plays a role in coordination of the cellcell and cellsubstrate cytoskeletal interactions. The analysis of LPP function demonstrates proof of principle that the proteomic analysis of E-cadherin proximal proteins expands the inventory of components and tools for understanding the function of E-cadherin. PMID:24338363

  14. 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

  15. Demonstration of adhesion activity of the soluble Ig-domain protein C-CAM4 by attachment to the plasma membrane.

    PubMed

    Lin, S H; Cheng, H; Earley, K; Luo, W; Chou, J

    1998-04-17

    The carcinoembryonic antigen (CEA) family is a large group of proteins with immunoglobulin (Ig)-like structures. The membrane-associated CEA-family proteins have been shown to mediate intercellular adhesion. In addition to these membrane-associated proteins, several secreted CEA-like proteins, such as C-CAM4, PSG1b, and PSG11s, have also been identified. The functions of these soluble proteins are not clear because they cannot support intercellular adhesion like the membrane-associated proteins can. A fundamental question important for understanding the functions of these soluble proteins is whether they can interact in a homophilic fashion as do many of their membrane-associated homologues. We found that the homophilic interactions between these soluble proteins were too weak to be detected by solution binding assays. This is not unexpected because interactions between adhesion molecules are usually transient and weak to allow for control of association and dissociation. By expressing these soluble CEA-family proteins, C-CAM4, PSG1b, and PSG11s, as membrane-anchored forms, we showed that C-CAM4 could mediate intercellular adhesion, whereas PSG1b and PSG11s, despite their 52% identity to C-CAM4, could not. These results suggest that C-CAM4, but not PSG1b and PSG11s, can probably form homodimers. Thus, these secretory CEA-family members most likely have different interaction mechanisms, i.e., C-CAM4 might function as dimers, while PSGs might function as monomers. PMID:9571177

  16. 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...

  17. 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

  18. Hydrogen-Rich Medium Attenuated Lipopolysaccharide-Induced Monocyte-Endothelial Cell Adhesion and Vascular Endothelial Permeability via Rho-Associated Coiled-Coil Protein Kinase.

    PubMed

    Xie, Keliang; Wang, Weina; Chen, Hongguang; Han, Huanzhi; Liu, Daquan; Wang, Guolin; Yu, Yonghao

    2015-07-01

    Sepsis is the leading cause of death in critically ill patients. In recent years, molecular hydrogen, as an effective free radical scavenger, has been shown a selective antioxidant and anti-inflammatory effect, and it is beneficial in the treatment of sepsis. Rho-associated coiled-coil protein kinase (ROCK) participates in junction between normal cells, and regulates vascular endothelial permeability. In this study, we used lipopolysaccharide to stimulate vascular endothelial cells and explored the effects of hydrogen-rich medium on the regulation of adhesion of monocytes to endothelial cells and vascular endothelial permeability. We found that hydrogen-rich medium could inhibit adhesion of monocytes to endothelial cells and decrease levels of adhesion molecules, whereas the levels of transepithelial/endothelial electrical resistance values and the expression of vascular endothelial cadherin were increased after hydrogen-rich medium treatment. Moreover, hydrogen-rich medium could lessen the expression of ROCK, as a similar effect of its inhibitor Y-27632. In addition, hydrogen-rich medium could also inhibit adhesion of polymorphonuclear neutrophils to endothelial cells. In conclusion, hydrogen-rich medium could regulate adhesion of monocytes/polymorphonuclear neutrophils to endothelial cells and vascular endothelial permeability, and this effect might be related to the decreased expression of ROCK protein. PMID:25895142

  19. Lipoxygenase metabolites of arachidonic and linoleic acids modulate the adhesion of tumor cells to endothelium via regulation of protein kinase C.

    PubMed Central

    Liu, B; Timar, J; Howlett, J; Diglio, C A; Honn, K V

    1991-01-01

    12(S)-hydroxyeicosatetraenoic acid (12[S]-HETE) and 13(S)-hydroxyoctadecadienoic acid (13[S]-HODE), lipoxygenase metabolites of arachidonic acid and linoleic acid, respectively, previously have been suggested to regulate tumor cell adhesion to endothelium during metastasis. Adhesion of rat Walker carcinosarcoma (W256) cells to a rat endothelial cell monolayer was enhanced after treatment with 12(S)-HETE and this 12(S)-HETE enhanced adhesion was blocked by 13(S)-HODE. Protein kinase inhibitors, staurosporine, calphostin C, and 1-(5-isoquinoline-sulfonyl)-2-methylpiperazine, inhibited the 12(S)-HETE enhanced W256 cell adhesion. Depleting W256 cells of protein kinase C (PKC) with phorbol 12-myristate-13-acetate abolished their ability to respond to 12(S)-HETE. Treatment of W256 cells with 12(S)-HETE induced a 100% increase in membrane-associated PKC activity whereas 13(S)-HODE inhibited the effect of 12(S)-HETE on PKC translocation. High-performance liquid chromatographic analysis revealed that in W256 cells 12-HETE and 13-HODE were two of the major lipoxygenase metabilites of arachidonic acid and linoleic acid, respectively. Therefore, these two metabolites may provide an alternative signaling pathway for the regulation of PKC. Further, these findings suggest that the regulation of tumor cell adhesion to endothelium by 12(S)-HETE and 13(S)-HODE may be a PKC-dependent process. Images PMID:1801923

  20. Adhesive protein-free synthetic hydrogels for retinal pigment epithelium cell culture with low ROS level.

    PubMed

    Chen, Yong Mei; Liu, Zhen Qi; Feng, Zhi Hui; Xu, Feng; Liu, Jian Kang

    2014-07-01

    Engineering of human retinal pigment epithelium (RPE) cell monolayer with low level of reactive oxygen species (ROS) is important for regenerative RPE-based therapies. However, it is still challenging to culture RPE monolayer with low ROS level on soft substrates in vitro. To address this, we developed cytocompatible hydrogels to culture human RPE cell monolayer for future use in regenerative RPE-based therapies. The cell adhesion, proliferation, monolayer formation, morphology, survival, and ROS level of human ARPE-19 cells cultured on the surfaces of negatively charged poly (2-acrylamido-2-methyl propane sulfonic sodium) (PNaAMPS) and neutral poly(N,N-dimethylacrylamide) (PDMAAm) hydrogels with different stiffness were investigated. The importance of hydrogel stiffness on the cell function was firstly highlighted on the base of determined optimal Young's modulus for cultivation of RPE cell monolayer with relatively low ROS level. The construction of RPE cell monolayer with low ROS level on the PNaAMPS hydrogel may hold great potential as promising candidates for transplantation of RPE cell monolayer-hydrogel construct into the subretinal space to repair retinal functions. PMID:23913900

  1. Physiological strains remodel extracellular matrix and cell-cell adhesion in osteoblastic cells cultured on alumina-coated titanium alloy.

    PubMed

    Di Palma, Fabrice; Chamson, Annette; Lafage-Proust, Marie-Hlne; Jouffray, Paul; Sabido, Odile; Peyroche, Sylvie; Vico, Laurence; Rattner, Aline

    2004-06-01

    The effects of mechanical strains on cellular activities were assessed in an in vitro model using human osteoblastic MG-63 cells grown on titanium alloy discs coated with porous alumina and exposed to chronic intermittent loading. Strain was applied with a Dynacell device for three 15-min sequences per day for several days with a magnitude of 600 microepsilon strain and a frequency of 0.25 Hz. We have previously demonstrated that this regimen increased alkaline phosphatase activity in confluent cultures on ceramic coated titanium (alumina and hydroxyapatite) (Biomaterials 24 (2003) 3139). In this study, we analysed the production of bone matrix proteins. Osteocalcin secretion quantified by ELISA between day 5 and 11 was not affected by mechanical strain. Strain had even no quantifiable effect on collagen production from day 1 to 5 as measured by carboxy terminal collagen type I propeptide release. On the other hand, stress stimulation resulted in increased expression of fibronectin (FN) measured by Western blot after 1 day stretching. This upregulation of FN production was followed by reorganisation of the FN network after 5 days stretching observed by immunostaining. The receptors for collagen and FN, alpha2beta1, alpha5beta1 and beta1 integrins were not quantitatively affected by the strains as measured by flow cytometry. A modification of cell morphology was seen after 5 days of loading that appeared to increase cell spreading, implying consequences on intercellular contacts. For this reason, N, C11 and E-adherins were examined. We noted a selective effect characterised by increased expression of N-cadherin using both RT-PCR and Western blot analyses. We concluded that reinforcement of cell-cell adhesion and remodelling of the FN network are important adaptive responses to physiological strains for human osteoblasts grown on alumina-coated biomaterials. PMID:14751742

  2. Novel barnacle underwater adhesive protein is a charged amino acid-rich protein constituted by a Cys-rich repetitive sequence.

    PubMed Central

    Kamino, K

    2001-01-01

    Barnacle cement is an underwater adhesive that is used for permanent settlement, and is an insoluble protein complex. A method for rendering soluble the cement of Megabalanus rosa has been developed, and three major proteins have been identified in a previous study. To survey the M. rosa cement proteins in a lower molecular mass range, the cement proteins were separated by reversed-phase HPLC and a previously unidentified protein named 20 kDa M. rosa cement protein (Mrcp-20k) was found. Mrcp-20k cDNA was cloned to reveal its primary structure. This cDNA was 902 bp long and encoded a 202 amino acid-long open reading frame, including 19 amino acids of the signal sequence. The molecular mass in the disulphide form was calculated to be 20357 Da and the isoelectric point of the mature polypeptide was 4.72. Mrcp-20k was characterized by an abundance of Cys residues and charged amino acids. The most common amino acid was Cys (17.5%), with Asp (11.5%), Glu (10.4%) and His (10.4%) following in order of magnitude. The alignment of the Cys residues indicated the primary structure of this protein to consist of six degenerated repeats, each about 30 residues long. Mrcp-20k has no intermolecular disulphide bonds and no free thiol groups of Cys in the insoluble cement complex. Abundant Cys is thought to play a role in maintaining the topology of charged amino acids on the molecular surface by intramolecular disulphide-bond formation. The possible function of abundant charged amino acids, including the interaction with a variety of surface metals on the substratum, is discussed. PMID:11368778

  3. Plasmodium falciparum adhesion domains linked to severe malaria differ in blockade of endothelial protein C receptor.

    PubMed

    Sampath, Sowmya; Brazier, Andrew Jay; Avril, Marion; Bernabeu, Maria; Vigdorovich, Vladimir; Mascarenhas, Anjali; Gomes, Edwin; Sather, D Noah; Esmon, Charles T; Smith, Joseph D

    2015-12-01

    Cytoadhesion of Plasmodium falciparum-infected erythrocytes to endothelial protein C receptor (EPCR) is associated with severe malaria. It has been postulated that parasite binding could exacerbate microvascular coagulation and endothelial dysfunction in cerebral malaria by impairing the protein C-EPCR interaction, but the extent of binding inhibition has not been fully determined. Here we expressed the cysteine-rich interdomain region (CIDR?1) domain from a variety of domain cassette (DC) 8 and DC13 P.?falciparum erythrocyte membrane protein 1 proteins and show they interact in a distinct manner with EPCR resulting in weak, moderate and strong inhibition of the activated protein C (APC)-EPCR interaction. Overall, there was a positive correlation between CIDR?1-EPCR binding activity and APC blockade activity. In addition, our analysis from a combination of mutagenesis and blocking antibodies finds that an Arg81 (R81) in EPCR plays a pivotal role in CIDR?1 binding, but domains with weak and strong APC blockade activity were distinguished by their sensitivity to inhibition by anti-EPCR mAb 1535, implying subtle differences in their binding footprints. These data reveal a previously unknown functional heterogeneity in the interaction between P.?falciparum and EPCR and have major implications for understanding the distinct clinical pathologies of cerebral malaria and developing new treatment strategies. PMID:26118955

  4. 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.

  5. 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 of surface immobilized protein and Trypsin/EDTA concentration. SPR results showed statistical differences in alpha5beta1 and alphanubeta3 integrin binding to RGD cell binding motifs introduced by bFN(9) and RGD-SA. Increase in binding specificity through these integrins lead to rapid cell binding and retention on Teflon-AF surfaces adsorbed with this protein formulation. This system appears to be the nexus at which the DL has proven its value. These results could have broader implications in augmenting EC attachment to SVG prior to implantation.

  6. 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.

  7. A tumor-suppressing function in the epithelial adhesion protein Trask.

    PubMed

    Spassov, D S; Wong, C H; Harris, G; McDonough, S; Phojanakong, P; Wang, D; Hann, B; Bazarov, A V; Yaswen, P; Khanafshar, E; Moasser, M M

    2012-01-26

    Trask/CDCP1 is a transmembrane glycoprotein widely expressed in epithelial tissues whose functions are just beginning to be understood, but include a role as an anti-adhesive effector of Src kinases. Early studies looking at RNA transcript levels seemed to suggest overexpression in some cancers, but immunostaining studies are now providing more accurate analyses of its expression. In an immuno-histochemical survey of human cancer specimens, we find that Trask expression is retained, reduced or sometimes lost in some tumors compared with their normal epithelial tissue counterparts. A survey of human cancer cell lines also show a similar wide variation in the expression of Trask, including some cell types with the loss of Trask expression, and additional cell types that have lost the physiological detachment-induced phosphorylation of Trask. Three experimental models were established to interrogate the role of Trask in tumor progression, including two gain-of-function models with tet-inducible expression of Trask in tumor cells lacking Trask expression, and one loss-of-function model to suppress Trask expression in tumor cells with abundant Trask expression. The induction of Trask expression and phosphorylation in MCF-7 cells and in 3T3v-src cells was associated with a reduction in tumor metastases while the shRNA-induced knockdown of Trask in L3.6pl cancer cells was associated with increased tumor metastases. The results from these three models are consistent with a tumor-suppressing role for Trask. These data identify Trask as one of several potential candidates for functionally relevant tumor suppressors on the 3p21.3 region of the genome frequently lost in human cancers. PMID:21706059

  8. 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

  9. A Tethered Agonist within the Ectodomain Activates the Adhesion G Protein-coupled Receptors GPR126 and GPR133

    PubMed Central

    Liebscher, Ines; Schn, Julia; Petersen, Sarah C.; Fischer, Liane; Auerbach, Nina; Demberg, Lilian Marie; Mogha, Amit; Cster, Maxi; Simon, Kay-Uwe; Rothemund, Sven; Monk, Kelly R.; Schneberg, Torsten

    2014-01-01

    Adhesion G protein-coupled receptors (aGPCRs) comprise the second largest yet least studied class of the GPCR superfamily. aGPCRs are involved in many developmental processes, immune and synaptic functions, but the mode of their signal transduction is unclear. Here, we show that a short peptide sequence (termed the Stachel sequence) within the ectodomain of two aGPCRs, GPR126 and GPR133, functions as a tethered agonist. Upon structural changes within the receptor ectodomain, this intramolecular agonist is exposed to the 7-transmembrane helix domain, which triggers G-protein activation. Our studies show high specificity of a given Stachel sequence for its receptor. Finally, the function of Gpr126 is abrogated in zebrafish with a mutated Stachel sequence, and signaling is restored in hypomorphic gpr126 zebrafish mutants upon exogenous Stachel peptide application. These findings illuminate a previously unknown mode of aGPCR activation, and can initiate the development of specific ligands for this currently untargeted GPCR family. PMID:25533341

  10. Laser- and UV-assisted modification of polystyrene surfaces for control of protein adsorption and cell adhesion

    NASA Astrophysics Data System (ADS)

    Pfleging, Wilhelm; Torge, Maika; Bruns, Michael; Trouillet, Vanessa; Welle, Alexander; Wilson, Sandra

    2009-03-01

    An appropriate choice of laser and process parameters enables new approaches for the fabrication of polymeric lab-on-chip devices with integrated functionalities. We will present our current research results in laser-assisted modification of polystyrene (PS) with respect to the fabrication of polymer devices for cell culture applications. For this purpose laser micro-patterning of PS and subsequent surface functionalization was investigated as function of laser and process parameters. A high power ArF-excimer laser radiation source with a pulse length of 19 ns as well as a high repetition ArF-excimer laser source with a pulse length of 5 ns were used in order to study the influence of laser pulse length on laser-induced surface oxidation. The change in surface chemistry was characterized by X-ray photoelectron spectroscopy and contact angle measurements. The difference between laser-assisted modification versus UV-lamp assisted modification was investigated. A photolytic activation of specific areas of the polymer surface and subsequent oxidization in oxygen or ambient air leads to a chemically modified polymer surface bearing carboxylic acid groups well-suited for controlled competitive protein adsorption or protein immobilization. Finally, distinct areas for cell growth and adhesion are obtained.

  11. Cell adhesion molecule contactin-associated protein 3 is expressed in the mouse basal ganglia during early postnatal stages.

    PubMed

    Hirata, Haruna; Umemori, Juzoh; Yoshioka, Hiroki; Koide, Tsuyoshi; Watanabe, Kazutada; Shimoda, Yasushi

    2016-01-01

    Cell adhesion molecules play important roles in the development of the nervous system. Among the contactin-associated protein (Caspr; also known as Cntnap) family, which belongs to the neurexin superfamily of proteins, Caspr and Caspr2 are indispensable for the formation and maintenance of myelinated nerves. In contrast, a physiological role for Caspr3 remains to be elucidated. This study examines the expression and localization of Caspr3 in the mouse brain using newly generated Caspr3 antibodies. Caspr3 was expressed abundantly between the first and the second postnatal weeks. During this period, Caspr3 was localized especially to the basal ganglia, including the striatum, external segment of the globus pallidus, and substantia nigra, and no gross abnormalities were apparent in the basal ganglia of Caspr3 knockout mice. In the striatum, Caspr3 was expressed by a subpopulation of medium spiny neurons that constitute the direct and indirect pathways. Caspr3 immunostaining was observed as punctate around the cell bodies as well as in the soma. These Caspr3 signals did not, however, overlap with those of synaptic markers. Our findings suggest that Caspr3 may play an important role in basal ganglia development during early postnatal stages. 2015 Wiley Periodicals, Inc. PMID:26389685

  12. Cell adhesion and cortex contractility determine cell patterning in the Drosophila retina.

    PubMed

    Kfer, Jos; Hayashi, Takashi; Mare, Athanasius F M; Carthew, Richard W; Graner, Franois

    2007-11-20

    Because of the resemblance of many epithelial tissues to densely packed soap bubbles, it has been suggested that surface minimization, which drives soap bubble packing, could be governing cell packing as well. We test this by modeling the shape of the cells in a Drosophila retina ommatidium. We use the observed configurations and shapes in wild-type flies, as well as in flies with different numbers of cells per ommatidia, and mutants with cells where E- or N-cadherin is either deleted or misexpressed. We find that surface minimization is insufficient to model the experimentally observed shapes and packing of the cells based on their cadherin expression. We then consider a model in which adhesion leads to a surface increase, balanced by cell cortex contraction. Using the experimentally observed distributions of E- and N-cadherin, we simulate the packing and cell shapes in the wild-type eye. Furthermore, by changing only the corresponding parameters, this model can describe the mutants with different numbers of cells or changes in cadherin expression. PMID:18003929

  13. Adhesion of adipose-derived mesenchymal stem cells to glycosaminoglycan surfaces with different protein patterns.

    PubMed

    Soares da Costa, Diana; Márquez-Posadas, Maria del Carmen; Araujo, Ana R; Yang, Yuan; Merino, Santos; Groth, Thomas; Reis, Rui L; Pashkuleva, Iva

    2015-05-13

    Proteins and glycosaminoglycans (GAGs) are the main constituents of the extracellular matrix (ECM). They act in synergism and are equally critical for the development, growth, function, or survival of an organism. In this work, we developed surfaces that display these two classes of biomacromolecules, namely, GAGs and proteins, in a spatially controlled fashion. The generated surfaces can be used as a minimalistic but straightforward model aiding the elucidation of cell-ECM interactions. GAGs (hyaluronic acid and heparin) were covalently bound to amino functionalized surfaces, and albumin or fibronectin was patterned by microcontact printing on top of them. We demonstrate that adipose-derived stem cells (ASCs) can adhere either on the protein or on the GAG pattern as a function of the patterned molecules. ASCs found on the GAG pattern had different morphology and expressed different surface markers than the cells adhered on the protein pattern. ASCs morphology and spreading were also dependent on the size of the pattern. These results show that the developed supports can also be used for ASCs differentiation into different lineages. PMID:25902379

  14. Blood group antigen loci demonstrate multivariate genetic associations with circulating cellular adhesion protein levels in the Multi-Ethnic Study of Atherosclerosis.

    PubMed

    Larson, Nicholas B; Decker, Paul A; Wassel, Christina L; Pankow, James S; Tang, Weihong; Hanson, Naomi Q; Tsai, Michael Y; Bielinski, Suzette J

    2016-04-01

    The cellular adhesion pathway is critical in the pathophysiology of atherosclerosis, and genetic factors contributing to regulation of circulating levels of related proteins may be relevant to risk prediction of cardiovascular disease. In contrast to conducting separate genome-wide protein quantitative trait loci (pQTL) mapping analyses of each individual protein, joint genetic association analyses of multiple quantitative traits can leverage cross-trait co-variation and identify simultaneous regulatory effects on protein levels across the pathway. We conducted a multi-pQTL (mpQTL) analysis of 15 proteins related to cellular adhesion assayed on 2313 participants from the Multi-Ethnic Study of Atherosclerosis (MESA). We applied the MQFAM multivariate association analysis method in PLINK on normalized protein level residuals derived from univariate linear regression, adjusting for age, sex, and principal components of ancestry. Race/ethnicity-stratified analyses identified nine genome-wide significant (P < 5e-08) loci associated with co-variation of protein levels. Although the majority of these SNPs were in proximity to structural genes of the assayed proteins, we discovered multiple loci demonstrating co-association with the circulation of at least two proteins. Of these, two significant loci specific to non-Hispanic white participants, rs17074898 at ALOX5AP (P = 1.78E-08) and rs7521237 at KIAA1614 (P = 2.2E-08), would not have met statistical significance using univariate analyses. Moreover, common patterns of multi-protein associations were discovered at the ABO locus across race/ethnicity. These results indicate the biological relevance of blood group antigens on regulation of circulating cellular adhesion pathway proteins while also demonstrating race/ethnicity-specific co-regulatory effects. PMID:26883866

  15. 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 37C than at 23C 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

  16. Dynamics of presynaptic protein recruitment induced by local presentation of artificial adhesive contacts.

    PubMed

    Suarez, Fernando; Thostrup, Peter; Colman, David; Grutter, Peter

    2013-01-01

    In this study, we introduce a novel approach to induce and observe the formation of presynaptic compartments in axons through a combination of atomic force microscopy (AFM) and fluorescence microscopy. First, we use a poly-D-lysine-coated bead attached to an AFM tip to induce the recruitment of two synaptic proteins, bassoon and synaptophysin, and measure their absolute arrival times to the presynaptic department. We find that bassoon arrives before synaptophysin. Second, we observe the formation of very long (several 10s of ?m), structured, protein-containing membranous strings as the AFM tip was withdrawn from the axon. It is conceivable that these strings might be a novel mechanism by which new neurites or branch points along existing neurites may be generated in situ. PMID:22648784

  17. Protein folding in bacterial adhesion: secretion and folding of classical monomeric autotransporters.

    PubMed

    van Ulsen, Peter

    2011-01-01

    Bacterial adhesins mediate the attachment of bacteria to their niches, such as the tissue of an infected host. Adhesins have to be transported across the cell envelope to become active and during this secretion process they fold into their final conformation. This chapter focuses on the biogenesis of the classical monomeric autotransporter proteins, which are the most ubiquitous class of secreted proteins in Gram-negative bacteria. They may function as adhesins, but other functions are also known. Autotransporter proteins have a modular structure and consist of an N-terminal signal peptide and a C-terminal translocator domain with in between the secreted passenger domain that harbours the functions. The signal peptide directs the transport across the inner membrane to the periplasm via the Sec machinery. The translocator domain inserts into the outer membrane and facilitates the transport of the passenger to the cell surface. In this chapter, I will review our current knowledge of the secretion of classical monomeric autotransporters and the methods that have been used to assess their folding during the translocation, both in vitro and in vivo. PMID:21557061

  18. Adrm1, a putative cell adhesion regulating protein, is a novel proteasome-associated factor.

    PubMed

    Jrgensen, Jakob Ploug; Lauridsen, Anne-Marie; Kristensen, Poul; Dissing, Karen; Johnsen, Anders H; Hendil, Klavs B; Hartmann-Petersen, Rasmus

    2006-07-28

    We have identified Adrm1 as a novel component of the regulatory ATPase complex of the 26 S proteasome: Adrm1 was precipitated with an antibody to proteasomes and vice versa. Adrm1 co-migrated with proteasomes on gel-filtration chromatography and non-denaturing polyacrylamide gel electrophoresis. Adrm1 has been described as an interferon-gamma-inducible, heavily glycosylated membrane protein of 110 kDa. However, we found Adrm1 in mouse tissues only as a 42 kDa peptide, corresponding to the mass of the non-glycosylated peptide chain, and it could not be induced in HeLa cells with interferon. Adrm1 was present almost exclusively in soluble 26 S proteasomes, albeit a small fraction was membrane-associated, like proteasomes. Adrm1 was found in cells in amounts equimolar with S6a, a 26 S proteasome subunit. HeLa cells contain no pool of free Adrm1 but recombinant Adrm1 could bind to pre-existing 26 S proteasomes in cell extracts. Adrm1 may be distantly related to the yeast proteasome subunit Rpn13, mutants of which are reported to display no obvious phenotype. Accordingly, knock-down of Adrm1 in HeLa cells had no effect on the amount of proteasomes, or on degradation of bulk cell protein, or accumulation of polyubiquitinylated proteins. This indicates that Adrm1 has a specialised role in proteasome function. PMID:16815440

  19. In-depth characterization of the secretome of colorectal cancer metastatic cells identifies key proteins in cell adhesion, migration, and invasion.

    PubMed

    Barderas, Rodrigo; Mendes, Marta; Torres, Sofia; Bartolomé, Rubén A; López-Lucendo, María; Villar-Vázquez, Roi; Peláez-García, Alberto; Fuente, Eduardo; Bonilla, Félix; Casal, J Ignacio

    2013-06-01

    Liver metastasis in colorectal cancer is the major cause of cancer-related deaths. To identify and characterize proteins associated with colon cancer metastasis, we have compared the conditioned serum-free medium of highly metastatic KM12SM colorectal cancer cells with the parental, poorly metastatic KM12C cells using quantitative stable isotope labeling by amino acids in cell culture (SILAC) analyses on a linear ion trap-Orbitrap Velos mass spectrometer. In total, 1337 proteins were simultaneously identified in SILAC forward and reverse experiments. For quantification, 1098 proteins were selected in both experiments, with 155 proteins showing >1.5-fold change. About 52% of these proteins were secreted directly or using alternative secretion pathways. GDF15, S100A8/A9, and SERPINI1 showed capacity to discriminate cancer serum samples from healthy controls using ELISAs. In silico analyses of deregulated proteins in the secretome of metastatic cells showed a major abundance of proteins involved in cell adhesion, migration, and invasion. To characterize the tumorigenic and metastatic properties of some top up- and down-regulated proteins, we used siRNA silencing and antibody blocking. Knockdown expression of NEO1, SERPINI1, and PODXL showed a significant effect on cellular adhesion. Silencing or blocking experiments with SOSTDC1, CTSS, EFNA3, CD137L/TNFSF9, ZG16B, and Midkine caused a significant decrease in migration and invasion of highly metastatic cells. In addition, silencing of SOSTDC1, EFNA3, and CD137L/TNFSF9 reduced liver colonization capacity of KM12SM cells. Finally, the panel of six proteins involved in invasion showed association with poor prognosis and overall survival after dataset analysis of gene alterations. In summary, we have defined a collection of proteins that are relevant for understanding the mechanisms underlying adhesion, migration, invasion, and metastasis in colorectal cancer. PMID:23443137

  20. 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.

  1. Myotactin, a Novel Hypodermal Protein Involved in Muscle–Cell Adhesion inCaenorhabditis elegans

    PubMed Central

    Hresko, Michelle Coutu; Schriefer, Lawrence A.; Shrimankar, Paresh; Waterston, Robert H.

    1999-01-01

    In C. elegans, assembly of hypodermal hemidesmosome-like structures called fibrous organelles is temporally and spatially coordinated with the assembly of the muscle contractile apparatus, suggesting that signals are exchanged between these cell types to position fibrous organelles correctly. Myotactin, a protein recognized by monoclonal antibody MH46, is a candidate for such a signaling molecule. The antigen, although expressed by hypodermis, first reflects the pattern of muscle elements and only later reflects the pattern of fibrous organelles. Confocal microscopy shows that in adult worms myotactin and fibrous organelles show coincident localization. Further, cell ablation studies show the bodywall muscle cells are necessary for normal myotactin distribution. To investigate myotactin's role in muscle-hypodermal signaling, we characterized the myotactin locus molecularly and genetically. Myotactin is a novel transmembrane protein of ∼500 kd. The extracellular domain contains at least 32 fibronectin type III repeats and the cytoplasmic domain contains unique sequence. In mutants lacking myotactin, muscle cells detach when embryonic muscle contraction begins. Later in development, fibrous organelles become delocalized and are not restricted to regions of the hypodermis previously contacted by muscle. These results suggest myotactin helps maintain the association between the muscle contractile apparatus and hypodermal fibrous organelles. PMID:10444073

  2. Influence of poly(ethylene oxide)-based copolymer on protein adsorption and bacterial adhesion on stainless steel: modulation by surface hydrophobicity.

    PubMed

    Yang, Yi; Rouxhet, Paul G; Chudziak, Dorota; Telegdi, Judit; Dupont-Gillain, Christine C

    2014-06-01

    The aim of the present work is to study the adhesion of Pseudomonas NCIMB 2021, a typical aerobic marine microorganism, on stainless steel (SS) substrate. More particularly, the potential effect on adhesion of adsorbed poly(ethylene oxide)-poly(propylene oxide)-poly(ethylene oxide) (PEO-PPO-PEO) triblock copolymer is investigated. Bacterial attachment experiments were carried out using a modified parallel plate flow chamber, allowing different surface treatments to be compared in a single experiment. The amount of adhering bacteria was determined via DAPI staining and fluorescence microscopy. X-ray photoelectron spectroscopy (XPS) was used to characterize the surface chemical composition of SS and hydrophobized SS before and after PEO-PPO-PEO adsorption. The adsorption of bovine serum albumin (BSA), a model protein, was investigated to test the resistance of PEO-PPO-PEO layers to protein adsorption. The results show that BSA adsorption and Pseudomonas 2021 adhesion are significantly reduced on hydrophobized SS conditioned with PEO-PPO-PEO. Although PEO-PPO-PEO is also found to adsorb on SS, it does not prevent BSA adsorption nor bacterial adhesion, which is attributed to different PEO-PPO-PEO adlayer structures on hydrophobic and hydrophilic surfaces. The obtained results open the way to a new strategy to reduce biofouling on metal oxide surfaces using PEO-PPO-PEO triblock copolymer. PMID:24650936

  3. Protein N-glycosylation in oral cancer: Dysregulated cellular networks among DPAGT1, E-cadherin adhesion and canonical Wnt signaling

    PubMed Central

    Varelas, Xaralabos; Bouchie, Meghan P; Kukuruzinska, Maria A

    2014-01-01

    N-Linked glycosylation (N-glycosylation) of proteins has long been associated with oncogenesis, but not until recently have the molecular mechanisms underlying this relationship begun to be unraveled. Here, we review studies describing how dysregulation of the N-glycosylation-regulating gene, DPAGT1, drives oral cancer. DPAGT1 encodes the first and rate-limiting enzyme in the assembly of the lipid-linked oligosaccharide precursor in the endoplasmic reticulum and thus mediates N-glycosylation of many cancer-related proteins. DPAGT1 controls N-glycosylation of E-cadherin, the major epithelial cell–cell adhesion receptor and a tumor suppressor, thereby affecting intercellular adhesion and cytoskeletal dynamics. DPAGT1 also regulates and is regulated by Wnt/β-catenin signaling, impacting the balance between proliferation and adhesion in homeostatic tissues. Thus, aberrant induction of DPAGT1 promotes a positive feedback network with Wnt/β-catenin that represses E-cadherin-based adhesion and drives tumorigenic phenotypes. Further, modification of receptor tyrosine kinases (RTKs) with N-glycans is known to control their surface presentation via the galectin lattice, and thus increased DPAGT1 expression likely contributes to abnormal activation of RTKs in oral cancer. Collectively, these studies suggest that dysregulation of the DPAGT1/Wnt/E-cadherin network underlies the etiology and pathogenesis of oral cancer. PMID:24742667

  4. A Comparative Study of Serum Level of Vascular Cell Adhesion Molecule-1 (sVCAM-1), Intercellular Adhesion Molecule-1(ICAM-1) and High Sensitive C - reactive protein (hs-CRP) in Normal and Pre-eclamptic Pregnancies

    PubMed Central

    Farzadnia, Mehdi; Ayatollahi, Hossein; Hasan-zade, Maliheh; Rahimi, Hamid Reza

    2013-01-01

    Objective(s): Pre-eclampsia is characterized by hypertension, dyslipidemia, and increased systemic inflammatory response and has been associated with an increased maternal risk of cardiovascular disease later in life. Endothelial dysfunction is thought to be a central pathogenic feature in pre-eclampsia on the basis of elevated adhesion molecules. The aim of this study was to determine the level of plasma serum level of vascular cell adhesion molecule-1 (sVCAM-1), intercellular adhesion molecule-1(ICAM-1), high sensitive C- reactive protein (hs-CRP) in pre-eclampsia and to compare hs-CRP levels between normal pregnant women, mild and severe pre-eclampsia. Materials and Methods : A cross-sectional study was conducted to determine the plasma concentrations of sVCAM-1, ICAM-1 and hs-CRP in peripheral blood obtained from normal pregnant women (n=40), mild pre-eclampsia (n=37) and severe pre-eclampsia (n=38). Concentrations of soluble adhesion molecule was determined with enzyme linked immunosorbent assay (ELISA). Results: There were significant difference in the means serum hs-CRP between normal pregnant women and mild pre-eclamptic women (P<0.05). Serum concentration of hs-CRP, sVCAM-1(ng.ml) and sICAM-1(ng.ml) were significantly higher in severe pre-eclampsia (P<0.05) than normal pregnancy. There were also significant differences in hs-CRP, s ICAM- 1 and in sVCAM- 1 levels between mild and severe pre-eclampsia (P<0.05). There was no difference in the mean plasma log sVCAM-1, sICAM-1 between normal pregnant women and mild pre-eclamptic women. Conclusion: We have determined the serum concentration of soluble adhesion molecule ICAM-1, VCAM-1 and hsCRP in normal pregnancy and pre-eclampsia. Adhesion molecule is elevated in severe pre-eclampsia compared with normal pregnancy, hsCRP are elevated in severe preeclampsia compared with mild preeclampsia and normal pregnancy and may be useful in predicting the severity of pre-eclampsia. PMID:23826490

  5. 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

  6. 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

  7. 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.

  8. A family of ROP proteins that suppresses actin dynamics, and is essential for polarized growth and cell adhesion.

    PubMed

    Burkart, Graham M; Baskin, Tobias I; Bezanilla, Magdalena

    2015-07-15

    In plants, the ROP family of small GTPases has been implicated in the polarized growth of tip-growing cells, such as root hairs and pollen tubes; however, most of the data derive from overexpressing ROP genes or constitutively active and dominant-negative isoforms, whereas confirmation by using loss-of-function studies has generally been lacking. Here, in the model moss Physcomitrella patens, we study ROP signaling during tip growth by using a loss-of-function approach based on RNA interference (RNAi) to silence the entire moss ROP family. We find that plants with reduced expression of ROP genes, in addition to failing to initiate tip growth, have perturbed cell wall staining, reduced cell adhesion and have increased actin-filament dynamics. Although plants subjected to RNAi against the ROP family also have reduced microtubule dynamics, this reduction is not specific to loss of ROP genes, as it occurs when actin function is compromised chemically or genetically. Our data suggest that ROP proteins polarize the actin cytoskeleton by suppressing actin-filament dynamics, leading to an increase in actin filaments at the site of polarized secretion. PMID:26045445

  9. 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

  10. A hybrid total internal reflection fluorescence and optical tweezers microscope to study cell adhesion and membrane protein dynamics of single living cells.

    PubMed

    Snijder-Van As, M I; Rieger, B; Joosten, B; Subramaniam, V; Figdor, C G; Kanger, J S

    2009-01-01

    The dynamics of cell surface membrane proteins plays an important role in cell-cell interactions. The onset of the interaction is typically not precisely controlled by current techniques, making especially difficult the visualization of early-stage dynamics. We have developed a novel method where optical tweezers are used to trap cells and precisely control in space and time the initiation of interactions between a cell and a functionalized surface. This approach is combined with total internal reflection fluorescence microscopy to monitor dynamics of membrane bound proteins. We demonstrate an accuracy of approximately 2 s in determining the onset of the interaction. Furthermore, we developed a data analysis method to determine the dynamics of cell adhesion and the organization of membrane molecules at the contact area. We demonstrate and validate this approach by studying the dynamics of the green fluorescent protein tagged membrane protein activated leukocyte cell adhesion molecule expressed in K562 cells upon interaction with its ligand CD6 immobilized on a coated substrate. The measured cell spreading is in excellent agreement with existing theoretical models. Active redistribution of activated leukocyte cell adhesion molecule is observed from a clustered to a more homogenous distribution upon contact initiation. This redistribution follows exponential decay behaviour with a characteristic time of 35 s. PMID:19196415

  11. 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.

  12. T cells respond to heat shock protein 60 via TLR2: activation of adhesion and inhibition of chemokine receptors.

    PubMed

    Zanin-Zhorov, Alexandra; Nussbaum, Gabriel; Franitza, Susanne; Cohen, Irun R; Lider, Ofer

    2003-08-01

    Soluble 60 kDa heat shock protein (HSP60) activates macrophages via TLR4. We now report that soluble HSP60 activates T cells via the innate receptor TLR2. HSP60 activated T cell adhesion to fibronectin to a degree similar to other activators: IL-2, SDF-1alpha, and RANTES. T cell type and state of activation was important; nonactivated CD45RA+ and IL-2-activated CD45RO+ T cells responded optimally (1 h) at low concentrations (0.1-1 ng/ml), but nonactivated CD45RO+ T cells required higher concentrations (approximately 1 microg/ml) of HSP60. T cell HSP60 signaling was inhibited specifically by monoclonal antibodies (mAb) to TLR2 but not by a mAb to TLR4. Indeed, T cells from mice with mutated TLR4 could still respond to HSP60, whereas Chinese hamster T cells with mutated TLR2 did not respond. The human T cell response to soluble HSP60 depended on phosphatidylinositol 3-kinase and protein kinase C signaling and involved the phosphorylation of Pyk-2. Soluble HSP60 also inhibited actin polymerization and T cell chemotaxis through extracellular matrix-like gels toward the chemokines SDF-1alpha (CXCL12) or ELC (CCL19). Exposure to HSP60 for longer times (18 h) down-regulated chemokine receptor expression: CXCR4 and CCR7. These results suggest that soluble HSP60, through TLR2-dependent interactions, can regulate T cell behavior in inflammation. PMID:12824285

  13. Structural and adhesive properties of the long polar fimbriae protein LpfD from adherent-invasive Escherichia coli.

    PubMed

    Coppens, Fanny; Iyyathurai, Jegan; Ruer, Sgolne; Fioravanti, Antonella; Taganna, Joemar; Vereecke, Lars; De Greve, Henri; Remaut, Han

    2015-08-01

    Crohn's disease (CD) is an inflammatory bowel disease characterized by an exaggerated immune response to commensal microbiota in the intestines of patients. Metagenomic studies have identified specific bacterial species and strains with increased prevalence in CD patients, amongst which is the adherent-invasive Escherichia coli (AIEC) strain LF82. AIEC strains express long polar fimbriae (LPF), which are known to target Peyer's patches in a mouse CD model. Here, the recombinant production of a soluble, self-complemented construct of the LpfD protein of E. coli LF82 is reported and it is demonstrated that it forms the adhesive tip subunit of LPF. The LpfD crystal reveals an N-terminal adhesin domain and a C-terminal pilin domain that connects the adhesin to the minor pilus subunit LpfE. Surface topology and sequence conservation in the adhesin domain hint at a putative receptor-binding pocket as found in the Klebsiella pneumoniae MrkD and E. coli F17-G (GafD) adhesins. Immunohistostaining of murine intestinal tissue sections revealed that LpfD specifically binds to the intestinal mucosa and submucosa. LpfD binding was found to be resistant to treatment with O- or N-glycosidases, but was lost in collagenase-treated tissue sections, indicating the possible involvement of an intestinal matrix-associated protein as the LpfD receptor. LpfD strongly adhered to isolated fibronectin in an in vitro assay, and showed lower levels of binding to collagen V and laminin and no binding to collagens I, III and IV. PMID:26249343

  14. Neurite outgrowth triggered by the cell adhesion molecule L1 requires activation and inactivation of the cytoskeletal protein cofilin.

    PubMed

    Figge, Carina; Loers, Gabriele; Schachner, Melitta; Tilling, Thomas

    2012-02-01

    Neurite outgrowth, an essential process for constructing nervous system connectivity, requires molecular cues which promote neurite extension and guide growing neurites. The neural cell adhesion molecule L1 is one of the molecules involved in this process. Growth of neurites depends on actin remodeling, but actin-remodeling proteins which act downstream of L1 signaling are not known. In this study, we investigated whether the actin-remodeling protein cofilin, which can be activated by dephosphorylation, is involved in neurite outgrowth stimulated by L1. Upon stimulation with an L1 monoclonal antibody which specifically triggers L1-dependent neurite outgrowth, cofilin phosphorylation in cultured cerebellar granule neurons and isolated growth cones was reduced to 47 13% or 58 9% of IgG control levels, respectively. We therefore investigated whether cofilin phosphorylation plays a role in L1-stimulated neurite outgrowth. Inhibition of calcineurin, a phosphatase acting upstream of cofilin dephosphorylation, impaired L1-dependent neurite extension in cultures of cerebellar granule neurons and led to an increase in cofilin phosphorylation. Moreover, when peptide S3, a competitive inhibitor of cofilin phosphorylation, or peptide pS3, a competitive inhibitor of cofilin dephosphorylation, were transferred into cerebellar neurons in culture, L1-stimulated neurite outgrowth was reduced from 173 15% to 103 4% of poly-L-lysine control levels in the presence of either peptide. Our findings suggest that both activation of cofilin by dephosphorylation and inactivation of cofilin by phosphorylation are essential for L1-stimulated neurite outgrowth. These results are in accordance with a cofilin activity cycle recently proposed for invasive tumor cells and inflammatory cells, indicating that a similar regulatory mechanism might be involved in neurite outgrowth. As L1 is expressed by invasive tumor cells, cofilin might also be a downstream actor of L1 in metastasis. PMID:22019611

  15. 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.

  16. Mechanism of a cytosolic O-glycosyltransferase essential for the synthesis of a bacterial adhesion protein.

    PubMed

    Chen, Yu; Seepersaud, Ravin; Bensing, Barbara A; Sullam, Paul M; Rapoport, Tom A

    2016-03-01

    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

  17. 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

  18. Lactobacillus Adhesion to Mucus

    PubMed Central

    Tassell, Maxwell L. Van; Miller, Michael J.

    2011-01-01

    Mucus provides protective functions in the gastrointestinal tract and plays an important role in the adhesion of microorganisms to host surfaces. Mucin glycoproteins polymerize, forming a framework to which certain microbial populations can adhere, including probiotic Lactobacillus species. Numerous mechanisms for adhesion to mucus have been discovered in lactobacilli, including partially characterized mucus binding proteins. These mechanisms vary in importance with the in vitro models studied, which could significantly affect the perceived probiotic potential of the organisms. Understanding the nature of mucus-microbe interactions could be the key to elucidating the mechanisms of probiotic adhesion within the host. PMID:22254114

  19. TgM2AP participates in Toxoplasma gondii invasion of host cells and is tightly associated with the adhesive protein TgMIC2.

    PubMed

    Rabenau, K E; Sohrabi, A; Tripathy, A; Reitter, C; Ajioka, J W; Tomley, F M; Carruthers, V B

    2001-08-01

    Like other members of the medically important phylum Apicomplexa, Toxoplasma gondii is an obligate intracellular parasite that secretes several classes of proteins involved in the active invasion of target host cells. Proteins in apical secretory organelles known as micronemes have been strongly implicated in parasite attachment to host cells. TgMIC2 is a microneme protein with multiple adhesive domains that bind target cells and is mobilized onto the parasite surface during parasite attachment. Here, we describe a novel parasite protein, TgM2AP, which is physically associated with TgMIC2. TgM2AP complexes with TgMIC2 within 15 min of synthesis and remains associated with TgMIC2 in the micronemes, on the parasite surface during invasion and in the culture medium after release from the parasite plasma membrane. TgM2AP is proteolytically processed initially when its propeptide is removed during transit through the golgi and later while it occupies the parasite surface after discharge from the micronemes. We show that TgM2AP is a member of a protein family expressed by coccidian parasites including Neospora caninum and Eimeria tenella. This phylogenic conservation and association with a key adhesive protein suggest that TgM2AP is a fundamental component of the T. gondii invasion machinery. PMID:11532123

  20. 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

  1. 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

  2. Enhanced protein adsorption and cellular adhesion using transparent titanate nanotube thin films made by a simple and inexpensive room temperature process: application to optical biochips.

    PubMed

    Nador, Judit; Orgovan, Norbert; Fried, Miklos; Petrik, Peter; Sulyok, Attila; Ramsden, Jeremy J; Korosi, Laszlo; Horvath, Robert

    2014-10-01

    A new type of titanate nanotube (TNT) coating is investigated for exploitation in biosensor applications. The TNT layers were prepared from stable but additive-free sols without applying any binding compounds. The simple, fast spin-coating process was carried out at room temperature, and resulted in well-formed films around 10nm thick. The films are highly transparent as expected from their nanostructure and may, therefore, be useful as coatings for surface-sensitive optical biosensors to enhance the specific surface area. In addition, these novel coatings could be applied to medical implant surfaces to control cellular adhesion. Their morphology and structure was characterized by spectroscopic ellipsometry (SE) and atomic force microscopy (AFM), and their chemical state by X-ray photoelectron spectroscopy (XPS). For quantitative surface adhesion studies, the films were prepared on optical waveguides. The coated waveguides were shown to still guide light; thus, their sensing capability remains. Protein adsorption and cell adhesion studies on the titanate nanotube films and on smooth control surfaces revealed that the nanostructured titanate enhanced the adsorption of albumin; furthermore, the coatings considerably enhanced the adhesion of living mammalian cells (human embryonic kidney and preosteoblast). PMID:25092586

  3. Serum Vascular Adhesion Protein-1 Predicts 10-Year Cardiovascular and Cancer Mortality in Individuals With Type 2 Diabetes

    PubMed Central

    Li, Hung-Yuan; Jiang, Yi-Der; Chang, Tien-Jyun; Wei, Jung-Nan; Lin, Mao-Shin; Lin, Cheng-Hsin; Chiang, Fu-Tien; Shih, Shyang-Rong; Hung, Chi Sheng; Hua, Cyue-Huei; Smith, David J.; Vanio, Jani; Chuang, Lee-Ming

    2011-01-01

    OBJECTIVE Vascular adhesion protein-1 (VAP-1) participates in inflammation and catalyzes the breakdown of amines to produce aldehyde, hydrogen peroxide, and ammonia. Serum VAP-1 correlates positively with both acute hyperglycemia and diabetes. We conducted a cohort study to evaluate whether serum VAP-1 predicts 10-year survival in type 2 diabetic patients. RESEARCH DESIGN AND METHODS Between July 1996 and June 2003, we enrolled 661 type 2 diabetic subjects at National Taiwan University Hospital. Serum VAP-1 in the samples obtained at enrollment was measured by time-resolved immunofluorometric assay. The vital status of all subjects was ascertained by linking their data with computerized death certificates in Taiwan. RESULTS The medium follow-up period was 10.4 years. Subjects with serum VAP-1 in the highest tertile had a hazard ratio (HR) of 2.19 (95% CI 1.174.11) for all-cause mortality adjusted for age, sex, smoking, history of cardiovascular disease, obesity, hypertension, hemoglobin A1c, diabetes duration, total cholesterol, use of statins, abnormal ankle-brachial index, estimated glomerular filtration rate (eGFR), and proteinuria. The adjusted HRs for logarithmically transformed serum VAP-1 were 5.83 (95% CI 1.1728.97) for cardiovascular mortality, 6.32 (95% CI 1.2532.00) for mortality from cardiovascular and diabetic causes, and 17.24 (95% CI 4.5765.07) for cancer mortality. There were four variables, including age, serum VAP-1, proteinuria, and eGFR, which could enhance mortality prediction significantly. CONCLUSIONS Serum VAP-1 can predict 10-year all-cause mortality, cardiovascular mortality, and cancer mortality independently in type 2 diabetic subjects. Serum VAP-1 is a novel biomarker that improves risk prediction over and above established risk factors. PMID:21282368

  4. 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-like cells while reducing bacterial adhesion to create a bioactive surface with potential for orthopaedic applications. PMID:26449451

  5. Rbt1 Protein Domains Analysis in Candida albicans Brings Insights into Hyphal Surface Modifications and Rbt1 Potential Role during Adhesion and Biofilm Formation

    PubMed Central

    Da Costa, Grgory; Chauvel, Muriel; Sautour, Marc; Bougnoux, Marie-Elisabeth; Bellon-Fontaine, Marie-Nolle; Dalle, Frdric; dEnfert, Christophe; Richard, Mathias L.

    2013-01-01

    Cell wall proteins are central to the virulence of Candida albicans. Hwp1, Hwp2 and Rbt1 form a family of hypha-associated cell surface proteins. Hwp1 and Hwp2 have been involved in adhesion and other virulence traits but Rbt1 is still poorly characterized. To assess the role of Rbt1 in the interaction of C. albicans with biotic and abiotic surfaces independently of its morphological state, heterologous expression and promoter swap strategies were applied. The N-terminal domain with features typical of the Flo11 superfamily was found to be essential for adhesiveness to polystyrene through an increase in cell surface hydrophobicity. A 42 amino acid-long domain localized in the central part of the protein was shown to enhance the aggregation function. We demonstrated that a VTTGVVVVT motif within the 42 amino acid domain displayed a high ?-aggregation potential and was responsible for cell-to-cell interactions by promoting the aggregation of hyphae. Finally, we showed through constitutive expression that while Rbt1 was directly accessible to antibodies in hyphae, it was not so in yeast. Similar results were obtained for another cell wall protein, namely Iff8, and suggested that modification of the cell wall structure between yeast and hyphae can regulate the extracellular accessibility of cell wall proteins independently of gene regulation. PMID:24349274

  6. Investigating the effects of bone cement, cyanoacrylate glue and marine mussel adhesive protein from Mytilus edulis on human osteoblasts and fibroblasts in vitro.

    PubMed

    Benthien, J P; Russlies, M; Behrens, P

    2004-12-01

    Bone cement is a widely used standard fixation substance in Orthopaedic Surgery. Cyanoacrylate glue is available for wound closure to supplement suturing. The mussel adhesive protein extracted from Mytilus edulis (Cell-Tak, BD Biosciences, Heidelberg, Germany) is an experimental fixation device used for in vitro purposes of cell adhesion. The aim of this study is to introduce a cell culture model investigating the effects of commonly applied and experimental glues on human fibroblasts and osteoblasts in vitro. Cells cultured without additives served as a control group. Microscopic examination was performed to evaluate the morphologic changes. An apoptosis test (Apo-Tag, Chemicon International, Temecula, CA, U. S. A.) was applied to determine the rate of natural cell death at the end of the study. It could be demonstrated that morphological changes in bone cement are different in fibroblasts and osteoblasts. Osteoblasts seem to grow on bone cement and develop an orderly formation. Fibroblasts grow in a confluent monolayer around bone cement but do not adhere to the cement itself. This is a desirable effect since most Orthopaedic applications aim at osteointegration as opposed to fibrous tissue overgrowth. Apoptosis attributed to bone cement is comparable to the respective natural rate of apoptosis. Cyanoacrylate glue and the mussel adhesive protein lead to an almost complete apoptosis in the investigated cells. Their routine application should be avoided. The developed cell culture model seems appropriate for performing further investigations. PMID:15646292

  7. 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.

  8. 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 (?) of approximately 16 min, whereas 4.1B and MPP3 are fitted to a double exponential function with two ?s of approximately 40-60 sec and 16 min. The longer ? 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

  9. 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 focal adhesion kinase activity. • Shb is critical for the long-term maintenance of the hematopoietic stem cell pool.

  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 and is a useful biomarker to improve risk stratification in type 2 diabetic subjects. PMID:26845338

  11. Bridging Adhesion of a Protein onto an Inorganic Surface Using Self-Assembled Dual-Functionalized Spheres.

    PubMed

    Sato, Sota; Ikemi, Masatoshi; Kikuchi, Takashi; Matsumura, Sachiko; Shiba, Kiyotaka; Fujita, Makoto

    2015-10-14

    For the bridging adhesion of different classes of materials in their intact functional states, the adhesion of biomolecules onto inorganic surfaces is a necessity. A new molecular design strategy for bridging adhesion was demonstrated by the introduction of two independent recognition groups on the periphery of spherical complexes self-assembled from metal ions (M) and bidentate ligands (L). These dual-functionalized M12L24 spheres were quantitatively synthesized in one step from two ligands, bearing either a biotin for streptavidin recognition or a titania-binding aptamer, and Pd(II) ions. The selective recognition of titania surfaces was achieved by ligands with hexapeptide aptamers (Arg-Lys-Leu-Pro-Asp-Ala: minTBP-1), whose fixation ability was enhanced by the accumulation effect on the surface of the M12L24 spheres. These well-defined spherical structures can be specifically tailored to promote interactions with both titania and streptavidin simultaneously without detrimentally affecting either recognition motif. The irreversible immobilization of the spheres onto titania was revealed quantitatively by quartz crystal microbalance measurements, and the adhesion of streptavidin to the titania surface mediated by the biotin surrounding the spheres was visually demonstrated by lithographic patterning experiments. PMID:26190770

  12. Evaluation of adhesion force between functionalized microbeads and protein-coated stainless steel using shear-flow-induced detachment.

    PubMed

    Mercier-Bonin, Muriel; Adoue, Mathieu; Zanna, Sandrine; Marcus, Philippe; Combes, Didier; Schmitz, Philippe

    2009-10-01

    Spherical microbeads functionalized with two types of chemical groups (NH(2), OH) were chosen as a simplified bacterial model, in order to elucidate the role of macromolecular interactions between specific biopolymers and 316 L stainless steel, in the frame of biofilm formation in the marine environment. NH(2) microbeads were used in their native form or after covalent binding to BSA or different representative poly-amino acids. OH microbeads were used in their native form. Adhesion force between microbeads and bare or BSA-coated stainless steel was quantified at nanoscale. Shear-flow-induced detachment experiments were combined with a simplified version of a theoretical model, based on the balance of hydrodynamic forces and torque exerted on microbeads. A maximal adhesion force of 27.6+/-8.5 nN was obtained for BSA-coated NH(2) microbeads. The high reactivity of OH functional groups was assessed (adhesion force of 15.6+/-4.8 nN for large microbeads). When charge-conducting stainless steel was coated with BSA, adhesion force was significantly lower than the one estimated with the bare surface, probably due to an increase in hydrophilic surface properties or suppression of charge transfer. The mechanism for microbead detachment was established (mainly rolling). The flow chamber and the associated theoretical modelling were demonstrated to be a relevant approach to quantify nanoscale forces between interacting surfaces. PMID:19586637

  13. A functionalized poly(ethylene glycol)-based bioassay surface chemistry that facilitates bio-immobilization and inhibits non-specific protein, bacterial, and mammalian cell adhesion

    PubMed Central

    Harbers, Gregory M.; Emoto, Kazunori; Greef, Charles; Metzger, Steven W.; Woodward, Heather N.; Mascali, James J.; Grainger, David W.; Lochhead, Michael J.

    2008-01-01

    This paper describes a new bioassay surface chemistry that effectively inhibits non-specific biomolecular and cell binding interactions, while providing a capacity for specific immobilization of desired biomolecules. Poly(ethylene glycol) (PEG) as the primary component in nonfouling film chemistry is well-established, but the multicomponent formulation described here is unique in that it (1) is applied in a single, reproducible, solution-based coating step; (2) can be applied to diverse substrate materials without the use of special primers; and (3) is readily functionalized to provide specific attachment chemistries. Surface analysis data are presented, detailing surface roughness, polymer film thickness, and film chemistry. Protein non-specific binding assays demonstrate significant inhibition of serum, fibrinogen, and lysozyme adsorption to coated glass, indium tin oxide, and tissue culture polystyrene dishes. Inhibition of S. aureus and K. pneumoniae microbial adhesion in a microfluidic flow cell, and inhibition of fibroblast cell adhesion from serum-based cell culture is shown. Effective functionalization of the coating is demonstrated by directing fibroblast adhesion to polymer surfaces activated with an RGD peptide. Batch-to-batch reproducibility data are included. The in situ cross-linked PEG-based coating chemistry is unique in its formulation, and its surface properties are attractive for a broad range of in vitro bioassay applications. PMID:18815622

  14. Baicalein suppresses 17-?-estradiol-induced migration, adhesion and invasion of breast cancer cells via the G protein-coupled receptor 30 signaling pathway.

    PubMed

    Shang, Dandan; Li, Zheng; Zhu, Zhuxia; Chen, Huamei; Zhao, Lujun; Wang, Xudong; Chen, Yan

    2015-04-01

    Flavonoids are structurally similar to steroid hormones, particularly estrogens, and therefore have been studied for their potential effects on hormone-dependent cancers. Baicalein is the primary flavonoid derived from the root of Scutellaria baicalensis Georgi. In the present study, we investigated the effects of baicalein on 17?-estradiol (E2)-induced migration, adhesion and invasion of MCF-7 and SK-BR-3 breast cancer cells. The results demonstrated that baicalein suppressed E2-stimulated wound-healing migration and cell?Matrigel adhesion, and ameliorated E2-promoted invasion across a Matrigel-coated Transwell membrane. Furthermore, baicalein interfered with E2-induced novel Gprotein-coupled estrogen receptor (GPR30)-related signaling, including a decrease in tyrosine phosphorylation of epidermal growth factor receptor (EGFR) as well as phosphorylation of extracellular signal-regulated kinase (ERK) and serine/threonine kinase Akt, without affecting GPR30 expression. The results also showed that baicalein suppressed the expression of GPR30 target genes, cysteine-rich61 (CYR61) and connective tissue growth factor (CTGF) induced by E2. Furthermore, baicalein prevented GPR30-related signaling activation and upregulation of CYR61 and CTGF mRNA levels induced by G1, a specific GPR30 agonist. The results suggest that baicalein inhibits E2-induced migration, adhesion and invasion through interfering with GPR30 signaling pathway activation, which indicates that it may act as a therapeutic candidate for the treatment of GPR30-positive breast cancer metastasis. PMID:25672442

  15. kakapo, a gene required for adhesion between and within cell layers in Drosophila, encodes a large cytoskeletal linker protein related to plectin and dystrophin.

    PubMed

    Gregory, S L; Brown, N H

    1998-11-30

    Mutations in kakapo were recovered in genetic screens designed to isolate genes required for integrin-mediated adhesion in Drosophila. We cloned the gene and found that it encodes a large protein (>5,000 amino acids) that is highly similar to plectin and BPAG1 over the first 1,000-amino acid region, and contains within this region an alpha-actinin type actin-binding domain. A central region containing dystrophin-like repeats is followed by a carboxy domain that is distinct from plectin and dystrophin, having neither the intermediate filament-binding domain of plectin nor the dystroglycan/syntrophin-binding domain of dystrophin. Instead, Kakapo has a carboxy terminus similar to the growth arrest-specific protein Gas2. Kakapo is strongly expressed late during embryogenesis at the most prominent site of position-specific integrin adhesion, the muscle attachment sites. It is concentrated at apical and basal surfaces of epidermal muscle attachment cells, at the termini of the prominent microtubule bundles, and is required in these cells for strong attachment to muscles. Kakapo is also expressed more widely at a lower level where it is essential for epidermal cell layer stability. These results suggest that the Kakapo protein forms essential links among integrins, actin, and microtubules. PMID:9832555

  16. The influence of N- and O-glycosylation inhibitors on the glycosylation profile of cellular membrane proteins and adhesive properties of carcinoma cell lines.

    PubMed

    Paszkiewicz-Gadek, Anna; Porowska, Halina; Lemancewicz, Dorota; Wolczynski, Slawomir; Gindzienski, Andrzej

    2006-04-01

    The effects of N- and O-glycosylation inhibitors on the expression of membrane proteins (MUC1 and some integrins) were evaluated in human endometrial (Ishikawa) and breast (MCF-7) cancer cells. Subconfluent cells were treated with 1-3 mg% concentration of tunicamycin and 2-10 mM of benzyl-N-acetyl-alpha-galactosaminide for 1-2 days, and used for flow cytometry, immunohistochemical staining, adhesion test and Western blotting. Benzyl-N-acetyl-alpha-galactosaminide inhibits MUC1 expression on the surface of breast more than endometrial cancer cells. Tunicamycin reduces MUC1 concentration on the cellular surface more than benzylglycoside, and greatly reduces glycosylation of glycoproteins, causing an increase in cell adhesion in both types of cancer cells. The expression of alpha2beta1 integrins on the surface of these cells was weak and decreased after treatment with inhibitors. Two different glycoforms of MUC1 proteins in endometrial cells and three in breast cancer cells were expressed and their molecular weights were reduced after treatment with glycosylation inhibitors. It was confirmed with lectin detection of carbohydrate epitopes (Tn and T) in MUC1 proteins. These observations show that glycosylation inhibitors altered the N- and O-glycan patterns in a sufficient manner, and positively modified the biological features of cancer cells. PMID:16525726

  17. The 18-kDa Translocator Protein Inhibits Vascular Cell Adhesion Molecule-1 Expression via Inhibition of Mitochondrial Reactive Oxygen Species

    PubMed Central

    Joo, Hee Kyoung; Lee, Yu Ran; Kang, Gun; Choi, Sunga; Kim, Cuk-Seong; Ryoo, Sungwoo; Park, Jin Bong; Jeon, Byeong Hwa

    2015-01-01

    Translocator protein 18 kDa (TSPO) is a mitochondrial outer membrane protein and is abundantly expressed in a variety of organ and tissues. To date, the functional role of TSPO on vascular endothelial cell activation has yet to be fully elucidated. In the present study, the phorbol 12-myristate 13-acetate (PMA, 250 nM), an activator of protein kinase C (PKC), was used to induce vascular endothelial activation. Adenoviral TSPO overexpression (10100 MOI) inhibited PMA-induced vascular cell adhesion molecule-1 (VCAM-1) and intracellular cell adhesion molecule-1 (ICAM-1) expression in a dose dependent manner. PMA-induced VCAM-1 expressions were inhibited by Mito-TEMPO (0.10.5 ?M), a specific mitochondrial antioxidants, and cyclosporin A (15 ?M), a mitochondrial permeability transition pore inhibitor, implying on an important role of mitochondrial reactive oxygen species (ROS) on the endothelial activation. Moreover, adenoviral TSPO overexpression inhibited mitochondrial ROS production and manganese superoxide dismutase expression. On contrasts, gene silencing of TSPO with siRNA increased PMA-induced VCAM-1 expression and mitochondrial ROS production. Midazolam (150 ?M), TSPO ligands, inhibited PMA-induced VCAM-1 and mitochondrial ROS production in endothelial cells. These results suggest that mitochondrial TSPO can inhibit PMA-induced endothelial inflammation via suppression of VCAM-1 and mitochondrial ROS production in endothelial cells. PMID:26608360

  18. The 18-kDa Translocator Protein Inhibits Vascular Cell Adhesion Molecule-1 Expression via Inhibition of Mitochondrial Reactive Oxygen Species.

    PubMed

    Joo, Hee Kyoung; Lee, Yu Ran; Kang, Gun; Choi, Sunga; Kim, Cuk-Seong; Ryoo, Sungwoo; Park, Jin Bong; Jeon, Byeong Hwa

    2015-12-31

    Translocator protein 18 kDa (TSPO) is a mitochondrial outer membrane protein and is abundantly expressed in a variety of organ and tissues. To date, the functional role of TSPO on vascular endothelial cell activation has yet to be fully elucidated. In the present study, the phorbol 12-myristate 13-acetate (PMA, 250 nM), an activator of protein kinase C (PKC), was used to induce vascular endothelial activation. Adenoviral TSPO overexpression (10-100 MOI) inhibited PMA-induced vascular cell adhesion molecule-1 (VCAM-1) and intracellular cell adhesion molecule-1 (ICAM-1) expression in a dose dependent manner. PMA-induced VCAM-1 expressions were inhibited by Mito-TEMPO (0.1-0.5 ?M), a specific mitochondrial antioxidants, and cyclosporin A (1-5 ?M), a mitochondrial permeability transition pore inhibitor, implying on an important role of mitochondrial reactive oxygen species (ROS) on the endothelial activation. Moreover, adenoviral TSPO overexpression inhibited mitochondrial ROS production and manganese superoxide dismutase expression. On contrasts, gene silencing of TSPO with siRNA increased PMA-induced VCAM-1 expression and mitochondrial ROS production. Midazolam (1-50 ?M), TSPO ligands, inhibited PMA-induced VCAM-1 and mitochondrial ROS production in endothelial cells. These results suggest that mitochondrial TSPO can inhibit PMA-induced endothelial inflammation via suppression of VCAM-1 and mitochondrial ROS production in endothelial cells. PMID:26608360

  19. Isolation and characterization of pollen coat proteins of Brassica campestris that interact with S locus-related glycoprotein 1 involved in pollenstigma adhesion

    PubMed Central

    Takayama, Seiji; Shiba, Hiroshi; Iwano, Megumi; Asano, Kosuke; Hara, Minoru; Che, Fang-Sik; Watanabe, Masao; Hinata, Kokichi; Isogai, Akira

    2000-01-01

    Adhesion of pollen grains to the stigmatic surface is a critical step during sexual reproduction in plants. In Brassica, S locus-related glycoprotein 1 (SLR1), a stigma-specific protein belonging to the S gene family of proteins, has been shown to be involved in this step. However, the identity of the interacting counterpart in pollen and the molecular mechanism of this interaction have not been determined. Using an optical biosensor immobilized with S gene family proteins, we detected strong SLR1-binding activity in pollen coat extracts of Brassica campestris. Two SLR1-binding proteins, named SLR1-BP1 and SLR1-BP2, were identified and purified by the combination of SLR1 affinity column chromatography and reverse-phase HPLC. Sequence analyses revealed that these two proteins (i) differ only in that a proline residue near the N terminus is hydroxylated in SLR1-BP1 but not in SLR1-BP2, and (ii) are members of the class A pollen coat protein (PCP) family, which includes PCP-A1, an SLG (S locus glycoprotein)-binding protein isolated from Brassica oleracea. Kinetic analysis showed that SLR1-BP1 and SLR1-BP2 specifically bound SLR1 with high affinity (Kd = 5.6 and 4.4 nM, respectively). The SLR1-BP gene was specifically expressed in pollen at late stages of development, and its sequence is highly conserved in Brassica species with the A genome. PMID:10716697

  20. 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.

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

    PubMed Central

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

    2016-01-01

    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

  2. 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

  3. Identification of a Monoclonal Antibody Against Pneumococcal Pilus 1 Ancillary Protein Impairing Bacterial Adhesion to Human Epithelial Cells.

    PubMed

    Amerighi, Fulvia; Valeri, Maria; Donnarumma, Danilo; Maccari, Silvia; Moschioni, Monica; Taddei, Annarita; Lapazio, Lucia; Pansegrau, Werner; Buccato, Scilla; De Angelis, Gabriella; Ruggiero, Paolo; Masignani, Vega; Soriani, Marco; Pezzicoli, Alfredo

    2016-02-15

    The adhesion of Streptococcus pneumoniae is a key step during colonization of human respiratory tract mucosae. Here we demonstrate that pneumococcal type I pilus significantly increases the adhesiveness of poorly adhering highly capsulated strains in vitro. Interestingly, preincubation of bacteria with antibodies against the major pilus backbone subunit (RrgB) or the adhesin component (RrgA) impaired pneumococcal association to human epithelial cells. Screening for anti-RrgA monoclonal antibodies specifically affecting the adhesive capacity of S. pneumoniae led to the identification of the monoclonal 11B9/61 antibody, which greatly reduced pilus-dependent cell contact. Proteomic-based epitope mapping of 11B9/61 monoclonal antibody revealed a well-exposed epitope on the D2 domain of RrgA as the target of this functional antibody. The data presented here confirm the importance of pilus I for S. pneumoniae pathogenesis and the potential use of antipilus antibodies to prevent bacterial colonization. PMID:26401026

  4. ?2-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 ?2-macroglobulin (?2M), which is both required and sufficient for rosetting mediated by the PfEMP1 protein HB3VAR06 and some other rosette-mediating PfEMP1 proteins. We map the ?2M binding site to the C terminal end of HB3VAR06, and demonstrate that ?2M 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 ?2M and probably due to the more limited cross-linking potential of IgM. Nevertheless, we show that IgM works synergistically with ?2M and markedly lowers the concentration of ?2M required for rosetting. Finally, HB3VAR06+ IEs share the capacity to bind ?2M 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 ?2M (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 ?2M(and IgM-) binding domains of PfEMP1, which would be independent of the host receptor specificity of clinically important PfEMP1 antigens. PMID:26134405

  5. Cell adhesion as a novel approach to determining the cellular binding motif on the severe acute respiratory syndrome coronavirus spike protein.

    PubMed

    Chang, Hsin-Hou; Chen, Po-Kong; Lin, Guan-Ling; Wang, Chun-Jen; Liao, Chih-Hsien; Hsiao, Yu-Cheng; Dong, Jing-Hua; Sun, Der-Shan

    2014-06-01

    Emerging life threatening pathogens such as severe acute aspiratory syndrome-coronavirus (SARS-CoV), avian-origin influenzas H7N9, and the Middle East respiratory syndrome coronavirus (MERS-CoV) have caused a high case-fatality rate and psychological effects on society and the economy. Therefore, a simple, rapid, and safe method to investigate a therapeutic approach against these pathogens is required. In this study, a simple, quick, and safe cell adhesion inhibition assay was developed to determine the potential cellular binding site on the SARS-CoV spike protein. Various synthetic peptides covering the potential binding site helped to minimize further the binding motif to 10-25 residues. Following analyses, 2 peptides spanning the 436-445 and 437-461 amino acids of the spike protein were identified as peptide inhibitor or peptide vaccine candidates against SARS-CoV. PMID:24530430

  6. Identification of a new isoform of cell-cell adhesion molecule 105 (C-CAM), C-CAM4: a secretory protein with only one Ig domain.

    PubMed Central

    Earley, K; Luo, W; Qiu, Y; Thompson, N L; Chou, J; Hixson, D C; Lin, S H

    1996-01-01

    A series of Southern blot hybridization experiments using probes derived from different regions of the rat liver cell-cell adhesion molecule 105 (C-CAM) cDNA revealed the presence of a 9.6 kb EcoRI genomic fragment that seemed to encode a unique C-CAM isoform. An RNase protection study showed that this c-CAM transcript was expressed in placenta, spleen, lung and large intestine. In contrast, the other C-CAM isoforms, C-CAM1 and C-CAM2, are expressed in liver and small intestine. This result also suggests that the new isoform, which we named C-CAM4, was indeed encoded by a new C-CAM gene. A rat placenta cDNA library was then screened and the full-length cDNA coding for C-CAM4 was isolated. The deduced protein contained 142 amino acids and had a calculated molecular mass of 15 kDa. C-CAM4 was composed of a leader sequence and the first V-like Ig domain typical of C-CAM-family proteins. However, C-CAM4 lacked the C-like Ig domains, the transmembrane domain, and the cytoplasmic domain found in other C-CAM isoforms. Thus, C-CAM4 is different from the other known C-CAMs in that it is a secreted protein. We have previously shown that the first Ig domain of C-CAM1 is crucial for its adhesion function. The V-like Ig domain of C-CAM4 had 92% and 89% sequence identity with the corresponding regions of C-CAM1 and C-cam2 respectively. Together these results suggest that C-CAM4 may play a role in regulating the function of other C-CAM family proteins. PMID:8645160

  7. Identification of a new isoform of cell-cell adhesion molecule 105 (C-CAM), C-CAM4: a secretory protein with only one Ig domain.

    PubMed

    Earley, K; Luo, W; Qiu, Y; Thompson, N L; Chou, J; Hixson, D C; Lin, S H

    1996-05-01

    A series of Southern blot hybridization experiments using probes derived from different regions of the rat liver cell-cell adhesion molecule 105 (C-CAM) cDNA revealed the presence of a 9.6 kb EcoRI genomic fragment that seemed to encode a unique C-CAM isoform. An RNase protection study showed that this c-CAM transcript was expressed in placenta, spleen, lung and large intestine. In contrast, the other C-CAM isoforms, C-CAM1 and C-CAM2, are expressed in liver and small intestine. This result also suggests that the new isoform, which we named C-CAM4, was indeed encoded by a new C-CAM gene. A rat placenta cDNA library was then screened and the full-length cDNA coding for C-CAM4 was isolated. The deduced protein contained 142 amino acids and had a calculated molecular mass of 15 kDa. C-CAM4 was composed of a leader sequence and the first V-like Ig domain typical of C-CAM-family proteins. However, C-CAM4 lacked the C-like Ig domains, the transmembrane domain, and the cytoplasmic domain found in other C-CAM isoforms. Thus, C-CAM4 is different from the other known C-CAMs in that it is a secreted protein. We have previously shown that the first Ig domain of C-CAM1 is crucial for its adhesion function. The V-like Ig domain of C-CAM4 had 92% and 89% sequence identity with the corresponding regions of C-CAM1 and C-cam2 respectively. Together these results suggest that C-CAM4 may play a role in regulating the function of other C-CAM family proteins. PMID:8645160

  8. CPNA-1, a copine domain protein, is located at integrin adhesion sites and is required for myofilament stability in Caenorhabditis elegans

    PubMed Central

    Warner, Adam; Xiong, Ge; Qadota, Hiroshi; Rogalski, Teresa; Vogl, A. Wayne; Moerman, Donald G.; Benian, Guy M.

    2013-01-01

    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-1null 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

  9. Participation of heparin binding proteins from the surface of Leishmania (Viannia) braziliensis promastigotes in the adhesion of parasites to Lutzomyia longipalpis cells (Lulo) in vitro

    PubMed Central

    2012-01-01

    Background Leishmania (V.) braziliensis is a causative agent of cutaneous leishmaniasis in Brazil. During the parasite life cycle, the promastigotes adhere to the gut of sandflies, to avoid being eliminated with the dejection. The Lulo cell line, derived from Lutzomyia longipalpis (Diptera: Psychodidae), is a suitable in vitro study model to understand the features of parasite adhesion. Here, we analyze the role of glycosaminoglycans (GAGs) from Lulo cells and proteins from the parasites in this event. Methods Flagellar (Ff) and membrane (Mf) fractions from promastigotes were obtained by differential centrifugation and the purity of fractions confirmed by western blot assays, using specific antibodies for cellular compartments. Heparin-binding proteins (HBP) were isolated from both fractions using a HiTrap-Heparin column. In addition, binding of promastigotes to Lulo cells or to a heparin-coated surface was assessed by inhibition assays or surface plasmon resonance (SPR) analysis. Results The success of promastigotes subcellular fractionation led to the obtainment of Ff and Mf proteins, both of which presented two main protein bands (65.0 and 55.0kDa) with affinity to heparin. The contribution of HBPs in the adherence of promastigotes to Lulo cells was assessed through competition assays, using HS or the purified HBPs fractions. All tested samples presented a measurable inhibition rate when compared to control adhesion rate (17??2.0% of culture cells with adhered parasites): 30% (for HS 20?g/ml) and 16% (for HS 10?g/ml); HBP Mf (35.2% for 10?g/ml and 25.4% for 20?g/ml) and HBP Ff (10.0% for 10?g/ml and 31.4% for 20?g/ml). Additionally, to verify the presence of sulfated GAGs in Lulo cells surface and intracellular compartment, metabolic labeling with radioactive sulfate was performed, indicating the presence of an HS and chondroitin sulfate in both cell sections. The SPR analysis performed further confirmed the presence of GAGs ligands on L. (V.) braziliensis promastigote surfaces. Conclusions The data presented here point to evidences that HBPs present on the surface of L. (V.) braziliensis promastigotes participate in adhesion of these parasites to Lulo cells through HS participation. PMID:22805335

  10. Adhesive dynamics.

    PubMed

    Hammer, Daniel A

    2014-02-01

    Adhesive dynamics (AD) is a method for simulating the dynamic response of biological systems in response to force. Biological bonds are mechanical entities that exert force under strain, and applying forces to biological bonds modulates their rate of dissociation. Since small numbers of events usually control biological interactions, we developed a simple method for sampling probability distributions for the formation or failure of individual bonds. This method allows a simple coupling between force and strain and kinetics, while capturing the stochastic response of biological systems. Biological bonds are dynamically reconfigured in response to applied mechanical stresses, and a detailed spatio-temporal map of molecules and the forces they exert emerges from AD. The shape or motion of materials bearing the molecules is easily calculated from a mechanical energy balance provided the rheology of the material is known. AD was originally used to simulate the dynamics of adhesion of leukocytes under flow, but new advances have allowed the method to be extended to many other applications, including but not limited to the binding of viruses to surface, the clustering of adhesion molecules driven by stiff substrates, and the effect of cell-cell interaction on cell capture and rolling dynamics. The technique has also been applied to applications outside of biology. A particular exciting recent development is the combination of signaling with AD (so-called integrated signaling adhesive dynamics, or ISAD), which allows facile integration of signaling networks with mechanical models of cell adhesion and motility. Potential opportunities in applying AD are summarized. PMID:24384944

  11. Ric-8a, a guanine nucleotide exchange factor for heterotrimeric G proteins, regulates bergmann glia-basement membrane adhesion during cerebellar foliation.

    PubMed

    Ma, Shang; Kwon, Hyo Jun; Huang, Zhen

    2012-10-24

    The cerebellum consists of an intricate array of lobules that arises during the process of foliation. Foliation not only increases surface area, but may also facilitate organization of cerebellar neural circuitry. Defects in cerebellar foliation are associated with a number of diseases. Yet, little is known about how foliation, a process involving large-scale and simultaneous movement of several different cell types, is coordinated by cell-cell signaling at the molecular level. Here we show that Ric-8a, a guanine nucleotide exchange factor in the G-protein-coupled receptor pathway, is specifically required in Bergmann glia during cerebellar foliation. We find that ric-8a mutation in mice results in disorganized Bergmann glial scaffolding, defective granule cell migration, and disrupted Purkinje cell positioning. These abnormalities result from primary defects in Bergmann glia since mutations in granule cells do not show similar effects. They first arise during late embryogenesis, at the onset of foliation, when ric-8a mutant Bergmann glia fail to maintain adhesion to the basement membrane specifically at emerging fissures. This suggests that Ric-8a is essential for the enhanced Bergmann glia-basement membrane adhesion required for fissure formation. Indeed, we find that ric-8a-deficient cerebellar glia show decreased affinity for basement membrane components. We also find that weakening Bergmann glia-basement membrane interaction by β1 integrin deletion results in a similar phenotype. These results thus reveal a novel role of Ric-8a in modulating Bergmann glia-basement membrane adhesion during foliation, and provide new insights into the signaling pathways that coordinate cellular movement during cerebellar morphogenesis. PMID:23100420

  12. Monocyte chemotactic protein-1 and soluble adhesion molecules as possible prognostic markers of the efficacy of antiviral treatment in chronic hepatitis C

    PubMed Central

    Panasiuk, Anatol; Prokopowicz, Danuta; Panasiuk, Bozena

    2004-01-01

    AIM: To explain the role of Monocyte chemotactic protein-1 (MCP-1) and soluble adhesion molecules in chronic hepatitis C during the treatment of interferon alpha (IFN? ) 2 b and ribavirin (RBV). METHODS: Concentrations of MCP-1, soluble adhesion molecules intercellular adhesion molecule-1 (sICAM-1), sP-selectin, interleukin (IL) 6, and IL10 in serum were estimated in the group of 40 patients with chronic hepatitis C treated with IFNalpha2 b and RBV in 0, 16, 32, 48 wk of the therapy. RESULTS: In chronic hepatitis C, before and during the treatment, the serum levels of MCP-1 and sP-selectin in responders were similar to those of healthy subjects. In non-responders (NR), MCP-1 increased in the course of IFN? + RBV treatment, differences were statistically significant as compared to responders. MCP-1 correlated statistically with the activity of periportal inflammation (r = 0.35, P < 0.05) but not with staging of liver fibrosis. sICAM-1 positively correlated with inflammatory activity and fibrosis in NR. sP-selectin did not correlate with histological findings in the liver. The MCP-1 correlated with the soluble form of sP-selectin concentrations (r = 6, P < 0.001) and with IL-10 level in NR (r = 0.4, P < 0.05). There was no correlation observed between the concentration of MCP-1 and sICAM-1, IL-6 during the treatment. CONCLUSION: MCP-1 concentration may be a prognostic marker of the efficacy of IFN + RBV therapy in patients with chronic hepatitis C. PMID:15534921

  13. 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

  14. Intranasal immunization with a non-adjuvanted adhesive protein descended from Pasteurella pneumotropica and its preventive efficacy against opportunistic infection in mice.

    PubMed

    Sasaki, Hiraku; Ishikawa, Hiroki; Kojima, Ken; Itoh, Masahiro; Matsumoto, Tetsuya; Itoh, Takumi; Hosomi, Osamu; Kawamoto, Eiichi

    2013-11-19

    Intranasal vaccination is one of the most effective means of protecting against invading and colonizing pathogens because the vaccine elicits a mucosal immune response. The exploitation of vaccine adjuvants and delivery systems for intranasal vaccines is an important way to evoke antigen immunogenicity and elicit a better immune response at the mucosal sites. In the present study, we assessed the potential of intranasal immunization using a non-adjuvanted bacterial adhesive protein toward the host organs. We evaluated intranasal immunization with modified recombinant PnxIIIA (MP3) from Pasteurella pneumotropica and its preventive efficacy against opportunistic infection caused by P. pneumotropica, without using any adjuvants or delivery systems. The 100-kDa MP3 was confirmed to retain its immunogenicity and binding activity to collagen type I similar to the parent PnxIIIA. When MP3 was fused to green-fluorescent protein and inoculated into C57BL/6J mice intranasally, fluorescence intensity in the intranasal airway could be observed until 3 h after inoculation. Mice were intranasally immunized with MP3 at a maximum of 4 doses, with 7-day intervals. The antibody titer of serum IgG and IgA specific for MP3, as well as that of bronchoalveolar lavage fluid IgA, showed more than 9 (log₂) after 3 or 4 rounds of immunization. Experimentally infecting immunized mice with P. pneumotropica resulted in the inability to isolate the bacterium from the nasal cavity, trachea, conjunctiva, or cecum with more than 3 doses in the immunized mice. Although the detection in each organ seldom changed with less than 2 rounds of immunization, unlike that observed in the non-immunized mice, the detection remarkably decreased with 3 or more rounds of immunization. These results suggest that intranasal immunization with a non-adjuvanted adhesive protein could have preventive effects against opportunistic infection by P. pneumotropica. PMID:24091313

  15. AmpA protein functions by different mechanisms to influence early cell type specification and to modulate cell adhesion and actin polymerization in Dictyostelium discoideum

    PubMed Central

    Cost, Hoa N.; Noratel, Elizabeth F.; Blumberg, Daphne D.

    2013-01-01

    The Dictyostelium discoideum ampA gene encodes a multifunctional regulator protein that modulates cellcell and cellsubstrate adhesions and actin polymerization during growth and is necessary for correct cell type specification and patterning during development. Insertional inactivation of the ampA gene results in defects that define two distinct roles for the ampA gene during development. AmpA is necessary in a non-cell autonomous manner to prevent premature expression of a prespore gene marker. It is also necessary in a cell autonomous manner for the anterior like cells, which express the ampA gene, to migrate to the upper cup during culmination. It is also necessary to prevent excessive cellcell agglutination when cells are developed in a submerged suspension culture. Here, we demonstrate that a supernatant source of AmpA protein, added extracellularly, can prevent the premature mis-expression of the prespore marker. Synthetic oligopeptides are used to identify the domain of the AmpA protein that is important for preventing cells from mis-expressing the prespore gene. We further demonstrate that a factor capable of inducing additional cells to express the prespore gene marker accumulates extracellularly in the absence of AmpA protein. While the secreted AmpA acts extracellularly to suppress prespore gene expression, the effects of AmpA on cell agglutination and on actin polymerization in growing cells are not due to an extracellular role of secreted AmpA protein. Rather, these effects appear to reflect a distinct cell autonomous role of the ampA gene. Finally, we show that secretion of AmpA protein is brought about by elevating the levels of expression of ampA so that the protein accumulates to an excessive level. PMID:23911723

  16. 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

  17. Haloarchaeal myovirus ?Ch1 harbours a phase variation system for the production of protein variants with distinct cell surface adhesion specificities.

    PubMed

    Klein, R; Rssler, N; Iro, M; Scholz, H; Witte, A

    2012-01-01

    The ?Ch1 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 ?Ch1 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 ?-D-galactose to purified gp34 or whole virions prevented their attachment to Nab. magadii. PMID:22111759

  18. 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.

  19. 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.

  20. The Orphan Adhesion G Protein-coupled Receptor GPR97 Regulates Migration of Lymphatic Endothelial Cells via the Small GTPases RhoA and Cdc42*

    PubMed Central

    Valtcheva, Nadejda; Primorac, Adriana; Jurisic, Giorgia; Hollmn, Maija; Detmar, Michael

    2013-01-01

    The important role of the lymphatic vascular system in pathological conditions such as inflammation and cancer has been increasingly recognized, but its potential as a pharmacological target is poorly exploited. Our study aimed at the identification and molecular characterization of lymphatic-specific G protein-coupled receptors (GPCRs) to assess new targets for pharmacological manipulation of the lymphatic vascular system. We used a TaqMan quantitative RT-PCR-based low density array to determine the GPCR expression profiles of ex vivo isolated intestinal mouse lymphatic (LECs) and blood vascular endothelial cells (BECs). GPR97, an orphan adhesion GPCR of unknown function, was the most highly and specifically expressed GPCR in mouse lymphatic endothelium. Using siRNA silencing, we found that GPR97-deficient primary human LECs displayed increased adhesion and collective cell migration, whereas single cell migration was decreased as compared with nontargeting siRNA-transfected control LECs. Loss of GPR97 shifted the ratio of active Cdc42 and RhoA and initiated cytoskeletal rearrangements, including F-actin redistribution, paxillin and PAK4 phosphorylation, and ?1-integrin activation. Our data suggest a possible role of GPR97 in lymphatic remodeling and furthermore provide the first insights into the biological functions of GPR97. PMID:24178298

  1. Receptor Protein-tyrosine Phosphatase α Regulates Focal Adhesion Kinase Phosphorylation and ErbB2 Oncoprotein-mediated Mammary Epithelial Cell Motility*

    PubMed Central

    Boivin, Benoit; Chaudhary, Fauzia; Dickinson, Bryan C.; Haque, Aftabul; Pero, Stephanie C.; Chang, Christopher J.; Tonks, Nicholas K.

    2013-01-01

    We investigated the role of protein-tyrosine phosphatase α (PTPα) in regulating signaling by the ErbB2 oncoprotein in mammary epithelial cells. Using this model, we demonstrated that activation of ErbB2 led to the transient inactivation of PTPα, suggesting that attenuation of PTPα activity may contribute to enhanced ErbB2 signaling. Furthermore, RNAi-induced suppression of PTPα led to increased cell migration in an ErbB2-dependent manner. The ability of ErbB2 to increase cell motility in the absence of PTPα was characterized by prolonged interaction of GRB7 with ErbB2 and increased association of ErbB2 with a β1-integrin-rich complex, which depended on GRB7-SH2 domain interactions. Finally, suppression of PTPα resulted in increased phosphorylation of focal adhesion kinase on Tyr-407, which induced the recruitment of vinculin and the formation of a novel focal adhesion kinase complex in response to ErbB2 activation in mammary epithelial cells. Collectively, these results reveal a new role for PTPα in the regulation of motility of mammary epithelial cells in response to ErbB2 activation. PMID:24217252

  2. Adhesion dynamics: mechanisms and measurements.

    PubMed

    Worth, Daniel C; Parsons, Maddy

    2008-01-01

    Adhesion to the extracellular matrix (ECM) is a fundamental requirement for survival, differentiation and migration of numerous cell types during both embryonic development and adult homeostasis. Different types of adhesion structures have been classified in different cell types or tissue environments. The best studied of these are focal adhesions which are found on a wide variety of cell types and will be the main focus of this review. Many years of research into the control of adhesion has yielded a wealth of information regarding the complexity of protein composition of these critical points of cell:ECM contact. Moreover, it has emerged that adhesions are not only highly ordered, but also dynamic structures under tight spatial control at the subcellular level to enable localised responses to extracellular cues. However, it is only in the last decade that the relative dynamics of these adhesion proteins have been closely studied. Here we provide an overview of the imaging strategies that have been developed and implemented to study the intricacies and hierarchy of protein turnover within focal adhesions. The caveats of employing these imaging techniques, as well as future directions will also be discussed. PMID:18485788

  3. Rho-associated protein kinase inhibitor, Y-27632, significantly enhances cell adhesion and induces a delay in G1 to S phase transition in rabbit corneal endothelial cells.

    PubMed

    Diao, Yu-Mei; Hong, Jing

    2015-08-01

    Human corneal endothelial cells are a non-proliferative cell type. As a result of the increase in corneal endothelium disease, increasing numbers of studies have been conducted in order to promote corneal endothelial cell proliferation. The aim of the present study was to investigate the proliferative effects of Rho-associated protein kinase inhibitor, Y-27632, on rabbit corneal endothelial cells (rCECs). Y-27632 (1, 10 or 30 ?M) was added at two different time points to two groups of rCECs. The first group received Y-27632 when rCECs were initially plated, and the second following 72 h of cell growth. Cell morphology and cell adhesion ratios were subsequently observed using light microscopy. A cell counting kit was used to measure the number of viable cells that adhered to culture plates. Cell cycle transitions and levels of Annexin V-positive apoptotic cells were detected using flow cytometry. Cells treated with 1 ?M Y-27632 and 10 ?M Y-27632 retained their cell shape. At a concentration of 30 ?M Y-27632, the cell shape became irregular. Cell adhesion ratios, in 1 ?M Y-27632 (36.84%), 10 ?M Y-27632 (84.21%) and 30 ?M Y-27632 (84.21%) were higher than the adhesion ratio in the control group (P<0.01). The optical densities of rCECs treated with 10 ?M or 30 ?M Y-27632 following 72 h of cell growth was less than that of the control cells (P<0.01), but higher than that of cells which received Y-27632 at the time of plating (P<0.01). Flow cytometry results also demonstrated that there was a delay in G1 to S phase cell cycle progression in rCECs following administration of 10 ?M Y-27632 (P<0.01). Cell apoptosis was inhibited when 10 ?M Y-27632 was added, at the time of cell plating, as well as when added following 72 h of cell growth (P<0.01). At a concentration of 10 ?M Y-27632, there was an improvement in cell adhesion and an inhibition of the cell cycle in rabbit corneal endothelial cells. In conclusion, Y-27632 has different effects on rCECs when administered at varying concentrations and at particular stages of cell growth. PMID:25891854

  4. [Transcriptional factor Xanf1 interacts with the focal adhesion protein zyxin in the early development of the Xenopus laevis brain].

    PubMed

    Martynova, N Iu; Ermolina, L V; Eroshkin, F M; Gioeva, F K; Zara?ski?, A G

    2008-01-01

    We searched for potential protein partners of the homeodomain transcription repressor Xanf1, a key transcriptional regulator of the early development of the forebrain, using a yeast two-hybrid system and identified for the first time the LIM domain protein zyxin from the African clawed frog Xenopus laevis. This protein is interesting because it can play the role of a signal transmitter from cell receptors to the cell nucleus and, thus, participate in the regulation of cell morphogenetic movements and gene expression. The interaction of zyxin with Xanf1 was confirmed by the immunoprecipitation of a complex of endogenous zyxin with the hybrid myc-Xanf1 protein from the lysate of X. laevis embryos. Using a set of deletion mutants of both proteins, we found that binding of the LIM2 domain of zyxin with the Engrailed Homology 1 repressor domain of Xanf1 is responsible for the interaction of these proteins. PMID:18695732

  5. Novel 1H-imidazol-2-amine derivatives as potent and orally active vascular adhesion protein-1 (VAP-1) inhibitors for diabetic macular edema treatment.

    PubMed

    Inoue, Takayuki; Morita, Masataka; Tojo, Takashi; Nagashima, Akira; Moritomo, Ayako; Miyake, Hiroshi

    2013-07-01

    Novel thiazole derivatives were synthesized and evaluated as vascular adhesion protein-1 (VAP-1) inhibitors. Although we previously identified a compound (2) with potent VAP-1 inhibitory activity in rats, the human activity was relatively weak. Here, to improve the human VAP-1 inhibitory activity of compound 2, we first evaluated the structure-activity relationships of guanidine bioisosteres as simple small molecules and identified a 1H-benzimidazol-2-amine (5) with potent activity compared to phenylguanidine (1). Based on the structure of compound 5, we synthesized a highly potent VAP-1 inhibitor (37b; human IC50=0.019 ?M, rat IC50=0.0051 ?M). Orally administered compound 37b also markedly inhibited ocular permeability in streptozotocin-induced diabetic rats after oral administration, suggesting it is a promising compound for the treatment of diabetic macular edema. PMID:23664164

  6. Synthesis and SAR study of new thiazole derivatives as vascular adhesion protein-1 (VAP-1) inhibitors for the treatment of diabetic macular edema.

    PubMed

    Inoue, Takayuki; Morita, Masataka; Tojo, Takashi; Yoshihara, Kousei; Nagashima, Akira; Moritomo, Ayako; Ohkubo, Mitsuru; Miyake, Hiroshi

    2013-03-01

    Vascular adhesion protein-1 (VAP-1), an amine oxidase that is also known as a semicarbazide-sensitive amine oxidase (SSAO), is present in particularly high levels in human plasma, and is considered a potential therapeutic target for various inflammatory diseases, including diabetes complications such as macular edema. In our VAP-1 inhibitor program, structural modifications following high-throughput screening (HTS) of our compound library resulted in the discovery that thiazole derivative 10, which includes a guanidine group, shows potent human VAP-1 inhibitory activity (IC(50) of 230 nM; rat IC(50) of 14 nM). Moreover, compound 10 exhibited significant inhibitory effects on ocular permeability in STZ-induced diabetic rats. PMID:23337801

  7. Abdominal Adhesions

    MedlinePLUS

    ... found that eating, diet, and nutrition play a role in causing or preventing abdominal adhesions. A person with a partial intestinal obstruction may relieve symptoms with a liquid or low-fiber diet, which is more easily broken down into smaller ...

  8. Regulation of cell adhesion to collagen via beta1 integrins is dependent on interactions of filamin A with vimentin and protein kinase C epsilon.

    PubMed

    Kim, Hugh; Nakamura, Fumihiko; Lee, Wilson; Hong, Claire; Prez-Sala, Dolores; McCulloch, Christopher A

    2010-07-01

    Cell adhesion and spreading on collagen, which are essential processes for development and wound healing in mammals, are mediated by beta1 integrins and the actin and intermediate filament cytoskeletons. The mechanisms by which these separate cytoskeletal systems interact to regulate beta1 integrins and cell spreading are poorly defined. We previously reported that the actin cross-linking protein filamin A binds the intermediate filament protein vimentin and that these two proteins co-regulate cell spreading. Here we used deletional mutants of filamin A to define filamin A-vimentin interactions and the subsequent phosphorylation and re-distribution of vimentin during cell spreading on collagen. Imaging of fixed and live cell preparations showed that phosphorylated vimentin is translocated to the cell membrane during spreading. Knockdown of filamin A inhibited cell spreading and the phosphorylation and re-distribution of vimentin. Knockdown of filamin A and/or vimentin reduced the cell surface expression and activation of beta1 integrins, as indicated by immunoblotting of plasma membrane-associated proteins and shear force assays. In vitro pull-down assays using filamin A mutants showed that both vimentin and protein kinase Cvarepsilon bind to repeats 1-8 of filamin A. Reconstitution of filamin-A-deficient cells with full-length filamin A or filamin A repeats 1-8 restored cell spreading, vimentin phosphorylation, and the cell surface expression of beta1 integrins. We conclude that the binding of filamin A to vimentin and protein kinase Cepsilon is an essential regulatory step for the trafficking and activation of beta1 integrins and cell spreading on collagen. PMID:20171211

  9. West Nile virus infection modulates human brain microvascular endothelial cells tight junction proteins and cell adhesion molecules: Transmigration across the in vitro blood-brain barrier

    PubMed Central

    Verma, Saguna; Lo, Yeung; Chapagain, Moti; Lum, Stephanie; Kumar, Mukesh; Gurjav, Ulziijargal; Luo, Haiyan; Nakatsuka, Austin; Nerurkar, Vivek R.

    2009-01-01

    Neurological complications such as inflammation, failure of the blood-brain barrier (BBB), and neuronal death contribute to the mortality and morbidity associated with WNV-induced meningitis. Compromised BBB indicates the ability of the virus to gain entry into the CNS via the BBB, however, the underlying mechanisms, and the specific cell types associated with WNV-CNS trafficking are not well understood. Brain microvascular endothelial cells, main component of the BBB, represent a barrier to virus dissemination into the CNS and could play key role in WNV spread via hematogenous route. To investigate WNV entry into the CNS, we infected primary human brain microvascular endothelial (HBMVE) cells with the neurovirulent strain of WNV (NY99) and examined WNV replication kinetics together with the changes in the expressions of key tight junction proteins (TJP) and cell adhesion molecules (CAM). WNV infection of HBMVE cells was productive as analyzed by plaque assay and qRT-PCR, and did not induce cytopathic effect. Increased mRNA and protein expressions of TJP (claudin-1) and CAM (vascular cell adhesion molecule and E-selectin) were observed at days 2 and 3 after infection, respectively, which coincided with the peak in WNV replication. Further, using an in vitro BBB model comprised of HBMVE cells, we demonstrate that cell-free WNV can cross the BBB, without compromising the BBB integrity. These data suggest that infection of HBMVE cells can facilitate entry of cell-free virus into the CNS without disturbing the BBB, and increased CAM may assist in the trafficking of WNV-infected immune cells into the CNS, via Trojan horse mechanism, thereby contributing to WNV dissemination in the CNS and associated pathology. PMID:19135695

  10. Adhesion molecules and transplantation.

    PubMed Central

    Heemann, U W; Tullius, S G; Azuma, H; Kupiec-Weglinsky, J; Tilney, N L

    1994-01-01

    OBJECTIVE: Accessory adhesion molecules are thought to influence the first interaction between host leukocytes and graft vascular endothelial cells. Their role in transplantation is reviewed. SUMMARY: Adhesion molecules have been divided into three major families: the selectins, the integrins, and the immunoglobulin superfamily. Selectins are small proteins that mediate the first contact between stimulated endothelial cells and leukocytes. Integrins interact with cytoskeletal components of cells, presumably coordinating extracellular stimuli with cytoskeleton dependent actions, such as motility, shape change, and phagocytic responses. Members of the immunoglobulin superfamily are structurally homologous, although they do not necessarily share similar functions. They are involved in T-cell proliferation and intracellular events. METHODS: Various groups of investigators have studied the influence and expression of adhesion molecules following transplantation. The authors of this article have reviewed and summarized the available literature. RESULTS: Many different adhesion molecules are up-regulated during the rejection event. Treatment of transplant recipients with monoclonal antibodies against accessory molecules, such as leukocyte function associated antigen 1 (LFA-1) and intercellular adhesion molecule 1 (ICAM-1), has resulted in either a prolongation of transplant survival or the induction of tolerance in some models. Other interventions are under study. CONCLUSION: By mediating the initial leukocyte/endothelial cell interactions, adhesion molecules may play an important role in graft rejection, mediation of infiltration into the graft, and dissemination of the antigenic message to the lymphoid tissues of the host. Future studies will have to deal not only with conceptualizing their function and mechanisms of action, but also with manipulating their interrelationships to the benefit of the graft recipient. PMID:8297174

  11. 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 curing. PMID:21643511

  12. 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

  13. Cytohesin-2/ARNO, through its interaction with focal adhesion adaptor protein paxillin, regulates preadipocyte migration via the downstream activation of Arf6.

    PubMed

    Torii, Tomohiro; Miyamoto, Yuki; Sanbe, Atsushi; Nishimura, Kohji; Yamauchi, Junji; Tanoue, Akito

    2010-07-30

    The formation of primitive adipose tissue is the initial process in adipose tissue development followed by the migration of preadipocytes into adipocyte clusters. Comparatively little is known about the molecular mechanism controlling preadipocyte migration. Here, we show that cytohesin-2, the guanine-nucleotide exchange factor for the Arf family GTP-binding proteins, regulates migration of mouse preadipocyte 3T3-L1 cells through Arf6. SecinH3, a specific inhibitor of the cytohesin family, markedly inhibits migration of 3T3-L1 cells. 3T3-L1 cells express cytohesin-2 and cytohesin-3, and knockdown of cytohesin-2 with its small interfering RNA effectively decreases cell migration. Cytohesin-2 preferentially acts upstream of Arf6 in this signaling pathway. Furthermore, we find that the focal adhesion protein paxillin forms a complex with cytohesin-2. Paxillin colocalizes with cytohesin-2 at the leading edges of migrating cells. This interaction is mediated by the LIM2 domain of paxillin and the isolated polybasic region of cytohesin-2. Importantly, migration is inhibited by expression of the constructs containing these regions. These results suggest that cytohesin-2, through a previously unexplored complex formation with paxillin, regulates preadipocyte migration and that paxillin plays a previously unknown role as a scaffold protein of Arf guanine-nucleotide exchange factor. PMID:20525696

  14. 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.

  15. Protein Aggregation Formed by Recombinant cp19k Homologue of Balanus albicostatus Combined with an 18 kDa N-Terminus Encoded by pET-32a(+) Plasmid Having Adhesion Strength Comparable to Several Commercial Glues.

    PubMed

    Liang, Chao; Li, Yunqiu; Liu, Zhiming; Wu, Wenjian; Hu, Biru

    2015-01-01

    The barnacle is well known for its tenacious and permanent attachment to a wide variety of underwater substrates, which is accomplished by synthesizing, secreting and curing a mixture of adhesive proteins termed "barnacle cement". In order to evaluate interfacial adhesion abilities of barnacle cement proteins, the cp19k homologous gene in Balanus albicostatus (Balcp19k) was cloned and expressed in Escherichia coli. Here, we report an intriguing discovery of a gel-like super adhesive aggregation produced by Trx-Balcp19k, a recombinant Balcp19k fusion protein. The Trx-Balcp19k consists of an 18 kDa fragment at the N-terminus, which is encoded by pET-32a(+) plasmid and mainly comprised of a thioredoxin (Trx) tag, and Balcp19k at the C-terminus. The sticky aggregation was designated as "Trx-Balcp19k gel", and the bulk adhesion strength, biochemical composition, as well as formation conditions were all carefully investigated. The Trx-Balcp19k gel exhibited strong adhesion strength of 2.10 0.67 MPa, which was approximately fifty folds higher than that of the disaggregated Trx-Balcp19k (40 8 kPa) and rivaled those of commercial polyvinyl acetate (PVA) craft glue (Mont Marte, Australia) and UHU glue (UHU GmbH & Co. KG, Germany). Lipids were absent from the Trx-Balcp19k gel and only a trace amount of carbohydrates was detected. We postulate that the electrostatic interactions play a key role in the formation of Trx-Balcp19k gel, by mediating self-aggregation of Trx-Balcp19k based on its asymmetric distribution pattern of charged amino acids. Taken together, we believe that our discovery not only presents a promising biological adhesive with potential applications in both biomedical and technical fields, but also provides valuable paradigms for molecular design of bio-inspired peptide- or protein-based materials. PMID:26317205

  16. 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

  17. Morphogenesis, Adhesive Properties, and Antifungal Resistance Depend on the Pmt6 Protein Mannosyltransferase in the Fungal Pathogen Candida albicans

    PubMed Central

    Timpel, Claudia; Zink, Sigrid; Strahl-Bolsinger, Sabine; Schröppel, Klaus; Ernst, Joachim

    2000-01-01

    Protein mannosyltransferases (Pmt proteins) initiate O glycosylation of secreted proteins in fungi. We have characterized PMT6, which encodes the second Pmt protein of the fungal pathogen Candida albicans. The residues of Pmt6p are 21 and 42% identical to those of C. albicans Pmt1p and S. cerevisiae Pmt6p, respectively. Mutants lacking one or two PMT6 alleles grow normally and contain normal Pmt enzymatic activities in cell extracts but show phenotypes including a partial block of hyphal formation (dimorphism) and a supersensitivity to hygromycin B. The morphogenetic defect can be suppressed by overproduction of known components of signaling pathways, including Cek1p, Cph1p, Tpk2p, and Efg1p, suggesting a specific Pmt6p target protein upstream of these components. Mutants lacking both PMT1 and PMT6 are viable and show pmt1 mutant phenotypes and an additional sensitivity to the iron chelator ethylenediamine-di(o-hydroxyphenylacetic acid). The lack of Pmt6p significantly reduces adherence to endothelial cells and overall virulence in a mouse model of systemic infection. The results suggest that Pmt6p regulates a more narrow subclass of proteins in C. albicans than Pmt1p, including secreted proteins responsible for morphogenesis and antifungal sensitivities. PMID:10809683

  18. The vaccinia virus kelch-like protein C2L affects calcium-independent adhesion to the extracellular matrix and inflammation in a murine intradermal model.

    PubMed

    Pires de Miranda, Marta; Reading, Patrick C; Tscharke, David C; Murphy, Brendan J; Smith, Geoffrey L

    2003-09-01

    Chordate poxviruses encode several uncharacterized POZ-kelch proteins and three of these are present in Vaccinia virus (VV) strain Western Reserve. VV gene C2L is predicted to encode a protein of 512 amino acid residues with a POZ/BTB domain in the N-terminal region and three kelch motifs in the C-terminal half of the protein. We have identified the C2L gene product as an intracellular protein of 56 kDa and constructed and characterized a VV mutant lacking the C2L gene (v Delta C2L). Compared to wild-type and revertant viruses, v Delta C2L had unaltered growth in vitro, but had a different plaque morphology due to an altered cytopathic effect (CPE) of infected cells. Deleting C2L had no effect on VV-induced formation of actin tails or enhanced cell motility, but affected the development of VV-induced cellular projections and the Ca(2+)-independent cell/extracellular matrix adhesion late during infection. In an intranasal mouse model, C2L did not contribute to virus virulence. However, in an intradermal mouse model, infection with v Delta C2L resulted in larger lesions and more cell infiltration into the infected ears during recovery from infection. Thus, in this model, C2L protein inhibits inflammation and reduces immunopathology. In summary, we found that C2L is not required for virus replication in vitro but contributes to aspects of VV-induced CPE and reduces immunopathology in vivo. PMID:12917467

  19. 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

  20. The conserved LIM domain-containing focal adhesion protein ZYX-1 regulates synapse maintenance in Caenorhabditis elegans

    PubMed Central

    Luo, Shuo; Schaefer, Anneliese M.; Dour, Scott; Nonet, Michael L.

    2014-01-01

    We describe the identification of zyxin as a regulator of synapse maintenance in mechanosensory neurons in C. elegans. zyx-1 mutants lacked PLM mechanosensory synapses as adult animals. However, most PLM synapses initially formed during development but were subsequently lost as the animals developed. Vertebrate zyxin regulates cytoskeletal responses to mechanical stress in culture. Our work provides in vivo evidence in support of such a role for zyxin. In particular, zyx-1 mutant synaptogenesis phenotypes were suppressed by disrupting locomotion of the mutant animals, suggesting that zyx-1 protects mechanosensory synapses from locomotion-induced forces. In cultured cells, zyxin is recruited to focal adhesions and stress fibers via C-terminal LIM domains and modulates cytoskeletal organization via the N-terminal domain. The synapse-stabilizing activity was mediated by a short isoform of ZYX-1 containing only t