Science.gov

Sample records for adhesion fa proteins

  1. Adhesives from modified soy protein

    DOEpatents

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

    2008-08-26

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

  2. Beta-cell hypertrophy in fa/fa rats is associated with basal glucose hypersensitivity and reduced SNARE protein expression.

    PubMed

    Chan, C B; MacPhail, R M; Sheu, L; Wheeler, M B; Gaisano, H Y

    1999-05-01

    In normal isolated beta-cells, the response to glucose is heterogeneous and characterized by an increasing number of secretory cells as glucose concentration rises (Pipeleers DG, Kiekens R, Ling Z, Wilikens A, Schuit F: Physiologic relevance of heterogeneity in the pancreatic beta-cell population. Diabetologia 37 (Suppl. 2):S57-S64, 1994). We hypothesized that fasting hyperinsulinemia in obesity might be explained by altered beta-cell heterogeneity of signal transduction mechanisms, possibly involving exocytotic proteins. Insulin secretion from individual beta-cells sorted according to the size of the islet donor (<125 microm, >250 microm, and intermediate diameter) was measured by reverse hemolytic plaque assay. Beta-cells from fa/fa rats were hypertrophied 25-40%, independent of donor islet size. This was accompanied by an increased proportion of secretory cells (recruitment) at 5.5-11.0 mmol/l glucose, increased secretion per cell at 2.8 mmol/l glucose, and decreased insulin content after acute glucose exposure without an increase in secretion per cell. Decreased expression of exocytotic (soluble N-ethylmaleimide-sensitive fusion protein receptor [SNARE]) proteins, vesicle-associated membrane protein isoform 2 (VAMP-2), synaptosomal protein of 25 kDa (SNAP-25), and syntaxin-1 and -2 in fa/fa beta-cells may contribute to the failure to sustain excessive plaque size at higher glucose concentrations. Fasting hyperinsulinemia may be maintained by increased recruitment and an exaggerated secretory response in all fa-derived islet populations. Glucose regulates beta-cell responsiveness in the short term, and these effects may involve altered expression of SNARE proteins.

  3. Mixed compared with single-source proteins in high-protein diets affect kidney structure and function differentially in obese fa/fa Zucker rats.

    PubMed

    Devassy, Jessay G; Wojcik, Jennifer L; Ibrahim, Naser H M; Zahradka, Peter; Taylor, Carla G; Aukema, Harold M

    2017-02-01

    Questions remain regarding the potential negative effects of dietary high protein (HP) on kidney health, particularly in the context of obesity in which the risk for renal disease is already increased. To examine whether some of the variability in HP effects on kidney health may be due to source of protein, obese fa/fa Zucker rats were given HP (35% of energy from protein) diets containing either casein, soy protein, or a mixed source of animal and plant proteins for 12 weeks. Control lean and obese rats were given diets containing casein at normal protein (15% of energy from protein) levels. Body weight and blood pressure were measured, and markers of renal structural changes, damage, and function were assessed. Obesity alone resulted in mild renal changes, as evidenced by higher kidney weights, proteinuria, and glomerular volumes. In obese rats, increasing the protein level using the single, but not mixed, protein sources resulted in higher renal fibrosis compared with the lean rats. The mixed-protein HP group also had lower levels of serum monocyte chemoattractant protein-1, even though this diet further increased kidney and glomerular size. Soy and mixed-protein HP diets also resulted in a small number of damaged glomeruli, while soy compared with mixed-protein HP diet delayed the increase in blood pressure over time. Since obesity itself confers added risk of renal disease, an HP diet from mixed-protein sources that enables weight loss but has fewer risks to renal health may be advantageous.

  4. Focal adhesion protein abnormalities in myelodysplastic mesenchymal stromal cells

    SciTech Connect

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

    2011-11-01

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

  5. The adhesive properties of coacervated recombinant hybrid mussel adhesive proteins.

    PubMed

    Lim, Seonghye; Choi, Yoo Seong; Kang, Dong Gyun; Song, Young Hoon; Cha, Hyung Joon

    2010-05-01

    Marine mussels attach to substrates using adhesive proteins. It has been suggested that complex coacervation (liquid-liquid phase separation via concentration) might be involved in the highly condensed and non-water dispersed adhesion process of mussel adhesive proteins (MAPs). However, as purified natural MAPs are difficult to obtain, it has not been possible to experimentally validate the coacervation model. In the present work, we demonstrate complex coacervation in a system including recombinant MAPs and hyaluronic acid (HA). Our recombinant hybrid MAPs, fp-151 and fp-131, can be produced in large quantities, and are readily purified. We observed successful complex coacervation using cationic fp-151 or fp-131, and an anionic HA partner. Importantly, we found that highly condensed complex coacervates significantly increased the bulk adhesive strength of MAPs in both dry and wet environments. In addition, oil droplets were successfully engulfed using a MAP-based interfacial coacervation process, to form microencapsulated particles. Collectively, our results indicate that a complex coacervation system based on MAPs shows superior adhesive properties, combined with additional valuable features including liquid/liquid phase separation and appropriate viscoelasticity. Our microencapsulation system could be useful in the development of new adhesive biomaterials, including self-adhesive microencapsulated drug carriers, for use in biotechnological and biomedical applications.

  6. ZF21 protein regulates cell adhesion and motility.

    PubMed

    Nagano, Makoto; Hoshino, Daisuke; Sakamoto, Takeharu; Kawasaki, Noritaka; Koshikawa, Naohiko; Seiki, Motoharu

    2010-07-02

    Cell migration on an extracellular matrix (ECM) requires continuous formation and turnover of focal adhesions (FAs) along the direction of cell movement. However, our knowledge of the components of FAs and the mechanism of their regulation remains limited. Here, we identify ZF21, a member of a protein family characterized by the presence of a phosphatidylinositol 3-phosphate-binding FYVE domain, to be a new regulator of FAs and cell movement. Knockdown of ZF21 expression in cells increased the number of FAs and suppressed cell migration. Knockdown of ZF21 expression also led to a significant delay in FA disassembly following induction of synchronous disassembly of FAs by nocodazole treatment. ZF21 bound to focal adhesion kinase, localized to FAs, and was necessary for dephosphorylation of FAK at Tyr(397), which is important for disassembly of FAs. Thus, ZF21 represents a new component of FAs, mediates disassembly of FAs, and thereby regulates cell motility.

  7. Wood adhesives containing proteins and carbohydrates

    Technology Transfer Automated Retrieval System (TEKTRAN)

    In recent years there has been resurgent interest in using biopolymers as sustainable and environmentally friendly ingredients in wood adhesive formulations. Among them, proteins and carbohydrates are the most commonly used. In this chapter, an overview is given of protein-based and carbohydrate-...

  8. Molecular mechanics of mussel adhesion proteins

    NASA Astrophysics Data System (ADS)

    Qin, Zhao; Buehler, Markus J.

    2014-01-01

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

  9. Purification of adhesive proteins from mussels.

    PubMed

    Pardo, J; Gutierrez, E; Sáez, C; Brito, M; Burzio, L O

    1990-11-01

    The adhesive polyphenolic proteins from the mussels Mytilus chilensis and Choromytilus chorus have been purified based on their solubility in dilute perchloric acid and on differential precipitation with acetone containing about 0.3 N HCl. The specific activity of the proteins obtained was 0.16 mg of 3,4-dihydroxyphenylalanine per milligram of protein, or higher. The proteins have an apparent molecular weight of about 100,000 and they contain a high proportion of 3,4-dihydroxyphenylalanine, lysine, and proline.

  10. Soy and cottonseed protein blends as wood adhesives

    Technology Transfer Automated Retrieval System (TEKTRAN)

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

  11. Protein conformation as a regulator of cell-matrix adhesion.

    PubMed

    Hytönen, Vesa P; Wehrle-Haller, Bernhard

    2014-04-14

    The dynamic regulation of cell-matrix adhesion is essential for tissue homeostasis and architecture, and thus numerous pathologies are linked to altered cell-extracellular matrix (ECM) interaction and ECM scaffold. The molecular machinery involved in cell-matrix adhesion is complex and involves both sensory and matrix-remodelling functions. In this review, we focus on how protein conformation controls the organization and dynamics of cell-matrix adhesion. The conformational changes in various adhesion machinery components are described, including examples from ECM as well as cytoplasmic proteins. The discussed mechanisms involved in the regulation of protein conformation include mechanical stress, post-translational modifications and allosteric ligand-binding. We emphasize the potential role of intrinsically disordered protein regions in these processes and discuss the role of protein networks and co-operative protein interactions in the formation and consolidation of cell-matrix adhesion and extracellular scaffolds.

  12. 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

    2015-10-28

    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.

  13. 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.

  14. 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

  15. Urea modified cottonseed protein adhesive for wood composite products

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Cottonseed protein has the potential to be used as renewable and environmentally friendly adhesives in wood products industry. However, the industry application was limited by its low mechanical properties, low water resistance and viscosity. In this work, urea modified cottonseed protein adhesive w...

  16. 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.

  17. Insights into the Utility of the Focal Adhesion Scaffolding Proteins in the Anaerobic Fungus Orpinomyces sp. C1A

    PubMed Central

    Calkins, Shelby; Youssef, Noha H.

    2016-01-01

    Focal adhesions (FAs) are large eukaryotic multiprotein complexes that are present in all metazoan cells and function as stable sites of tight adhesion between the extracellular matrix (ECM) and the cell’s cytoskeleton. FAs consist of anchor membrane protein (integrins), scaffolding proteins (e.g. α-actinin, talin, paxillin, and vinculin), signaling proteins of the IPP complex (e.g. integrin-linked kinase, α-parvin, and PINCH), and signaling kinases (e.g. focal adhesion kinase (FAK) and Src kinase). While genes encoding complete focal adhesion machineries are present in genomes of all multicellular Metazoa; incomplete machineries were identified in the genomes of multiple non-metazoan unicellular Holozoa, basal fungal lineages, and amoebozoan representatives. Since a complete FA machinery is required for functioning, the putative role, if any, of these incomplete FA machineries is currently unclear. We sought to examine the expression patterns of FA-associated genes in the anaerobic basal fungal isolate Orpinomyces sp. strain C1A under different growth conditions and at different developmental stages. Strain C1A lacks clear homologues of integrin, and the two signaling kinases FAK and Src, but encodes for all scaffolding proteins, and the IPP complex proteins. We developed a protocol for synchronizing growth of C1A cultures, allowing for the collection and mRNA extraction from flagellated spores, encysted germinating spores, active zoosporangia, and late inactive sporangia of strain C1A. We demonstrate that the genes encoding the FA scaffolding proteins α-actinin, talin, paxillin, and vinculin are indeed transcribed under all growth conditions, and at all developmental stages of growth. Further, analysis of the observed transcriptional patterns suggests the putative involvement of these components in alternative non-adhesion-specific functions, such as hyphal tip growth during germination and flagellar assembly during zoosporogenesis. Based on these results

  18. 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...

  19. Protein Kinase D1 regulates focal adhesion dynamics and cell adhesion through Phosphatidylinositol-4-phosphate 5-kinase type-l γ

    PubMed Central

    Durand, Nisha; Bastea, Ligia I.; Long, Jason; Döppler, Heike; Ling, Kun; Storz, Peter

    2016-01-01

    Focal adhesions (FAs) are highly dynamic structures that are assembled and disassembled on a continuous basis. The balance between the two processes mediates various aspects of cell behavior, ranging from cell adhesion and spreading to directed cell migration. The turnover of FAs is regulated at multiple levels and involves a variety of signaling molecules and adaptor proteins. In the present study, we show that in response to integrin engagement, a subcellular pool of Protein Kinase D1 (PKD1) localizes to the FAs. PKD1 affects FAs by decreasing turnover and promoting maturation, resulting in enhanced cell adhesion. The effects of PKD1 are mediated through direct phosphorylation of FA-localized phosphatidylinositol-4-phosphate 5-kinase type-l γ (PIP5Klγ) at serine residue 448. This phosphorylation occurs in response to Fibronectin-RhoA signaling and leads to a decrease in PIP5Klγs’ lipid kinase activity and binding affinity for Talin. Our data reveal a novel function for PKD1 as a regulator of FA dynamics and by identifying PIP5Klγ as a novel PKD1 substrate provide mechanistic insight into this process. PMID:27775029

  20. Protein Kinase D1 regulates focal adhesion dynamics and cell adhesion through Phosphatidylinositol-4-phosphate 5-kinase type-l γ.

    PubMed

    Durand, Nisha; Bastea, Ligia I; Long, Jason; Döppler, Heike; Ling, Kun; Storz, Peter

    2016-10-24

    Focal adhesions (FAs) are highly dynamic structures that are assembled and disassembled on a continuous basis. The balance between the two processes mediates various aspects of cell behavior, ranging from cell adhesion and spreading to directed cell migration. The turnover of FAs is regulated at multiple levels and involves a variety of signaling molecules and adaptor proteins. In the present study, we show that in response to integrin engagement, a subcellular pool of Protein Kinase D1 (PKD1) localizes to the FAs. PKD1 affects FAs by decreasing turnover and promoting maturation, resulting in enhanced cell adhesion. The effects of PKD1 are mediated through direct phosphorylation of FA-localized phosphatidylinositol-4-phosphate 5-kinase type-l γ (PIP5Klγ) at serine residue 448. This phosphorylation occurs in response to Fibronectin-RhoA signaling and leads to a decrease in PIP5Klγs' lipid kinase activity and binding affinity for Talin. Our data reveal a novel function for PKD1 as a regulator of FA dynamics and by identifying PIP5Klγ as a novel PKD1 substrate provide mechanistic insight into this process.

  1. 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

  2. Adhesion family of G protein-coupled receptors and cancer.

    PubMed

    Lin, Hsi-Hsien

    2012-01-01

    The adhesion-class G protein-coupled receptors (adhesion-GPCRs) constitute the second largest GPCR sub-family in humans. Adhesion-GPCRs are defined by the chimeric structure of an unusually large extracellular cell-adhesion domain and a GPCR-like seven-pass transmembrane domain. Adhesion-GPCRs are hence expected to display both cellular adhesion and signaling functions in many biological systems. Adhesion-GPCRs are normally expressed in the central nervous, immune, and reproductive systems in a cell type- or tissue-restricted fashion. However, aberrant expression of distinct adhesion-GPCR molecules has been identified in various human cancers with some of the receptors closely associated with cancer development. Tumor-associated adhesion-GPCRs are thought to involve in tumorigenesis by affecting the growth of tumor cells, angiogenesis, tumor cell migration, invasion and metastasis either positively or negatively. Furthermore, some adhesion-GPCRs are considered potential biomarkers for specific types of cancers. In this review article, the expressional characteristics and functional role of cancer-associated adhesion-GPCRs are discussed in depth.

  3. 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

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

    PubMed

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

    2016-01-01

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

  5. 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.

  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. Hypomorphic mutations in the gene encoding a key Fanconi anemia protein, FANCD2, sustain a significant group of FA-D2 patients with severe phenotype.

    PubMed

    Kalb, Reinhard; Neveling, Kornelia; Hoehn, Holger; Schneider, Hildegard; Linka, Yvonne; Batish, Sat Dev; Hunt, Curtis; Berwick, Marianne; Callen, Elsa; Surralles, Jordi; Casado, Jose A; Bueren, Juan; Dasi, Angeles; Soulier, Jean; Gluckman, Eliane; Zwaan, C Michel; van Spaendonk, Rosalina; Pals, Gerard; de Winter, Johan P; Joenje, Hans; Grompe, Markus; Auerbach, Arleen D; Hanenberg, Helmut; Schindler, Detlev

    2007-05-01

    FANCD2 is an evolutionarily conserved Fanconi anemia (FA) gene that plays a key role in DNA double-strand-type damage responses. Using complementation assays and immunoblotting, a consortium of American and European groups assigned 29 patients with FA from 23 families and 4 additional unrelated patients to complementation group FA-D2. This amounts to 3%-6% of FA-affected patients registered in various data sets. Malformations are frequent in FA-D2 patients, and hematological manifestations appear earlier and progress more rapidly when compared with all other patients combined (FA-non-D2) in the International Fanconi Anemia Registry. FANCD2 is flanked by two pseudogenes. Mutation analysis revealed the expected total of 66 mutated alleles, 34 of which result in aberrant splicing patterns. Many mutations are recurrent and have ethnic associations and shared allelic haplotypes. There were no biallelic null mutations; residual FANCD2 protein of both isotypes was observed in all available patient cell lines. These analyses suggest that, unlike the knockout mouse model, total absence of FANCD2 does not exist in FA-D2 patients, because of constraints on viable combinations of FANCD2 mutations. Although hypomorphic mutations arie involved, clinically, these patients have a relatively severe form of FA.

  8. Nanoscale adhesion forces between enamel pellicle proteins and hydroxyapatite.

    PubMed

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

    2014-05-01

    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.

  9. GNRs@SiO2-FA in combination with radiotherapy induces the apoptosis of HepG2 cells by modulating the expression of apoptosis-related proteins

    PubMed Central

    GAO, BIN; SHEN, LEI; HE, KE-WU; XIAO, WEI-HUA

    2015-01-01

    The aim of the present study was to examine the apoptosis of the hepatocellular carcinoma cell line, HepG2, induced by treatment with folic acid-conjugated silica-coated gold nanorods (GNRs@SiO2-FA) in combination with radiotherapy, and to determine the involvement of apoptosis-related proteins. An MTT colorimetric assay was used to assess the biocompatibility of GNRs@SiO2-FA. The distribution of GNRs@SiO2-FA into the cells was observed using transmission electron microscopy (TEM). HepG2 cells cultured in vitro were divided into the following 4 groups: i)the control group (untreated), ii) the GNRs@SiO2-FA group, iii) the radiotherapy group (iodine 125 seeds) and iv) the combination group (treated with GNRs@SiO2-FA and iodine 125 seeds) groups. The apoptosis of the HepG2 cells was detected by flow cytometry. The concentration range of <40 µg/ml GNRs@SiO2-FA was found to be safe for the biological activity of the HepG2 cells. GNRs@SiO2-FA entered the cytoplasm through endocytosis. The apoptotic rates of the HepG2 cells were higher in the GNRs@SiO2-FA and radiotherapy groups than in the control group (P<0.05). The apoptotic rate was also significantly higher in the combination group than the GNRs@SiO2-FA and radiotherapy groups (P<0.05). Taken together, these findings demonstrate that the combination of GNRs@SiO2-FA and radiotherapy more effectively induces the apoptosis of HepG2 cells. These apoptotic effects are achieved by increasing the protein expression of Bax and caspase-3, and inhibiting the protein expression of Bcl-2 and Ki-67. The combination of GNRs@SiO2-FA and radiotherapy may thus prove to be a new approach in the treatment of primary liver cancer. PMID:26648274

  10. The MRL proteins: adapting cell adhesion, migration and growth.

    PubMed

    Coló, Georgina P; Lafuente, Esther M; Teixidó, Joaquin

    2012-01-01

    MIG-10, RIAM and Lamellipodin (Lpd) are the founding members of the MRL family of multi-adaptor molecules. These proteins have common domain structures but display distinct functions in cell migration and adhesion, signaling, and in cell growth. The binding of RIAM with active Rap1 and with talin provides these MRL molecules with important regulatory roles on integrin-mediated cell adhesion and migration. Furthermore, RIAM and Lpd can regulate actin dynamics through their binding to actin regulatory Ena/VASP proteins. Recent data generated with the Drosophila MRL ortholog called Pico and with RIAM in melanoma cells indicate that these proteins can also regulate cell growth. As MRL proteins represent a relatively new family, many questions on their structure-function relationships remain unanswered, including regulation of their expression, post-translational modifications, new interactions, involvement in signaling and their knockout mice phenotype.

  11. 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

  12. Adhesions

    MedlinePlus

    Adhesions are bands of scar-like tissue. Normally, internal tissues and organs have slippery surfaces so they can shift easily as the body moves. Adhesions cause tissues and organs to stick together. They ...

  13. Adhesion

    MedlinePlus

    ... the intestines, adhesions can cause partial or complete bowel obstruction . Adhesions inside the uterine cavity, called Asherman syndrome , ... 1. Read More Appendicitis Asherman syndrome Glaucoma Infertility Intestinal obstruction Review Date 4/5/2016 Updated by: Irina ...

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

    PubMed

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

    2016-01-11

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

  15. The focal adhesion-associated proteins DOCK5 and GIT2 comprise a rheostat in control of epithelial invasion

    PubMed Central

    Frank, Scott R.; Köllmann, Clemens P.; van Lidth de Jeude, Jooske F.; Thiagarajah, Jay R.; Engelholm, Lars H.; Frödin, Morten; Hansen, Steen H.

    2016-01-01

    DOCK proteins are guanine nucleotide exchange factors for Rac and Cdc42 GTPases. DOCK1 is the founding member of the family and acts downstream of integrins via the canonical Crk-p130Cas complex to activate Rac GTPases in numerous contexts. In contrast, DOCK5, which possesses the greatest similarity to DOCK1, remains sparingly studied. Here we establish that DOCK5 plays a non-redundant role in regulating motile and invasive capacities of epithelial cells. DOCK1 is constitutively associated with sites of integrin attachment termed focal adhesions (FA). In contrast, we demonstrate that DOCK5 recruitment to FAs in Hela cells is restricted by GIT2, an established regulator of FA signaling. We determine that GIT2 is targeted to FAs in response to Rho-ROCK signaling and actomyosin contractility. Accordingly, inhibition of ROCK activity or MLC function promotes enrichment of DOCK5 in membrane protrusions and nascent cell-substratum adhesions. We further demonstrate that GIT2 inhibits the interaction of DOCK5 with Crk. Moreover, we show that depletion of GIT2 promotes DOCK5-dependent activation of the Crk-p130Cas signaling cascade to promote Rac1-mediated lamellipodial protrusion and FA turnover. The antagonism between GIT2 and DOCK5 extends to non-transformed MCF10A mammary epithelial cells, with DOCK5 “dialing-up” and GIT2 “dialing-down” invasiveness. Finally, we determine that DOCK5 inhibition attenuates invasion and metastasis of MDA-MB-231 cells and prolongs life span of mice injected with these cells. Collectively, our work identifies DOCK5 as a key regulator of epithelial invasion and metastasis, and demonstrates that suppression of DOCK5 by GIT2 represents a previously unappreciated mechanism for coordination of Rho and Rac GTPases. PMID:27669437

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

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

  17. Extracellular proteins from Lactobacillus plantarum BMCM12 prevent adhesion of enteropathogens to mucin.

    PubMed

    Sánchez, Borja; Urdaci, María C

    2012-06-01

    The aim of this study was to study the interference of the extracellular proteins produced by Lactobacillus plantarum BMCM12 with the adhesion of some well-known gut pathogens. The extracellular proteins secreted by L. plantarum BMCM12 in MRS broth were precipitated, resolved by SDS-PAGE, and identified by tandem mass spectrometry. Discordances between the observed and the theoretical molecular masses of several proteins suggested the presence of protein glycosylation, corroborated with specific glycoprotein staining after protein de-glycosylation using trifluoromethanesulfonic acid. Experiments of exclusion, competition, or prevention of the pathogen adhesion to mucin were performed using BMCM12 extracellular proteins, using Escherichia coli LMG2092 and Salmonella enterica subsp. enterica LMG15860. Extracellular proteins from BMCM12 reduced significantly the adhesion of the pathogens when they were added prior to adhesion assays. These proteins play thus important roles in preventing pathogen adhesion to the mucin layer.

  18. 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

  19. Connexin 43 expressed in endothelial cells modulates monocyte‑endothelial adhesion by regulating cell adhesion proteins.

    PubMed

    Yuan, Dongdong; Sun, Guoliang; Zhang, Rui; Luo, Chenfang; Ge, Mian; Luo, Gangjian; Hei, Ziqing

    2015-11-01

    Adhesion between circulating monocytes and vascular endothelial cells is a key initiator of atherosclerosis. In our previous studies, it was demonstrated that the expression of connexin (Cx)43 in monocytes modulates cell adhesion, however, the effects of the expression of Cx43 in endothelial cells remains to be elucidated. Therefore, the present study investigated the role of the expression of Cx43 in endothelial cells in the process of cell adhesion. A total of four different methods with distinct mechanisms were used to change the function and expression of Cx43 channels in human umbilical vein endothelial cells: Cx43 channel inhibitor (oleamide), enhancer (retinoic acid), overexpression of Cx43 by transfection with pcDNA‑Cx43 and knock‑down of the expression of Cx43 by small interfering RNA against Cx43. The results indicated that the upregulation of the expression of Cx43 enhanced monocyte‑endothelial adhesion and this was markedly decreased by downregulation of Cx43. This mechanism was associated with Cx43‑induced expression of vascular cell adhesion molecule‑1 and intercellular cell adhesion molecule‑1. The effects of Cx43 in endothelial cells was independent of Cx37 or Cx40. These experiments suggested that local regulation of endothelial Cx43 expression within the vasculature regulates monocyte‑endothelial adhesion, a critical event in the development of atherosclerosis and other inflammatory pathologies, with baseline adhesion set by the expression of Cx43. This balance may be crucial in controlling leukocyte involvement in inflammatory cascades.

  20. Shared midgut binding sites for Cry1A.105, Cry1Aa, Cry1Ab, Cry1Ac and Cry1Fa proteins from Bacillus thuringiensis in two important corn pests, Ostrinia nubilalis and Spodoptera frugiperda.

    PubMed

    Hernández-Rodríguez, Carmen Sara; Hernández-Martínez, Patricia; Van Rie, Jeroen; Escriche, Baltasar; Ferré, Juan

    2013-01-01

    First generation of insect-protected transgenic corn (Bt-corn) was based on the expression of Cry1Ab or Cry1Fa proteins. Currently, the trend is the combination of two or more genes expressing proteins that bind to different targets. In addition to broadening the spectrum of action, this strategy helps to delay the evolution of resistance in exposed insect populations. One of such examples is the combination of Cry1A.105 with Cry1Fa and Cry2Ab to control O. nubilalis and S. frugiperda. Cry1A.105 is a chimeric protein with domains I and II and the C-terminal half of the protein from Cry1Ac, and domain III almost identical to Cry1Fa. The aim of the present study was to determine whether the chimeric Cry1A.105 has shared binding sites either with Cry1A proteins, with Cry1Fa, or with both, in O. nubilalis and in S. frugiperda. Brush-border membrane vesicles (BBMV) from last instar larval midguts were used in competition binding assays with (125)I-labeled Cry1A.105, Cry1Ab, and Cry1Fa, and unlabeled Cry1A.105, Cry1Aa, Cry1Ab, Cry1Ac, Cry1Fa, Cry2Ab and Cry2Ae. The results showed that Cry1A.105, Cry1Ab, Cry1Ac and Cry1Fa competed with high affinity for the same binding sites in both insect species. However, Cry2Ab and Cry2Ae did not compete for the binding sites of Cry1 proteins. Therefore, according to our results, the development of cross-resistance among Cry1Ab/Ac, Cry1A.105, and Cry1Fa proteins is possible in these two insect species if the alteration of shared binding sites occurs. Conversely, cross-resistance between these proteins and Cry2A proteins is very unlikely in such case.

  1. A novel role for Fanconi anemia (FA) pathway effector protein FANCD2 in cell cycle progression of untransformed primary human cells.

    PubMed

    Song, Ihn Young; Barkley, Laura R; Day, Tovah A; Weiss, Robert S; Vaziri, Cyrus

    2010-06-15

    Fanconi Anemia (FA) is a cancer-susceptibility syndrome characterized by cellular sensitivity to DNA inter-strand cross-link (ICL)-inducing agents. The Fanconia Anemia D2 (FANCD2) protein is implicated in repair of various forms of DNA damage including ICLs. Studies with replicating extracts from Xenopus eggs indicate a role for FANCD2 in processing and repair of DNA replication-associated double stranded breaks (DSB). We have investigated the role of FANCD2 in cell cycle progression of cultured human cells. Similar to Xenopus cell-free extracts, we show that chromatin association of FANCD2 in human cells is coupled to ongoing DNA replication. siRNA depletion experiments demonstrate that FANCD2 is necessary for efficient DNA synthesis. However, in contrast with Xenopus extracts, FANCD2-deficiency does not elicit a DNA damage response, and does not affect the elongation phase of DNA synthesis, suggesting that FANCD2 is dispensable for repair of replication-associated DNA damage. Using synchronized cultures of primary untransformed human dermal fibroblasts we demonstrate that FANCD2 is necessary for efficient initiation of DNA synthesis. Taken together, our results suggest a novel role for the FA pathway in regulation of DNA synthesis and cell cycle progression. Inefficient DNA replication may contribute to the genome instability and cancer-propensity of FA patients.

  2. Protein-based underwater adhesives and the prospects for their biotechnological production

    PubMed Central

    Stewart, Russell J.

    2011-01-01

    Biotechnological approaches to practical production of biological protein-based adhesives have had limited success over the last several decades. Broader efforts to produce recombinant adhesive proteins may have been limited by early disappointments. More recent synthetic polymer approaches have successfully replicated some aspects of natural underwater adhesives. For example, synthetic polymers, inspired by mussels, containing the catecholic functional group of 3,4-L-dihydroxyphenylalanine adhere strongly to wet metal oxide surfaces. Synthetic complex coacervates inspired by the Sandcastle worm are water-borne adhesives that can be delivered underwater without dispersing. Synthetic approaches offer several advantages, including versatile chemistries and scalable production. In the future, more sophisticated mimetic adhesives may combine synthetic copolymers with recombinant or agriculture-derived proteins to better replicate the structural and functional organization of natural adhesives. PMID:20890598

  3. Protein-based underwater adhesives and the prospects for their biotechnological production.

    PubMed

    Stewart, Russell J

    2011-01-01

    Biotechnological approaches to practical production of biological protein-based adhesives have had limited success over the last several decades. Broader efforts to produce recombinant adhesive proteins may have been limited by early disappointments. More recent synthetic polymer approaches have successfully replicated some aspects of natural underwater adhesives. For example, synthetic polymers, inspired by mussels, containing the catecholic functional group of 3,4-L-dihydroxyphenylalanine adhere strongly to wet metal oxide surfaces. Synthetic complex coacervates inspired by the Sandcastle worm are water-borne adhesives that can be delivered underwater without dispersing. Synthetic approaches offer several advantages, including versatile chemistries and scalable production. In the future, more sophisticated mimetic adhesives may combine synthetic copolymers with recombinant or agriculture-derived proteins to better replicate the structural and functional organization of natural adhesives.

  4. Processing of mussel adhesive protein analog thin films by matrix assisted pulsed laser evaporation

    NASA Astrophysics Data System (ADS)

    Cristescu, R.; Patz, T.; Narayan, R. J.; Menegazzo, N.; Mizaikoff, B.; Mihaiescu, D. E.; Messersmith, P. B.; Stamatin, I.; Mihailescu, I. N.; Chrisey, D. B.

    2005-07-01

    Mussel adhesive proteins are a new class of biologically-derived materials that possess unique biocompatibility, bioactivity, and adhesion properties. We have demonstrated successful thin film growth of 3,4-dihydroxyphenyl- L-alanine modified poly(ethylene oxide)-poly(propylene oxide)-poly(ethylene oxide) (DOPA modified- PEO-PPO-PEO) block copolymer, a mussel adhesive protein analog, using matrix assisted pulsed laser evaporation. We have demonstrated that the main functional groups of the mussel adhesive protein analog are present in the transferred film. The effect of increasing of chain length of the mussel adhesive protein analog on film structure was also examined. These novel polymer thin films could have numerous medical and technological applications if their thin film properties are similar to what is found in bulk. This is the first report of successful MAPLE deposition of this material as thin films.

  5. Adhesion G-protein-coupled receptors: elusive hybrids come of age.

    PubMed

    Simundza, Julia; Cowin, Pamela

    2013-12-01

    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.

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

    PubMed

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

    2010-08-01

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

  7. 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

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

    NASA Astrophysics Data System (ADS)

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

    2012-06-01

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

  9. 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

  10. Modulation of cell adhesion complexes by surface protein patterns.

    PubMed

    Pesen, Devrim; Haviland, David B

    2009-03-01

    Cell adhesion is an important process in several biological phenomena. To investigate the formation and organization of focal adhesions, we developed a patterning approach based on electron beam lithography. Nanodots (radius <1230 nm) and nanorings (inner radius <320 nm) of fibronectin (FN) were patterned on a K-Casein background. Intracellular vinculin immunofluorescence mirrored the FN nanopatterns. Atomic force microscopy showed that FN nanodots and nanorings organize the immediate cytoskeleton into straight fibrils and diverging fibril bundles, respectively. Our results suggest that a minimum of approximately 40 FN molecules is required for a cell to form a focal adhesion.

  11. Monitoring in real-time focal adhesion protein dynamics in response to a discrete mechanical stimulus.

    PubMed

    von Bilderling, Catalina; Caldarola, Martín; Masip, Martín E; Bragas, Andrea V; Pietrasanta, Lía I

    2017-01-01

    The adhesion of cells to the extracellular matrix is a hierarchical, force-dependent, multistage process that evolves at several temporal scales. An understanding of this complex process requires a precise measurement of forces and its correlation with protein responses in living cells. We present a method to quantitatively assess live cell responses to a local and specific mechanical stimulus. Our approach combines atomic force microscopy with fluorescence imaging. Using this approach, we evaluated the recruitment of adhesion proteins such as vinculin, focal adhesion kinase, paxillin, and zyxin triggered by applying forces in the nN regime to live cells. We observed in real time the development of nascent adhesion sites, evident from the accumulation of early adhesion proteins at the position where the force was applied. We show that the method can be used to quantify the recruitment characteristic times for adhesion proteins in the formation of focal complexes. We also found a spatial remodeling of the mature focal adhesion protein zyxin as a function of the applied force. Our approach allows the study of a variety of complex biological processes involved in cellular mechanotransduction.

  12. 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.

  13. 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.

  14. Adhesion-Linked Protein Tyrosine Phosphatases, Morphogenesis and Breast Cancer Progression

    DTIC Science & Technology

    2004-07-01

    Award Number: DAMD17-03-1-0496 TITLE: Adhesion-linked Protein Tyrosine Phosphatases, Morphogenesis and Breast Cancer Progression PRINCIPAL...Adhesion-linked Protein Tyrosine Phosphatases, DAMD17-03-1-0496 Morphogenesis and Breast Cancer Progression 6. AUTHOR(S) Valerie M. Weaver, Ph.D. 7...we identified the Band 4.1 PTPs MEG1 and D1 as two candidate PTP metastasis suppressors. Our studies show that during MEC differentiation PTP MEG1

  15. A bioinspired elastin-based protein for a cytocompatible underwater adhesive.

    PubMed

    Brennan, M Jane; Kilbride, Bridget F; Wilker, Jonathan J; Liu, Julie C

    2017-04-01

    The development of adhesives that can be applied and create strong bonds underwater is a significant challenge for materials engineering. When the adhesive is intended for biomedical applications, further criteria, such as biocompatibility, must be met. Current biomedical adhesive technologies do not meet these needs. In response, we designed a bioinspired protein system that shows promise to achieve biocompatible underwater adhesion coupled with environmentally responsive behavior that is "smart" - that is, it can be tuned to suit a specific application. The material, ELY16, is constructed from an elastin-like polypeptide (ELP) that can be produced in high yields from Escherichia coli and can coacervate in response to environmental factors such as temperature, pH, and salinity. To confer wet adhesion, we utilized design principles from marine organisms such as mussels and sandcastle worms. When expressed, ELY16 is rich in tyrosine. Upon modification with the tyrosinase enzyme to form mELY16, the tyrosine residues are converted to 3,4-dihydroxyphenylalanine (DOPA). Both ELY16 and mELY16 exhibit cytocompatibility and significant dry adhesion strength (>2 MPa). Modification with DOPA increases protein adsorption to glass and provides moderate adhesion strength (∼240 kPa) in a highly humid environment. Furthermore, this ELP exhibits a tunable phase transition behavior that can be formulated to coacervate in physiological conditions and provides a convenient mechanism for application underwater. Finally, mELY16 possesses significantly higher adhesion strength in dry, humid, and underwater environments compared with a commercially available fibrin sealant. To our knowledge, mELY16 provides the strongest bonds of any rationally designed protein when used completely underwater, and its high yields make it more viable for commercial application compared to natural adhesive proteins. In conclusion, this ELP shows great potential to be a new "smart" underwater adhesive.

  16. Phosphoproteome reveals an atlas of protein signaling networks during osteoblast adhesion.

    PubMed

    Milani, Renato; Ferreira, Carmen V; Granjeiro, José M; Paredes-Gamero, Edgar J; Silva, Rodrigo A; Justo, Giselle Z; Nader, Helena B; Galembeck, Eduardo; Peppelenbosch, Maikel P; Aoyama, Hiroshi; Zambuzzi, Willian F

    2010-04-01

    Cell adhesion on surfaces is a fundamental process in the emerging biomaterials field and developmental events as well. However, the mechanisms regulating this biological process in osteoblasts are not fully understood. Reversible phosphorylation catalyzed by kinases is probably the most important regulatory mechanism in eukaryotes. Therefore, the goal of this study is to assess osteoblast adhesion through a molecular prism under a peptide array technology, revealing essential signaling proteins governing adhesion-related events. First, we showed that there are main morphological changes on osteoblast shape during adhesion up to 3 h. Second, besides classical proteins activated upon integrin activation, our results showed a novel network involving signaling proteins such as Rap1A, PKA, PKC, and GSK3beta during osteoblast adhesion on polystyrene. Third, these proteins were grouped in different signaling cascades including focal adhesion establishment, cytoskeleton rearrangement, and cell-cycle arrest. We have thus provided evidence that a global phosphorylation screening is able to yield a systems-oriented look at osteoblast adhesion, providing new insights for understanding of bone formation and improvement of cell-substratum interactions. Altogether, these statements are necessary means for further intervention and development of new approaches for the progress of tissue engineering.

  17. Are conformational changes, induced by osmotic pressure variations, the underlying mechanism of controlling the adhesive activity of mussel adhesive proteins?

    PubMed

    van der Leeden, Mieke C

    2005-11-22

    The mussel adhesive protein Mefp-1, under physiological conditions, presumably has a self-avoiding random walk conformation with helix-like or turned deca-peptide segments. Such a conformation may coil up under osmotic pressure induced by surrounding macromolecules. As a consequence, the orientation of the 3,4-dihydroxy-phenylalanine groups (dopa), essential for the adhesive strength as well as the cohesive strength in Mefp-1, will be altered. Changing the concentration of the protein itself or of different-type surrounding macromolecules may therefore be a tool to control the protein's adhesive activity. The effect of osmotic pressure on the conformation and dopa reactivity of Mefp-1 is studied by the addition of (poly)ethylene oxide (PEO) as a model macromolecule (Mw = 100 kD). From UV-spectroscopy measurements, it can be concluded that dopa reactivity in Mefp-1 changes with increasing PEO concentration. Fitting of the measured absorbance intensity data of the oxidation product dopaquinone versus time with a kinetic model points to the decreased accessibility of dopa groups in the Mefp-1 structure, a faster oxidation, and diminished cross linking under the influence of increasing PEO concentration up to 2.4 g/L, corresponding to an osmotic pressure of approximately 73 Pa. At higher PEO concentrations, the accessibility of the dopa groups for oxidation as well as cross-link formation decreases until about 20% of the dopa groups are oxidized at a PEO concentration of 3.8 g/L, corresponding to an osmotic pressure of approximately 113 Pa. FTIR measurements on the basis of amide I shifts qualitatively point to a transition to a more continuously turned structure of Mefp-1 in the presence of PEO. Therefore, it seems that conformational changes caused by variations of osmotic pressure determine the extent of steric hindrance of the dopa groups and hence the adhesive reactivity of Mefp-1.

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

    SciTech Connect

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

    2013-11-22

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

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

    PubMed

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

    2013-12-14

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

  20. A mucus adhesion promoting protein, MapA, mediates the adhesion of Lactobacillus reuteri to Caco-2 human intestinal epithelial cells.

    PubMed

    Miyoshi, Yukihiro; Okada, Sanae; Uchimura, Tai; Satoh, Eiichi

    2006-07-01

    Lactobacillus reuteri is one of the dominant lactobacilli found in the gastrointestinal tract of various animals. A surface protein of L. reuteri 104R, mucus adhesion promoting protein (MapA), is considered to be an adhesion factor of this strain. We investigated the relation between MapA and adhesion of L. reuteri to human intestinal (Caco-2) cells. Quantitative analysis of the adhesion of L. reuteri strains to Caco-2 cells showed that various L. reuteri strains bind not only to mucus but also to intestinal epithelial cells. In addition, purified MapA bound to Caco-2 cells, and this binding inhibited the adhesion of L. reuteri in a concentration-dependent manner. Based on these observations, the adhesion of L. reuteri appears due to the binding of MapA to receptor-like molecules on Caco-2 cells. Further, far-western analysis indicated the existence of multiple receptor-like molecules in Caco-2 cells.

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

    PubMed

    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.

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

    NASA Astrophysics Data System (ADS)

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

    2014-10-01

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

  3. Adhesive proteins of stalked and acorn barnacles display homology with low sequence similarities.

    PubMed

    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).

  4. 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

  5. Shared Midgut Binding Sites for Cry1A.105, Cry1Aa, Cry1Ab, Cry1Ac and Cry1Fa Proteins from Bacillus thuringiensis in Two Important Corn Pests, Ostrinia nubilalis and Spodoptera frugiperda

    PubMed Central

    Hernández-Rodríguez, Carmen Sara; Hernández-Martínez, Patricia; Van Rie, Jeroen; Escriche, Baltasar; Ferré, Juan

    2013-01-01

    First generation of insect-protected transgenic corn (Bt-corn) was based on the expression of Cry1Ab or Cry1Fa proteins. Currently, the trend is the combination of two or more genes expressing proteins that bind to different targets. In addition to broadening the spectrum of action, this strategy helps to delay the evolution of resistance in exposed insect populations. One of such examples is the combination of Cry1A.105 with Cry1Fa and Cry2Ab to control O. nubilalis and S. frugiperda. Cry1A.105 is a chimeric protein with domains I and II and the C-terminal half of the protein from Cry1Ac, and domain III almost identical to Cry1Fa. The aim of the present study was to determine whether the chimeric Cry1A.105 has shared binding sites either with Cry1A proteins, with Cry1Fa, or with both, in O. nubilalis and in S. frugiperda. Brush-border membrane vesicles (BBMV) from last instar larval midguts were used in competition binding assays with 125I-labeled Cry1A.105, Cry1Ab, and Cry1Fa, and unlabeled Cry1A.105, Cry1Aa, Cry1Ab, Cry1Ac, Cry1Fa, Cry2Ab and Cry2Ae. The results showed that Cry1A.105, Cry1Ab, Cry1Ac and Cry1Fa competed with high affinity for the same binding sites in both insect species. However, Cry2Ab and Cry2Ae did not compete for the binding sites of Cry1 proteins. Therefore, according to our results, the development of cross-resistance among Cry1Ab/Ac, Cry1A.105, and Cry1Fa proteins is possible in these two insect species if the alteration of shared binding sites occurs. Conversely, cross-resistance between these proteins and Cry2A proteins is very unlikely in such case. PMID:23861865

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

    PubMed

    Maier, Marcus; Ströbele, 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.

  7. Diatom Adhesive Mucilage Contains Distinct Supramolecular Assemblies of a Single Modular Protein

    PubMed Central

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

    2006-01-01

    A previous study used atomic force microscopy saw-tooth retraction curves to characterize the adhesive mucilage pads of the diatom Toxarium undulatum. The major mucilage component consisted of adhesive nanofibers (ANFs) made up of modular proteins arranged into cohesive units, each containing a set number of modular proteins aligned in parallel. This study shows that T. undulatum adhesive mucilage is a biocomposite containing four additional adhesive components, including single modular proteins that are likely to be the structural units from which the ANFs are assembled. Two further distinct supramolecular assemblies were observed to coexist with ANFs (ANFs II and III), along with a continuum of single modular proteins through oligomers made up of varying numbers of modular proteins arranged in parallel. All components of the adhesive biocomposite produce a characteristic force spectrum with the same interpeak distance (35.3 ± 0.3 (mean ± SE) nm), suggesting they are derived from discrete supramolecular assemblies of the same modular protein, but they are distinguishable from one another based on the rupture force, persistence length, and interpeak force measured from their saw-tooth curves. PMID:16443662

  8. Developmental regulation of the adhesive and enzymatic activity of vascular adhesion protein-1 (VAP-1) in humans.

    PubMed

    Salmi, Marko; Jalkanen, Sirpa

    2006-09-01

    Vascular adhesion protein-1 (VAP-1) is a homodimeric glycoprotein that belongs to a unique subgroup of cell-surface-expressed oxidases. In adults, endothelial VAP-1 supports leukocyte rolling, firm adhesion, and transmigration in both enzyme activity-dependent and enzyme activity-independent manner. Here we studied the induction and function of VAP-1 during human ontogeny. We show that VAP-1 is already found in the smooth muscle at embryonic week 7. There are marked time-dependent switches in VAP-1 expression in the sinusoids of the liver, in the peritubular capillaries of the kidney, in the capillaries of the heart, and in the venules in the lamina propria of the gut. Fetal VAP-1 is dimerized, and it is enzymatically active. VAP-1 in fetal-type venules is able to bind cord blood lymphocytes. Also, adenovirally transfected VAP-1 on human umbilical vein endothelial cells is involved in rolling and firm adhesion of cord blood lymphocytes under conditions of physiologic shear stress. We conclude that VAP-1 is synthesized from early on in human vessels and it is functionally intact already before birth. Thus, VAP-1 may contribute critically to the oxidase activities in utero, and prove important for lymphocyte trafficking during human ontogeny.

  9. Protein adhesion on dental surfaces-a combined surface analytical approach.

    PubMed

    Müller, Christine; Wald, Johanna; Hoth-Hannig, Wiebke; Umanskaya, Natalia; Scholz, Daniel; Hannig, Matthias; Ziegler, Christiane

    2011-05-01

    Protein adsorption is a field of huge interest in a number of application fields. Information on protein adhesion is accessible by a variety of methods. However, the results obtained are significantly influenced by the applied technique. The objective of this work was to understand the role of adhesion forces (obtained by scanning force spectroscopy, SFS) in the process of protein adsorption and desorption. In SFS, the protein is forced to and retracted from the surface, even under unfavorable conditions, in contrast to the natural situation. Furthermore, adhesion forces are correlated with adhesion energies, neglecting the entropic part in the Gibbs enthalpy. In this context, dynamic contact angle (DCA) measurements were performed to identify the potential of this method to complement SFS data. In DCA measurements, the protein diffuses voluntarily to the surface and information on surface coverage and reversibility of adsorption is obtained, including entropic effects (conformational changes and hydrophobic effect). It could be shown that the surface coverage (by DCA) of bovine serum albumin on dental materials correlates well with the adhesion forces (by SFS) if no hydrophobic surface is involved. On those, the entropic hydrophobic effect plays a major role. As a second task, the reversibility of the protein adsorption, i.e., the voluntary desorption as studied by DCA, was compared to the adhesion forces. Here, a correlation between low adhesion forces and good reversibility could be found as long as no covalent bonds were involved. The comparative study of DCA and SFS, thus, leads to a more detailed picture of the complete adsorption/desorption cycle.

  10. Both protein kinase A and exchange protein activated by cAMP coordinate adhesion of human vascular endothelial cells.

    PubMed

    Netherton, Stuart J; Sutton, Jayda A; Wilson, Lindsay S; Carter, Rhonda L; Maurice, Donald H

    2007-10-12

    cAMP regulates integrin-dependent adhesions of vascular endothelial cells (VECs) to extracellular matrix proteins, their vascular endothelial cadherin-dependent intercellular adhesions, and their proliferation and migration in response to growth and chemotactic factors. Previously, we reported that cAMP-elevating agents differentially inhibited migration of human VECs isolated from large vascular structures (macro-VECs, human aortic endothelial cells [HAECs]) or small vascular structures (micro-VECs, human microvascular endothelial cells [HMVECs]) and that cAMP hydrolysis by phosphodiesterase (PDE)3 and PDE4 enzymes was important in coordinating this difference. Here we report that 2 cAMP-effector enzymes, namely protein kinase (PK)A and exchange protein activated by cAMP (EPAC), each regulate extracellular matrix protein-based adhesions of both macro- and micro-VECs. Of interest and potential therapeutic importance, we report that although specific pharmacological activation of EPAC markedly stimulated adhesion of micro-VECs to extracellular matrix proteins when PKA was inhibited, this treatment only modestly promoted adhesion of macro-VECs. Consistent with an important role for cAMP PDEs in this difference, PDE3 or PDE4 inhibitors promoted EPAC-dependent adhesions in micro-VECs when PKA was inhibited but not in macro-VECs. At a molecular level, we identify multiple, nonoverlapping, PKA- or EPAC-based signaling protein complexes in both macro- and micro-VECs and demonstrate that each of these complexes contains either PDE3B or PDE4D but not both of these PDEs. Taken together, our data support the concept that adhesion of macro- and micro-VECs is differentially regulated by cAMP and that these differences are coordinated through selective actions of cAMP at multiple nonoverlapping signaling complexes that contain PKA or EPAC and distinct PDE variants.

  11. A hot water extract of Curcuma longa inhibits adhesion molecule protein expression and monocyte adhesion to TNF-α-stimulated human endothelial cells.

    PubMed

    Kawasaki, Kengo; Muroyama, Koutarou; Yamamoto, Norio; Murosaki, Shinji

    2015-01-01

    The recruitment of arterial leukocytes to endothelial cells is an important step in the progression of various inflammatory diseases. Therefore, its modulation is thought to be a prospective target for the prevention or treatment of such diseases. Adhesion molecules on endothelial cells are induced by proinflammatory cytokines, including tumor necrosis factor-α (TNF-α), and contribute to the recruitment of leukocytes. In the present study, we investigated the effect of hot water extract of Curcuma longa (WEC) on the protein expression of adhesion molecules, monocyte adhesion induced by TNF-α in human umbilical vascular endothelial cells (HUVECs). Treatment of HUVECs with WEC significantly suppressed both TNF-α-induced protein expression of adhesion molecules and monocyte adhesion. WEC also suppressed phosphorylation and degradation of nuclear factor of kappa light polypeptide gene enhancer in B-cells inhibitor, alpha (IκBα) induced by TNF-α in HUVECs, suggesting that WEC inhibits the NF-κB signaling pathway.

  12. Abnormal amounts of intracellular calcium regulatory proteins in SHRSP.Z-Lepr(fa)/IzmDmcr rats with metabolic syndrome and cardiac dysfunction.

    PubMed

    Kagota, Satomi; Maruyama, Kana; Tada, Yukari; Wakuda, Hirokazu; Nakamura, Kazuki; Kunitomo, Masaru; Shinozuka, Kazumasa

    2013-02-01

    Metabolic syndrome is known to increase the risk of abnormal cardiac structure and function, which are considered to contribute to increased incidence of cardiovascular disease and mortality. We previously demonstrated that ventricular hypertrophy and diastolic dysfunction occur in SHRSP.Z-Lepr(fa)/IzmDmcr (SHRSP fatty) rats with metabolic syndrome. The aim of this study was to investigate the possible mechanisms underlying abnormal heart function in SHRSP fatty rats. The amount of sarcoplasmic reticulum Ca(2+)-ATPase (SERCA) 2a, phospholamban (PLB) protein, and Ser(16)-phosphorylated PLB was decreased in cardiomyocytes from SHRSP fatty rats compared with those from control Wistar-Kyoto rats at 18 weeks of age, and the PLB-to-SERCA2a ratio was increased. Left ventricular developed pressure was unchanged, and coronary flow rate and maximum rate of left ventricular pressure decline (-dP/dt) was decreased in SHRSP fatty rats. Treatment with telmisartan reversed the abnormalities of PLB amount, coronary flow rate, and -dP/dt in SHRSP fatty rats. These results indicate that abnormal amounts of intracellular Ca(2+) regulatory proteins in cardiomyocytes, leading to reduced intracellular Ca(2+) reuptake into the sarcoplasmic reticulum, may play a role in the diastolic dysfunction in SHRSP fatty rats and that these effects are partially related to decreased coronary circulation. Telmisartan may be beneficial in protecting against disturbances in cardiac function associated with metabolic syndrome.

  13. Binding of the cell adhesive protein tropoelastin to PTFE through plasma immersion ion implantation treatment.

    PubMed

    Bax, Daniel V; Wang, Yiwei; Li, Zhe; Maitz, Peter K M; McKenzie, David R; Bilek, Marcela M M; Weiss, Anthony S

    2011-08-01

    The interaction of proteins and cells with polymers is critical to their use in scientific and medical applications. In this study, plasma immersion ion implantation (PIII) was used to modify the surface of polytetrafluorethylene (PTFE), enabling the covalent binding of a cell adhesive protein, tropoelastin, without employing chemical linking molecules. Tropoelastin coating of untreated or PIII treated PFTE simultaneously promoted and blocked cell interactions respectively, i.e. PIII treatment of the PTFE surface completely inverses the cell interactive properties of bound tropoelastin. This activity persisted over long term storage of the PIII treated surfaces. The integrin binding C-terminus of tropoelastin was markedly less solvent exposed when bound to PIII treated PTFE than untreated PTFE, accounting for the modulation of cell adhesive activity. This presents a new methodology to specifically modulate cell behavior on a polymer surface using a simple one step treatment process, by adjusting the adhesive activity of a single extracellular matrix protein.

  14. Recent approaches in designing bioadhesive materials inspired by mussel adhesive protein

    PubMed Central

    Kord Forooshani, Pegah

    2016-01-01

    ABSTRACT Marine mussels secret protein‐based adhesives, which enable them to anchor to various surfaces in a saline, intertidal zone. Mussel foot proteins (Mfps) contain a large abundance of a unique, catecholic amino acid, Dopa, in their protein sequences. Catechol offers robust and durable adhesion to various substrate surfaces and contributes to the curing of the adhesive plaques. In this article, we review the unique features and the key functionalities of Mfps, catechol chemistry, and strategies for preparing catechol‐functionalized polymers. Specifically, we reviewed recent findings on the contributions of various features of Mfps on interfacial binding, which include coacervate formation, surface drying properties, control of the oxidation state of catechol, among other features. We also summarized recent developments in designing advanced biomimetic materials including coacervate‐forming adhesives, mechanically improved nano‐ and micro‐composite adhesive hydrogels, as well as smart and self‐healing materials. Finally, we review the applications of catechol‐functionalized materials for the use as biomedical adhesives, therapeutic applications, and antifouling coatings. © 2016 The Authors. Journal of Polymer Science Part A: Polymer Chemistry Published by Wiley Periodicals, Inc. J. Polym. Sci., Part A: Polym. Chem. 2017, 55, 9–33 PMID:27917020

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

    PubMed Central

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

    2005-01-01

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

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

    PubMed Central

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

    2013-01-01

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

  17. Hydrophobic enhancement of Dopa-mediated adhesion in a mussel foot protein

    PubMed Central

    Wei, Wei; Yu, Jing; Broomell, Christopher; Israelachvili, Jacob N.; Waite, J. Herbert

    2013-01-01

    Dopa (3,4-dihydroxyphenylalanine) is recognized as a key chemical signature of mussel adhesion and has been adopted into diverse synthetic polymer systems. Dopa’s notorious susceptibility to oxidation, however, poses significant challenges to the practical translation of mussel adhesion. Using a Surface Forces Apparatus to investigate the adhesion of Mfp3 (mussel foot protein 3) slow, a hydrophobic protein variant of the Mfp3 family in the plaque, we have discovered a subtle molecular strategy correlated with hydrophobicity that appears to compensate for Dopa instability. At pH 3, where Dopa is stable, Mfp3 slow like Mfp3 fast adhesion to mica is directly proportional to the mol% of Dopa present in the protein. At pH 5.5 and 7.5, however, loss of adhesion in Mfp3 slow was less than half that occurring in Mfp3 fast, purportedly because Dopa in Mfp3 slow is less prone to oxidation. Indeed, cyclic voltammetry showed that the oxidation potential of Dopa in Mfp3 slow is significantly higher than in Mfp3 fast at pH 7.5. A much greater difference between the two variants was revealed in the interaction energy of two symmetric Mfp3 slow films (Ead = −3 mJ/m2). This energy corresponds to the energy of protein cohesion which is notable for its reversibility and pH-independence. Exploitation of aromatic hydrophobic sequences to protect Dopa against oxidation as well as to mediate hydrophobic and H-bonding interactions between proteins provides new insights for developing effective artificial underwater adhesives. PMID:23214725

  18. Hydrophobic enhancement of Dopa-mediated adhesion in a mussel foot protein.

    PubMed

    Wei, Wei; Yu, Jing; Broomell, Christopher; Israelachvili, Jacob N; Waite, J Herbert

    2013-01-09

    Dopa (3,4-dihydroxyphenylalanine) is recognized as a key chemical signature of mussel adhesion and has been adopted into diverse synthetic polymer systems. Dopa's notorious susceptibility to oxidation, however, poses significant challenges to the practical translation of mussel adhesion. Using a surface forces apparatus to investigate the adhesion of mussel foot protein 3 (Mfp3) "slow", a hydrophobic protein variant of the Mfp3 family in the plaque, we have discovered a subtle molecular strategy correlated with hydrophobicity that appears to compensate for Dopa instability. At pH 3, where Dopa is stable, Mfp3 slow, like Mfp3 "fast" adhesion to mica, is directly proportional to the mol % of Dopa present in the protein. At pH of 5.5 and 7.5, however, loss of adhesion in Mfp3 slow was less than half that occurring in Mfp3 fast, purportedly because Dopa in Mfp3 slow is less prone to oxidation. Indeed, cyclic voltammetry showed that the oxidation potential of Dopa in Mfp3 slow is significantly higher than in Mfp3 fast at pH of 7.5. A much greater difference between the two variants was revealed in the interaction energy of two symmetric Mfp3 slow films (E(ad) = -3 mJ/m(2)). This energy corresponds to the energy of protein cohesion which is notable for its reversibility and pH independence. Exploitation of aromatic hydrophobic sequences to protect Dopa against oxidation as well as to mediate hydrophobic and H-bonding interactions between proteins provides new insights for developing effective artificial underwater adhesives.

  19. Adhesion dynamics of porcine esophageal fibroblasts on extracellular matrix protein-functionalized poly(lactic acid).

    PubMed

    Cai, Ning; Gong, Yingxue; Chian, Kerm Sin; Chan, Vincent; Liao, Kin

    2008-03-01

    Effective attachment of esophageal cells on biomaterials is one important requirement in designing engineered esophagus substitute for esophageal cancer treatment. In this study, poly(lactic acid) (PLA) was subjected to surface modification by coupling extracellular matrix (ECM) proteins on its surface to promote cell adhesion. Two typical ECM proteins, collagen type I (COL) and fibronectin (FN), were immobilized on the PLA surface with the aid of glutaraldehyde as a cross linker between aminolyzed PLA and ECM proteins. By using confocal reflectance interference contrast microscopy (C-RICM) integrating with phase contrast microscopy, the long-term adhesion dynamics of porcine esophageal fibroblasts (PEFs) on four types of surfaces (unmodified PLA, PLA-COOH, PLA-COL and PLA-FN) was investigated during 24 h of culture. It is demonstrated by C-RICM results that PEFs form strong adhesion contact on all four types of surfaces at different stages of cell seeding. Among the four surfaces, PEFs on the PLA-FN surface reach the maximum adhesion energy (9.5 x 10(-7) J m(-2)) in the shortest time (20 min) during the initial stage of cell seeding. After adhesion energy reaches the maximum value, PEFs maintain their highly deformed geometries till they reached a steady state after 20 h of culture. F-actin immunostaining results show that the evolvement of spatial organization of F-actin is tightly correlated with the formation of adhesion contact and cell spreading. Furthermore, the cell attachment ratio of PEFs on PLA in 2 h is only 26% compared with 88% on PLA-FN, 73% on PLA-COL and 36% on PLA-COOH. All the results demonstrate the effect of surface functionalization on the biophysical responses of PEFs in cell adhesion. Fibronectin-immobilized PLA demonstrates promising potential for application as an engineered esophagus substitute.

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

    PubMed Central

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

    2014-01-01

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

  1. 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.

  2. Talin determines the nanoscale architecture of focal adhesions

    PubMed Central

    Liu, Jaron; Wang, Yilin; Goh, Wah Ing; Goh, Honzhen; Baird, Michelle A.; Ruehland, Svenja; Teo, Shijia; Bate, Neil; Critchley, David R.; Davidson, Michael W.; Kanchanawong, Pakorn

    2015-01-01

    Insight into how molecular machines perform their biological functions depends on knowledge of the spatial organization of the components, their connectivity, geometry, and organizational hierarchy. However, these parameters are difficult to determine in multicomponent assemblies such as integrin-based focal adhesions (FAs). We have previously applied 3D superresolution fluorescence microscopy to probe the spatial organization of major FA components, observing a nanoscale stratification of proteins between integrins and the actin cytoskeleton. Here we combine superresolution imaging techniques with a protein engineering approach to investigate how such nanoscale architecture arises. We demonstrate that talin plays a key structural role in regulating the nanoscale architecture of FAs, akin to a molecular ruler. Talin diagonally spans the FA core, with its N terminus at the membrane and C terminus demarcating the FA/stress fiber interface. In contrast, vinculin is found to be dispensable for specification of FA nanoscale architecture. Recombinant analogs of talin with modified lengths recapitulated its polarized orientation but altered the FA/stress fiber interface in a linear manner, consistent with its modular structure, and implicating the integrin–talin–actin complex as the primary mechanical linkage in FAs. Talin was found to be ∼97 nm in length and oriented at ∼15° relative to the plasma membrane. Our results identify talin as the primary determinant of FA nanoscale organization and suggest how multiple cellular forces may be integrated at adhesion sites. PMID:26283369

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

    PubMed

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

    2014-11-01

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

  4. Evidence for a positive regulatory role of strawberry (Fragaria x ananassa) Fa WRKY1 and Arabidopsis At WRKY75 proteins in resistance.

    PubMed

    Encinas-Villarejo, Sonia; Maldonado, Ana M; Amil-Ruiz, Francisco; de los Santos, Berta; Romero, Fernando; Pliego-Alfaro, Fernando; Muñoz-Blanco, Juan; Caballero, José L

    2009-01-01

    Knowledge of the molecular basis of plant resistance to pathogens in species other than Arabidopsis is limited. The function of Fa WRKY1, the first WRKY gene isolated from strawberry (Fragaria x ananassa), an important agronomical fruit crop, has been investigated here. Fa WRKY1 encodes a IIc WRKY transcription factor and is up-regulated in strawberry following Colletotrichum acutatum infection, treatments with elicitors, and wounding. Its Arabidopsis sequence homologue, At WRKY75, has been described as playing a role in regulating phosphate starvation responses. However, using T-DNA insertion mutants, a role for the At WRKY75 and Fa WRKY1 in the activation of basal and R-mediated resistance in Arabidopsis is demonstrated. At wrky75 mutants are more susceptible to virulent and avirulent isolates of Pseudomonas syringae. Overexpression of Fa WRKY1 in At wrky75 mutant and wild type reverts the enhanced susceptible phenotype of the mutant, and even increases resistance to avirulent strains of P. syringae. The resistance phenotype is uncoupled to PATHOGENESIS-RELATED (PR) gene expression, but it is associated with a strong oxidative burst and glutathione-S-transferase (GST) induction. Taken together, these results indicate that At WRKY75 and Fa WRKY1 act as positive regulators of defence during compatible and incompatible interactions in Arabidopsis and, very likely, Fa WRKY1 is an important element mediating defence responses to C. acutatum in strawberry. Moreover, these results provide evidence that Arabidopsis can be a useful model for functional studies in Rosacea species like strawberry.

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

    PubMed

    Demais, V; Audrain, C; Mabilleau, G; Chappard, D; Baslé, 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.

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

    PubMed Central

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

    1999-01-01

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

  7. 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...

  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. Comparison of the adhesive performances of soy meal, water washed meal fractions, and protein isolates

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Adhesive bonding of wood plays an increasing role in the forest products industry and is a key factor for efficiently utilizing timber and other lignocellulosic resources. In this work, we obtained five soy meal products through commercial sources or in-house preparations. The protein content was 49...

  10. Dominant-negative effect on adhesion by myelin Po protein truncated in its cytoplasmic domain

    PubMed Central

    1996-01-01

    The myelin Po protein is believed to hold myelin together via interactions of both its extracellular and cytoplasmic domains. We have already shown that the extracellular domains of Po can interact in a homophilic manner (Filbin, M.T., F.S. Walsh, B.D. Trapp, J.A. Pizzey, and G.I. Tennekoon. 1990. Nature (Lond.). 344:871-872). In addition, we have shown that for this homophilic adhesion to take place, the cytoplasmic domain of Po must be intact and most likely interacting with the cytoskeleton; Po proteins truncated in their cytoplasmic domains are not adhesive (Wong, M.H., and M.T. Filbin, 1994. J. Cell Biol. 126:1089-1097). To determine if the presence of these truncated forms of Po could have an effect on the functioning of the full-length Po, we coexpressed both molecules in CHO cells. The adhesiveness of CHO cells expressing both full-length Po and truncated Po was then compared to cells expressing only full-length Po. In these coexpressors, both the full-length and the truncated Po proteins were glycosylated. They reached the surface of the cell in approximately equal amounts as shown by an ELISA and surface labeling, followed by immunoprecipitation. Furthermore, the amount of full-length Po at the cell surface was equivalent to other cell lines expressing only full-length Po that we had already shown to be adhesive. Therefore, there should be sufficient levels of full-length Po at the surface of these coexpressors to measure adhesion of Po. However, as assessed by an aggregation assay, the coexpressors were not adhesive. By 60 min they had not formed large aggregates and were indistinguishable from the control transfected cells not expressing Po. In contrast, in the same time, the cells expressing only the full-length Po had formed large aggregates. This indicates that the truncated forms of Po have a dominant-negative effect on the adhesiveness of the full-length Po. Furthermore, from cross-linking studies, full-length Po, when expressed alone but not when

  11. Therapy with hydroxyurea is associated with reduced adhesion molecule gene and protein expression in sickle red cells with a concomitant reduction in adhesive properties.

    PubMed

    Gambero, Sheley; Canalli, Andreia A; Traina, Fabiola; Albuquerque, Dulcinéia M; Saad, Sara T O; Costa, Fernando F; Conran, Nicola

    2007-02-01

    Propagation of the vaso-occlusive process in sickle cell anaemia (SCA) is a complex process involving the adhesion of steady-state SCA patients red cells and reticulocytes to the vascular endothelium. The effect of hydroxyurea therapy (HUT) on the adhesive properties of sickle cells and the expression of adhesion molecule genes by erythroid cells of SCA individuals is not yet fully understood. The expressions of the CD36 gene and the VLA-4-integrin subunit genes, CD49d (alpha-subunit) and CD29 (beta-subunit), were compared in the reticulocytes of steady-state SCA patients and patients on HUT using real-time PCR. Basal adhesion of red cells from these subjects was also compared using static adhesion assays, as was surface protein expression, using flow cytometry. Basal sickle red cell adhesion to fibronectin was significantly greater than that of normal cells (P < 0.01); in contrast, HUT was associated with significantly lower levels (P < 0.01) of red cell adhesion that were similar to those of control cells; this decrease could not be justified solely by altered reticulocyte numbers in this population. Accordingly, flow cytometry demonstrated that reticulocytes from patients on HUT had significantly lower CD36 and CD49d surface expressions (P < 0.01) and, importantly, significantly lower expressions of the CD36, CD49d and CD29 genes (P < 0.05) than reticulocytes of SCA patients not on HUT. Taken together, data support the hypothesis that HUT reduces the adhesive properties of sickle cells and that this decrease appears to be mediated, at least in part, by a decrease in the gene and, consequently, surface protein expression of adhesion molecules such as VLA-4 and CD36.

  12. Nuclear magnetic resonance spectroscopy of mussel adhesive protein repeating peptide segment.

    PubMed

    Olivieri, M P; Wollman, R M; Alderfer, J L

    1997-12-01

    Mussel adhesive protein (MAP) is the adhesive agent used by the common blue sea mussel (Mytilus edulis) to attach the animal to various underwater surfaces. It is generally composed of 75 to 85 repeating decameric units with the reported primary sequence NH2-Ala(1)-Lyst(2)-Pro(3)-Ser(4)-Tyr(5)-Hyp(6)-Hyp(7)-Thr(8)-DOPA( 9)- Lys(10)-COOH. This study examines this peptide's solution-state conformation using proton nuclear magnetic resonance (NMR) spectroscopy. NMR and molecular modeling of the decamer before and after molecular dynamics calculations in water suggests a conformation that retains an overall bent helix.

  13. An Underwater Surface-Drying Peptide Inspired by a Mussel Adhesive Protein

    PubMed Central

    Wei, Wei; Petrone, Luigi; Tan, YerPeng; Cai, Hao; Israelachvili, Jacob N.; Miserez, Ali

    2016-01-01

    Water hampers the formation of strong and durable bonds between adhesive polymers and solid surfaces, in turn hindering the development of adhesives for biomedical and marine applications. Inspired by mussel adhesion, a mussel foot protein homologue (mfp3S-pep) is designed, whose primary sequence is designed to mimic the pI, polyampholyte, and hydrophobic characteristics of the native protein. Noticeably, native protein and synthetic peptide exhibit similar abilities to self-coacervate at given pH and ionic strength. 3,4-dihydroxy-l-phenylalanine (Dopa) proves necessary for irreversible peptide adsorption to both TiO2 (anatase) and hydroxyapatite (HAP) surfaces, as confirmed by quartz crystal microbalance measurements, with the coacervate showing superior adsorption. The adsorption of Dopa-containing peptides is investigated by attenuated total reflection infrared spectroscopy, revealing initially bidentate coordinative bonds on TiO2, followed by H-bonded and eventually long-ranged electrostatic and Van der Waals interactions. On HAP, mfp3s-pep-3Dopa adsorption occurs predominantly via H-bond and outer-sphere complexes of the catechol groups. Importantly, only the Dopa-bearing compounds are able to remove interfacial water from the target surfaces, with the coacervate achieving the highest water displacement arising from its superior wetting properties. These findings provide an impetus for developing coacervated Dopa-functionalized peptides/polymers adhesive formulations for a variety of applications on wet polar surfaces. PMID:27840600

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

    PubMed

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

    2015-10-01

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

  15. 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.

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

    PubMed

    Yang, Dayun; Lü, 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-β 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.

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

    PubMed

    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.

  18. 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

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

    PubMed

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

    2009-07-01

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

  20. OmpA-like protein influences cell shape and adhesive activity of Tannerella forsythia.

    PubMed

    Abe, T; Murakami, Y; Nagano, K; Hasegawa, Y; Moriguchi, K; Ohno, N; Shimozato, K; Yoshimura, F

    2011-12-01

    Tannerella forsythia, a gram-negative fusiform rod, is implicated in several types of oral anaerobic infections. Most gram-negative bacteria have OmpA-like proteins that are homologous to the OmpA protein in Escherichia coli. We identified an OmpA-like protein in T. forsythia encoded by the tf1331 gene as one of the major proteins by mass spectrometric analysis. Two-dimensional, diagonal electrophoresis showed that the OmpA-like protein formed a dimeric or trimeric structure via intermolecular disulfide bonds. A biotin labeling experiment revealed that a portion of the protein was exposed on the cell surface, even though T. forsythia possesses an S-layer at the outermost cell surface. Using a tf1331-deletion mutant, we showed that the OmpA-like protein affected cell morphology. The length of the mutant cell was reduced almost by half. Cell swelling was observed in more than 40% of the mutant cells. Moreover, the mutant exhibited decreased adhesion to fibronectin, retarded autoaggregation, and reduced cell surface hydrophobicity. These results suggest that the OmpA-like protein in T. forsythia plays an important role in cellular integrity and adhesive function.

  1. 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.

  2. 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.

  3. Extracellular matrix protein patterns guide human chondrocytes adhesion and alignment characterized by vimentin and matrilin-3.

    PubMed

    Pan, Chang-Jiang; Ding, Hong-Yan; Dong, Yun-Xiao

    2013-02-01

    The main purpose of the present study is to investigate the influences of collagen VI (col-VI) patterns on human chondrocytes behaviors. To this end, col-VI stripes with varying width and interstripe spacing are created on polystyrene (PS) surfaces by microcontact printing (μCP). Human chondrocytes are then seeded on these protein patterns and the cell adhesion and alignment are investigated by staining the vimentin and matrilin-3 secreted by seeded chondrocytes. The results indicate that the cells preferentially attach onto the protein areas, rendering cell patterns and the elongated cell shapes. The pattern dimensions can significantly influence cell adhesion, spreading and orientation. The stripe protein patterns can guide cell adhesion and alignment. The cell morphologies can be controlled by carefully designing the pattern shapes and sizes. Our results suggest that the protein patterns can be used to modify biomaterials' surfaces for selective cell-binding and cell alignment. It could provide some cues for the development of novel implantable biomaterials, such as tissue-engineered scaffolds for cartilage replacement, where specific cell alignment is needed.

  4. 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...

  5. TSOL18/HP6-Tsol, an immunogenic Taenia solium oncospheral adhesion protein and potential protective antigen.

    PubMed

    Parkhouse, R Michael E; Bonay, Pedro; González, Luis Miguel; Ferrer, Elizabeth; Gárate, Teresa; Aguilar, Cruz M; Cortez A, Milagros M; Harrison, Leslie J S

    2008-04-01

    In this study, we employed Taenia solium mRNA extracted from a tapeworm of Venezuelan origin to clone express and test the recombinant protein of the T. solium homologue of the 18-kDa oncospheral adhesion molecule of Taenia saginata (HP6-Tsag/TSA18). We first confirm the conserved nature of the sequence of the T. solium homologue (TSOL18/HP6-Tsol) and demonstrate that the recombinant protein, which, as with its T. saginata homologue, is characterised by a fibronectin type III homology region, functions as an adhesion molecule. This emphasises the possible importance of TSOL18/HP6-Tsol in tissue invasion, thus providing a rational explanation for its efficacy as a vaccine. As protection against Taenia spp., oncospheres is antibody mediated, logically, therefore, TSOL18/HP6-Tsol may also serve as a diagnostic antigen, as is indeed the case for recombinant HP6-Tsag/TSA18.

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

    NASA Astrophysics Data System (ADS)

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

    2008-12-01

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

  7. Adhesion of phospholipid vesicles to Chinese hamster fibroblasts: Role of cell surface proteins

    PubMed Central

    Pagano, RE; Takeichi, M

    1977-01-01

    The adhesion of artificially generated lipid membrane vesicles to Chinese hamster V79 fibroblasts in suspension was used as a model system for studying membrane interactions. Below their gel-liquid crystalline phase transition temperature, vesicles comprised of dipalmitoyl lecithin (DPL) or dimyristoyl lecithin (DML) absorbed to the surfaces of EDTA- dissociated cells. These adherent vesicles could not be removed by repeated washings of the treated cells but could be released into the medium by treatment with trypsin. EM autoradiographic studies of cells treated with[(3)H]DML or [(3)H]DPL vesicles showed that most of the radioactive lipids were confined to the cell periphery. Scanning electron microscopy and fluorescence microscopy further confirmed the presence of adherent vesicles at the cell surface. Adhesion of DML or DPL vesicles to EDTA-dissociated cells modified the lactoperoxidase-catalyzed iodination pattern of the cell surface proteins; the inhibition of labeling of two proteins with an approximately 60,000- dalton mol wt was particularly evident. Incubation of cells wit h (3)H-lipid vesicles followed by sodium dodecyl sulfate (SDS)- polyacrylamide gel electrophoresis showed that some of the (3)H-lipid migrated preferentially with these approximately 60,000-mol wt proteins. Studies of the temperature dependence of vesicle uptake and subsequent release by trypsin showed that DML or DPL vesicle adhesion to EDTA- dissociated cells increased with decreasing temperatures. In contrast, cells trypsinized before incubation with vesicles showed practically no temperature dependence of vesicle uptake. These results suggest two pathways for adhesion of lipid vesicles to the cell surface-a temperature-sensitive one involving cell surface proteins, and a temperature-independent one. These findings are discussed in terms of current models for cell-cell interactions. PMID:407233

  8. Tunable Adsorption and Film Formation of Mussel Adhesive Protein by Potential Control.

    PubMed

    Zhang, Fan; Xie, Guoxin; Pan, Jinshan

    2017-01-23

    Mussel adhesive proteins are of great interest in many applications because of their outstanding adhesive property and film-forming ability. Understanding and controlling the film formation and its performance is crucial for the effective use of such proteins. In this study, we focus on the potential controlled film formation and compaction of one mussel adhesive protein, Mefp-1. The adsorption and film-forming behavior of Mefp-1 on a platinum (Pt) substrate under applied potentials were investigated by cyclic voltammetry, potential-controlled electrochemical impedance spectroscopy (EIS), and quartz crystal microbalance with dissipation monitoring (QCM-D). Moreover, microfriction measurements were performed to evaluate the mechanical properties of the Mefp-1 films formed at selected potentials. The results led to the conclusion that Mefp-1 adsorbs on the Pt substrate through both electrostatic and nonelectrostatic interactions and shows an effective blocking effect for the electroactive sites on the substrate. The properties of the adsorbed Mefp-1 film vary with the applied potential, and the compactness of the adsorbed Mefp-1 film can be reversibly tuned by the applied potential.

  9. 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

  10. Lectin receptor kinases participate in protein-protein interactions to mediate plasma membrane-cell wall adhesions in Arabidopsis.

    PubMed

    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.

  11. Adhesion-modulating/matricellular ECM protein families: a structural, functional and evolutionary appraisal.

    PubMed

    Mosher, Deane F; Adams, Josephine C

    2012-04-01

    The thrombospondins are a family of secreted, oligomeric glycoproteins that interact with cell surfaces, multiple components of the extracellular matrix, growth factors and proteases. These interactions underlie complex roles in cell interactions and tissue homeostasis in animals. Thrombospondins have been grouped functionally with SPARCs, tenascins and CCN proteins as adhesion-modulating or matricellular components of the extracellular milieu. Although all these multi-domain proteins share various commonalities of domains, the grouping is not based on structural homologies. Instead, the terms emphasise the general observations that these proteins do not form large-scale ECM structures, yet act at cell surfaces and function in coordination with the structural ECM and associated extracellular proteins. The designation of adhesion-modulation thus depends on observed tissue and cell culture ECM distributions and on experimentally identified functional properties. To date, the evolutionary relationships of these proteins have not been critically compared: yet, knowledge of their evolutionary histories is clearly relevant to any consideration of functional similarities. In this article, we survey briefly the structural and functional knowledge of these protein families, consider the evolution of each family, and outline a perspective on their functional roles.

  12. The glue protein of ribbed mussels (Geukensia demissa): a natural adhesive with some features of collagen.

    PubMed

    Waite, J H; Hansen, D C; Little, K T

    1989-01-01

    The Atlantic ribbed mussel Geukensia (Modiolus) demissa attaches itself to the roots of cord grass and other hard objects in tidal salt marshes by spinning adhesive byssal threads. The precursor of a protein apparently present in the adhesive plaques of the threads was isolated in quantity from the foot of the mussel. The protein has an apparent molecular weight of 130,000, a pI of 8.1, and contains a high proportion of Gly, Glu/Gln, Lys and 3,4-dihydroxyphenyl-L-alanine (DOPA). Sequence of tryptic peptides suggests a pattern of repeated motifs, such as: Gly--DOPA--Lys, and X--Gly--DOPA--Y--Z--Gly--DOPA/Tyr--Lys, where X is Thr or Ala in octapeptides and Gln--Thr in nonapeptides. Y is variable, but more often than not hydrophobic; and Z is frequently Pro or 4-trans-hydroxyproline (Hyp). The presence of Pro--Gly and Hyp--Gly sequences of delta-hydroxylysine in the protein is reminiscent of typical collagens; however, the protein is not labile to clostridial collagenase, nor does collagen cross-react with antibodies raised against the mussel protein. Unlike typical collagens, Gly probably occurs only at every 4th or 5th residue in this unusual mussel protein.

  13. 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.

  14. Identification of surface proteins involved in the adhesion of a probiotic Bacillus cereus strain to mucin and fibronectin.

    PubMed

    Sánchez, B; Arias, S; Chaignepain, S; Denayrolles, M; Schmitter, J M; Bressollier, P; Urdaci, M C

    2009-05-01

    Several Bacillus strains isolated from commercial probiotic preparations were identified at the species level, and their adhesion capabilities to three different model intestinal surfaces (mucin, Matrigel and Caco-2 cells) were assessed. In general, adhesion of spores was higher than that of vegetative cells to the three matrices, and overall strain Bacillus cereus(CH) displayed the best adhesion. Different biochemical treatments revealed that surface proteins of B. cereus(CH) were involved in the adhesion properties of the strain. Surface-associated proteins from vegetative cells and spores of B. cereus(CH) were extracted and identified, and some proteins such as S-layer components, flagellin and cell-bound proteases were found to bind to mucin or fibronectin. These facts suggest that those proteins might play important roles in the interaction of this probiotic Bacillus strain within the human gastrointestinal tract.

  15. Control of high affinity interactions in the talin C terminus: how talin domains coordinate protein dynamics in cell adhesions.

    PubMed

    Himmel, Mirko; Ritter, Anett; Rothemund, Sven; Pauling, Björg V; Rottner, Klemens; Gingras, Alexandre R; Ziegler, Wolfgang H

    2009-05-15

    In cell-extracellular matrix junctions (focal adhesions), the cytoskeletal protein talin is central to the connection of integrins to the actin cytoskeleton. Talin is thought to mediate this connection via its two integrin, (at least) three actin, and several vinculin binding sites. The binding sites are cryptic in the head-to-rod autoinhibited cytoplasmic form of the protein and require (stepwise) conformational activation. This activation process, however, remains poorly understood, and there are contradictory models with respect to the determinants of adhesion site localization. Here, we report turnover rates and protein-protein interactions in a range of talin rod domain constructs varying in helix bundle structure. We conclude that several bundles of the C terminus cooperate to regulate targeting and concomitantly tailor high affinity interactions of the talin rod in cell adhesions. Intrinsic control of ligand binding activities is essential for the coordination of adhesion site function of talin.

  16. 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

  17. 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.

  18. 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.

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

    PubMed

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

    2015-01-01

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

  20. Processing of mussel-adhesive protein analog copolymer thin films by matrix-assisted pulsed laser evaporation

    NASA Astrophysics Data System (ADS)

    Patz, T.; Cristescu, R.; Narayan, R.; Menegazzo, N.; Mizaikoff, B.; Messersmith, P. B.; Stamatin, I.; Mihailescu, I. N.; Chrisey, D. B.

    2005-07-01

    We have demonstrated the successful thin film growth of a mussel-adhesive protein analog, DOPA-modified PEO-PPO-PEO block copolymer PF127, using matrix-assisted pulsed laser evaporation (MAPLE). The MAPLE-deposited thin films were examined using Fourier transform infrared spectroscopy, atomic force microscopy, X-ray photoelectron spectroscopy, and contact-angle measurements. We have found that the main functional groups of the mussel-adhesive protein analog are present in the transferred film. These adhesive materials have several potential electronic, medical, and marine applications.

  1. A genome-wide screen identifies conserved protein hubs required for cadherin-mediated cell–cell adhesion

    PubMed Central

    Toret, Christopher P.; D’Ambrosio, 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-cadherin–expressing Drosophila melanogaster S2 cells in suspension culture. This novel screen eliminated Ca2+-independent cell–cell adhesion, integrin-based adhesion, cell spreading, and cell migration. We identified 17 interconnected regulatory hubs, based on protein functions and protein–protein interactions that regulate the levels of the core cadherin–catenin complex and coordinate cadherin-mediated cell–cell adhesion. Representative proteins from these hubs were analyzed further in Drosophila oogenesis, using targeted germline RNAi, and adhesion was analyzed in Madin–Darby canine kidney mammalian epithelial cell–cell adhesion. These experiments reveal roles for a diversity of cellular pathways that are required for cadherin function in Metazoa, including cytoskeleton organization, cell–substrate interactions, and nuclear and cytoplasmic signaling. PMID:24446484

  2. 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 VWF’s 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.

  3. GEF-H1 controls focal adhesion signaling that regulates mesenchymal stem cell lineage commitment

    PubMed Central

    Huang, I-Husan; Hsiao, Cheng-Te; Wu, Jui-Chung; Liu, Ching-Yi; Wang, Yang-Kao; Chen, Yu-Chen; Huang, Chi-Ming; del álamo, Juan C.; Chang, Zee-Fen; Tang, Ming-Jer; Khoo, Kay-Hooi; Kuo, Jean-Cheng

    2014-01-01

    ABSTRACT Focal adhesions (FAs) undergo maturation that culminates in size and composition changes that modulate adhesion, cytoskeleton remodeling and differentiation. Although it is well recognized that stimuli for osteogenesis of mesenchymal stem cells (MSCs) drive FA maturation, actin organization and stress fiber polarization, the extent to which FA-mediated signals regulated by the FA protein composition specifies MSC commitment remains largely unknown. Here, we demonstrate that, upon dexamethasone (osteogenic induction) treatment, guanine nucleotide exchange factor H1 (GEF-H1, also known as Rho guanine nucleotide exchange factor 2, encoded by ARHGEF2) is significantly enriched in FAs. Perturbation of GEF-H1 inhibits FA formation, anisotropic stress fiber orientation and MSC osteogenesis in an actomyosin-contractility-independent manner. To determine the role of GEF-H1 in MSC osteogenesis, we explore the GEF-H1-modulated FA proteome that reveals non-muscle myosin-II heavy chain-B (NMIIB, also known as myosin-10, encoded by MYH10) as a target of GEF-H1 in FAs. Inhibition of targeting NMIIB into FAs suppresses FA formation, stress fiber polarization, cell stiffness and osteogenic commitments in MSCs. Our data demonstrate a role for FA signaling in specifying MSC commitment. PMID:25107365

  4. Aire knockdown in medullary thymic epithelial cells affects Aire protein, deregulates cell adhesion genes and decreases thymocyte interaction.

    PubMed

    Pezzi, Nicole; Assis, Amanda Freire; Cotrim-Sousa, Larissa Cotrim; Lopes, Gabriel Sarti; Mosella, Maritza Salas; Lima, Djalma Sousa; Bombonato-Prado, Karina F; Passos, Geraldo Aleixo

    2016-09-01

    We demonstrate that even a partial reduction of Aire mRNA levels by siRNA-induced Aire knockdown (Aire KD) has important consequences to medullary thymic epithelial cells (mTECs). Aire knockdown is sufficient to reduce Aire protein levels, impair its nuclear location, and cause an imbalance in large-scale gene expression, including genes that encode cell adhesion molecules. These genes drew our attention because adhesion molecules are implicated in the process of mTEC-thymocyte adhesion, which is critical for T cell development and the establishment of central self-tolerance. Accordingly, we consider the following: 1) mTECs contribute to the elimination of self-reactive thymocytes through adhesion; 2) Adhesion molecules play a crucial role during physical contact between these cells; and 3) Aire is an important transcriptional regulator in mTECs. However, its role in controlling mTEC-thymocyte adhesion remains unclear. Because Aire controls adhesion molecule genes, we hypothesized that the disruption of its expression could influence mTEC-thymocyte interaction. To test this hypothesis, we used a murine Aire(+) mTEC cell line as a model system to reproduce mTEC-thymocyte adhesion in vitro. Transcriptome analysis of the mTEC cell line revealed that Aire KD led to the down-modulation of more than 800 genes, including those encoding for proteins involved in cell adhesion, i.e., the extracellular matrix constituent Lama1, the CAM family adhesion molecules Vcam1 and Icam4, and those that encode peripheral tissue antigens. Thymocytes co-cultured with Aire KD mTECs had a significantly reduced capacity to adhere to these cells. This finding is the first direct evidence that Aire also plays a role in controlling mTEC-thymocyte adhesion.

  5. A small fibronectin-mimicking protein from bacteria induces cell spreading and focal adhesion formation.

    PubMed

    Tegtmeyer, Nicole; Hartig, Roland; Delahay, Robin M; Rohde, Manfred; Brandt, Sabine; Conradi, Jens; Takahashi, Seiichiro; Smolka, Adam J; Sewald, Norbert; Backert, Steffen

    2010-07-23

    Fibronectin, a 250-kDa eukaryotic extracellular matrix protein containing an RGD motif plays crucial roles in cell-cell communication, development, tissue homeostasis, and disease development. The highly complex fibrillar fibronectin meshwork orchestrates the functions of other extracellular matrix proteins, promoting cell adhesion, migration, and intracellular signaling. Here, we demonstrate that CagL, a 26-kDa protein of the gastric pathogen and type I carcinogen Helicobacter pylori, mimics fibronectin in various cellular functions. Like fibronectin, CagL contains a RGD motif and is located on the surface of the bacterial type IV secretion pili as previously shown. CagL binds to the integrin receptor alpha(5)beta(1) and mediates the injection of virulence factors into host target cells. We show that purified CagL alone can directly trigger intracellular signaling pathways upon contact with mammalian cells and can complement the spreading defect of fibronectin(-/-) knock-out cells in vitro. During interaction with various human and mouse cell lines, CagL mimics fibronectin in triggering cell spreading, focal adhesion formation, and activation of several tyrosine kinases in an RGD-dependent manner. Among the activated factors are the nonreceptor tyrosine kinases focal adhesion kinase and Src but also the epidermal growth factor receptor and epidermal growth factor receptor family member Her3/ErbB3. Interestingly, fibronectin activates a similar range of tyrosine kinases but not Her3/ErbB3. These findings suggest that the bacterial protein CagL not only exhibits functional mimicry with fibronectin but is also capable of activating fibronectin-independent signaling events. We thus postulate that CagL may contribute directly to H. pylori pathogenesis by promoting aberrant signaling cross-talk within host cells.

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

    PubMed

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

    2016-04-05

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

  7. 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.

  8. Glutamate Receptor Interacting Protein 1 Mediates Platelet Adhesion and Thrombus Formation

    PubMed Central

    Modjeski, Kristina L.; Ture, Sara K.; Field, David J.; Cameron, Scott J.; Morrell, Craig N.

    2016-01-01

    Thrombosis-associated pathologies, such as myocardial infarction and stroke, are major causes of morbidity and mortality worldwide. Because platelets are necessary for hemostasis and thrombosis, platelet directed therapies must balance inhibiting platelet function with bleeding risk. Glutamate receptor interacting protein 1 (GRIP1) is a large scaffolding protein that localizes and organizes interacting proteins in other cells, such as neurons. We have investigated the role of GRIP1 in platelet function to determine its role as a molecular scaffold in thrombus formation. Platelet-specific GRIP1-/- mice were used to determine the role of GRIP1 in platelets. GRIP1-/- mice had normal platelet counts, but a prolonged bleeding time and delayed thrombus formation in a FeCl3-induced vessel injury model. In vitro stimulation of WT and GRIP1-/- platelets with multiple agonists showed no difference in platelet activation. However, in vivo platelet rolling velocity after endothelial stimulation was significantly greater in GRIP1-/- platelets compared to WT platelets, indicating a potential platelet adhesion defect. Mass spectrometry analysis of GRIP1 platelet immunoprecipitation revealed enrichment of GRIP1 binding to GPIb-IX complex proteins. Western blots confirmed the mass spectrometry findings that GRIP1 interacts with GPIbα, GPIbβ, and 14-3-3. Additionally, in resting GRIP1-/- platelets, GPIbα and 14-3-3 have increased interaction compared to WT platelets. GRIP1 interactions with the GPIb-IX binding complex are necessary for normal platelet adhesion to a stimulated endothelium. PMID:27631377

  9. 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.

  10. Organization of pp60src and selected cytoskeletal proteins within adhesion plaques and junctions of Rous sarcoma virus-transformed rat cells

    PubMed Central

    1981-01-01

    The localization of pp60src within adhesion structures of epithelioid rat kidney cells transformed by the Schmidt-Ruppin strain of Rous sarcoma virus was compared to the organization of actin, alpha-actinin, vinculin (a 130,000-dalton protein), tubulin, and the 58,000-dalton intermediate filament protein. The adhesion structures included both adhesion plaques and previously uncharacterized adhesive regions formed at cell-cell junctions. We have termed these latter structures "adhesion junctions." Both adhesion plaques and adhesion junctions were identified by interference-reflection microscopy and compared to the location of pp60src and the various cytoskeletal proteins by double fluorescence. The results demonstrated that the src gene product was found within both adhesion plaques and the adhesion junctions. In addition, actin, alpha-actinin, and vinculin were also localized within the same pp60src-containing adhesion structures. In contrast, tubulin and the 58,000-dalton intermediate filament protein were not associated with either adhesion plaques or adhesion junctions. Both adhesion plaques and adhesion junctions were isolated as substratum-bound structures and characterized by scanning electron microscopy. Immunofluorescence revealed that pp60src, actin, alpha-actinin, and vinculin were organized within specific regions of the adhesion junctions. Heavy accumulations of actin and alpha-actinin were found on both sides of the junctions with a narrow gap of unstained material at the midline, whereas pp60src stain was more intense in this central region. Antibody to vinculin stained double narrow lines defining the periphery of the junctional complexes but was excluded from the intervening region. In addition, the distribution of vinculin relative to pp60src within adhesion plaques suggested an inverse relationship between the presence of these two proteins. Overall, these results establish a close link between the src gene product and components of the

  11. Proper Control of Caulobacter crescentus Cell Surface Adhesion Requires the General Protein Chaperone DnaK

    PubMed Central

    Eaton, Daniel S.; Crosson, Sean

    2016-01-01

    ABSTRACT Growth in a surface-attached bacterial community, or biofilm, confers a number of advantages. However, as a biofilm matures, high-density growth imposes stresses on individual cells, and it can become less advantageous for progeny to remain in the community. Thus, bacteria employ a variety of mechanisms to control attachment to and dispersal from surfaces in response to the state of the environment. The freshwater oligotroph Caulobacter crescentus can elaborate a polysaccharide-rich polar organelle, known as the holdfast, which enables permanent surface attachment. Holdfast development is strongly inhibited by the small protein HfiA; mechanisms that control HfiA levels in the cell are not well understood. We have discovered a connection between the essential general protein chaperone, DnaK, and control of C. crescentus holdfast development. C. crescentus mutants partially or completely lacking the C-terminal substrate binding “lid” domain of DnaK exhibit enhanced bulk surface attachment. Partial or complete truncation of the DnaK lid domain increases the probability that any single cell will develop a holdfast by 3- to 10-fold. These results are consistent with the observation that steady-state levels of an HfiA fusion protein are significantly diminished in strains that lack the entire lid domain of DnaK. While dispensable for growth, the lid domain of C. crescentus DnaK is required for proper chaperone function, as evidenced by observed dysregulation of HfiA and holdfast development in strains expressing lidless DnaK mutants. We conclude that DnaK is an important molecular determinant of HfiA stability and surface adhesion control. IMPORTANCE Regulatory control of cell adhesion ensures that bacterial cells can transition between free-living and surface-attached states. We define a role for the essential protein chaperone, DnaK, in the control of Caulobacter crescentus cell adhesion. C. crescentus surface adhesion is mediated by an envelope

  12. Adhesion of Type 1-Fimbriated Escherichia coli to Abiotic Surfaces Leads to Altered Composition of Outer Membrane Proteins

    PubMed Central

    Otto, Karen; Norbeck, Joakim; Larsson, Thomas; Karlsson, Karl-Anders; Hermansson, Malte

    2001-01-01

    Phenotypic differences between planktonic bacteria and those attached to abiotic surfaces exist, but the mechanisms involved in the adhesion response of bacteria are not well understood. By the use of two-dimensional (2D) polyacrylamide gel electrophoresis, we have demonstrated that attachment of Escherichia coli to abiotic surfaces leads to alteration in the composition of outer membrane proteins. A major decrease in the abundance of resolved proteins was observed during adhesion of type 1-fimbriated E. coli strains, which was at least partly caused by proteolysis. Moreover, a study of fimbriated and nonfimbriated mutants revealed that these changes were due mainly to type 1 fimbria-mediated surface contact and that only a few changes occurred in the outer membranes of nonfimbriated mutant strains. Protein synthesis and proteolytic degradation were involved to different extents in adhesion of fimbriated and nonfimbriated cells. While protein synthesis appeared to affect adhesion of only the nonfimbriated strain, proteolytic activity mostly seemed to contribute to adhesion of the fimbriated strain. Using matrix-assisted laser desorption ionization–time of flight mass spectrometry, six of the proteins resolved by 2D analysis were identified as BtuB, EF-Tu, OmpA, OmpX, Slp, and TolC. While the first two proteins were unaffected by adhesion, the levels of the last four were moderately to strongly reduced. Based on the present results, it may be suggested that physical interactions between type 1 fimbriae and the surface are part of a surface-sensing mechanism in which protein turnover may contribute to the observed change in composition of outer membrane proteins. This change alters the surface characteristics of the cell envelope and may thus influence adhesion. PMID:11274103

  13. Measurement of adhesive forces between bacteria and protein-coated surfaces using optical tweezers

    NASA Astrophysics Data System (ADS)

    Simpson, Kathryn H.; Bowden, Gabriela; Hook, Magnus; Anvari, Bahman

    2002-05-01

    Bacterial adhesion is a primary cause of failure in implanted medical devices. Bacteria commonly found in device-related infections, such as S. aureus, have multiple cell surface adhesins which mediate specific adhesion to molecules found in extracellular matrix and blood plasma. Adhesins recognizing fibrinogen, fibronectin, collagen, and elastin molecules have been isolated in S. aureus. We have used optical tweezers to measure the adhesive force between a single bacterium and a protein-coated surface. Various concentrations of fibronectin, fibrinogen, or whole plasma were immobilized onto 10-micrometers diameter polystyrene microspheres. We optically trapped a bacterium with a titanium-sapphire laser tuned to 830 nm and contacted the cell with a coated bead. We determined the minimum force necessary to separate the cell and bead. For beads coated with fibronectin and fibrinogen, detachment force values occurred as approximate integer multiples of an estimated single bond detachment force. With plasma-coated beads, only cells lacking the fibrinogen adhesin could be detached; therefore, we believe that either this adhesin is prevalent on wilde-type cells, or it is preferentially adsorbed onto the beads. Additionally, the whole plasma detachment forces appeared random; therefore, we believe that many adhesins participate in boding to plasma.

  14. 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

  15. Nanometer polymer surface features: the influence on surface energy, protein adsorption and endothelial cell adhesion

    NASA Astrophysics Data System (ADS)

    Carpenter, Joseph; Khang, Dongwoo; Webster, Thomas J.

    2008-12-01

    Current small diameter (<5 mm) synthetic vascular graft materials exhibit poor long-term patency due to thrombosis and intimal hyperplasia. Tissue engineered solutions have yielded functional vascular tissue, but some require an eight-week in vitro culture period prior to implantation—too long for immediate clinical bedside applications. Previous in vitro studies have shown that nanostructured poly(lactic-co-glycolic acid) (PLGA) surfaces elevated endothelial cell adhesion, proliferation, and extracellular matrix synthesis when compared to nanosmooth surfaces. Nonetheless, these studies failed to address the importance of lateral and vertical surface feature dimensionality coupled with surface free energy; nor did such studies elicit an optimum specific surface feature size for promoting endothelial cell adhesion. In this study, a series of highly ordered nanometer to submicron structured PLGA surfaces of identical chemistry were created using a technique employing polystyrene nanobeads and poly(dimethylsiloxane) (PDMS) molds. Results demonstrated increased endothelial cell adhesion on PLGA surfaces with vertical surface features of size less than 18.87 nm but greater than 0 nm due to increased surface energy and subsequently protein (fibronectin and collagen type IV) adsorption. Furthermore, this study provided evidence that the vertical dimension of nanometer surface features, rather than the lateral dimension, is largely responsible for these increases. In this manner, this study provides key design parameters that may promote vascular graft efficacy.

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

    PubMed Central

    Koetsier, Jennifer L.; Amargo, Evangeline V.; Todorović, 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 cell–cell 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, β4 and β1 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 β1 integrin. However, integrin expression levels are not restored by RhoA signaling inhibition. These data uncover a potential role for PKP2 upstream of β1 integrin and RhoA in integrating cell–cell and cell–substrate contact signaling in basal keratinocytes necessary for the morphogenesis, homeostasis, and reepithelialization of the stratified epidermis. PMID:23884246

  17. 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.

  18. 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.

  19. Sticky Situation: An Investigation of Robust Aqueous-Based Recombinant Spider Silk Protein Coatings and Adhesives.

    PubMed

    Harris, Thomas I; Gaztambide, Danielle A; Day, Breton A; Brock, Cameron L; Ruben, Ashley L; Jones, Justin A; Lewis, Randolph V

    2016-11-14

    The mechanical properties and biocompatibility of spider silks have made them one of the most sought after and studied natural biomaterials. A biomimetic process has been developed that uses water to solvate purified recombinant spider silk proteins (rSSps) prior to material formation. The absence of harsh organic solvents increases cost effectiveness, safety, and decreases the environmental impact of these materials. This development allows for the investigation of aqueous-based rSSps as coatings and adhesives and their potential applications. In these studies it was determined that fiber-based rSSps in nonfiber formations have the capability to coat and adhere numerous substrates, whether rough, smooth, hydrophobic, or hydrophilic. Further, these materials can be functionalized for a variety of processes. Drug-eluting coatings have been made with the capacity to release a variety of compounds in addition to their inherent ability to prevent blood clotting and biofouling. Additionally, spider silk protein adhesives are strong enough to outperform some conventional glues and still display favorable tissue implantation properties. The physical properties, corresponding capabilities, and potential applications of these nonfibrous materials were characterized in this study. Mechanical properties, ease of manufacturing, biodegradability, biocompatibility, and functionality are the hallmarks of these revolutionary spider silk protein materials.

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

    PubMed Central

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

    2014-01-01

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

  1. Amigo adhesion protein regulates development of neural circuits in zebrafish brain.

    PubMed

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

    2014-07-18

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

  2. Adhesion of Candida albicans to various dental implant surfaces and the influence of salivary pellicle proteins.

    PubMed

    Bürgers, Ralf; Hahnel, Sebastian; Reichert, Torsten E; Rosentritt, Martin; Behr, Michael; Gerlach, Till; Handel, Gerhard; Gosau, Martin

    2010-06-01

    Dental implants may be considered a potential reservoir for (re)infection with oral Candida albicans. Our aim was to evaluate initial fungal adhesion to three differentially textured titanium and one zirconia implant surface, and to correlate these findings to differences in specific surface characteristics (surface roughness (R(a)) and surface free energy (SFE)). Additionally, we investigated the influence of salivary protein films and two pellicle proteins (mucin and albumin). Implant surfaces were characterized by perthometer (R(a)) and goniometer (SFE) measurements. Implant specimens were rinsed with human whole saliva, mucin, albumin, or phosphate buffered saline and incubated in C. albicans suspension for 2.5h. Adherent fungi were quantified by means of a bioluminometric assay. The lowest amount of fungal cells was found on sand-blasted titanium, whereas zirconia implants did not show any reduced potential to adhere C. albicans. The influence of the implant SFE on fungal biofilm formation appears to be more important than the influence of R(a). The protein mucin enhanced C. albicans accumulation. In contrast, albumin is unlikely to be involved in the adhesion process of C. albicans.

  3. The effect of surface microtopography of poly(dimethylsiloxane) on protein adsorption, platelet and cell adhesion.

    PubMed

    Chen, Hong; Song, Wei; Zhou, Feng; Wu, Zhongkui; Huang, He; Zhang, Junhu; Lin, Quan; Yang, Bai

    2009-07-01

    Chemical homogeneous poly(dimethylsiloxane) (PDMS) surface with dot-like protrusion pattern was used to investigate the individual effect of surface microtopography on protein adsorption and subsequent biological responses. Fibrinogen (Fg) and fibronectin (Fn) were chosen as model proteins due to their effect on platelet and cell adhesion, respectively. Fg labeled with (125)I and fluorescein isothiocyanate (FITC) was used to study its adsorption on flat and patterned surfaces. Patterned surface has a 46% increase in the adsorption of Fg when compared with flat surface. However, the surface area of the patterned surface was only 8% larger than that of the flat surface. Therefore, the increase in the surface area was not the only factor responsible for the increase in protein adsorption. Clear fluorescent pattern was visualized on patterned surface, indicating that adsorbed Fg regularly distributed and adsorbed most on the flanks and valleys of the protrusions. Such distribution and local enrichment of Fg presumably caused the specific location of platelets adhered from platelet-rich plasma (PRP) and flowing whole blood (FWB) on patterned surface. Furthermore, the different combination of surface topography and pre-adsorbed Fn could influence the adhesion of L929 cells. The flat surface with pre-adsorbed Fn was the optimum substrate while the virgin patterned surface was the poor substrate in terms of L929 cells spread.

  4. The effects of cell adhesion on the growth and protein productivity of animal cells.

    PubMed

    Nishijima, K; Fujiki, T; Kojima, H; Iijima, S

    2000-07-01

    We investigated the effect of cell adhesion on cellgrowth and productivity of recombinant protein inChinese hamster ovary (CHO) cells. Cells cultured innormal tissue culture dishes attached to the dishsurfaces and grew as a monolayer, while cells culturedin non-treated dishes proliferated in suspension assingle cells without adhering to the dish surfaces. On an agarose-coated dish surface, cell aggregatesformed without attaching to the dish. Growth rates inboth suspension cultures were slightly lower thanthose in monolayer culture. Cell cycle analysisindicated that the duration of the G(1) phase insuspension cultures was longer than that in monolayerculture, suggesting that attachment to the substratummainly affected the transition from the G(1) to theS phase. Consistent with this, CDK inhibitor p27,that inhibits the G(1)S transition, was induced inthe cells cultured in suspension.To assess the productivity of recombinant proteins,CHO cells were transfected with a plasmid containingmurine interferon gamma (mIFN-gamma) under thecontrol of the cytomegalovirus promoter. Insuspension culture, mIFN-gamma productivity wasslightly lower than that in the monolayer culture. When protein kinase C was activated by phorbol ester,mIFN-gamma production was enhanced in both themonolayer and suspension cultures. However, theproductivity in the suspension culture was lower thanthat in the adherent culture even in the presence ofhigh concentrations of phorbol ester. These resultssuggested that cell adhesion to the substratum affectsvarious features of CHO cells.

  5. Quantum dots as bio-labels for the localization of a small plant adhesion protein

    NASA Astrophysics Data System (ADS)

    Ravindran, Sathyajith; Kim, Sunran; Martin, Rebecca; Lord, Elizabeth M.; Ozkan, Cengiz S.

    2005-01-01

    Recently, semiconducting nanoparticles have been successfully applied in live mammalian cell cultures, as alternative biological labels for multicolour imaging, by verifying known physiological processes. Here, we report the application of semiconducting nanoparticles to live plant cells in culture. Utilizing this technique, we have uncovered new knowledge regarding the localization of a plant pollen tube adhesion protein, stigma/stylar cysteine-rich adhesin (SCA). The potential of these nanoparticles is evident when the results were compared with conventional immunolocalization methods using fluorescently labelled antibodies.

  6. Parallel Control over Surface Charge and Wettability Using Polyelectrolyte Architecture: Effect on Protein Adsorption and Cell Adhesion.

    PubMed

    Guo, Shanshan; Zhu, Xiaoying; Li, Min; Shi, Liya; Ong, June Lay Ting; Jańczewski, Dominik; Neoh, Koon Gee

    2016-11-09

    Surface charge and wettability, the two prominent physical factors governing protein adsorption and cell adhesion, have been extensively investigated in the literature. However, a comparison between these driving forces in terms of their independent and cooperative effects in affecting adhesion is rarely explored on a systematic and quantitative level. Herein, we formulate a protocol that features two-dimensional control over both surface charge and wettability with limited cross-parameter influence. This strategy is implemented by controlling both the polyion charge density in the layer-by-layer (LbL) assembly process and the polyion side-chain chemical structures. The 2D property matrix spans surface isoelectric points ranging from 5 to 9 and water contact angles from 35 to 70°, with other interferential factors (e.g., roughness) eliminated. The interplay between these two surface variables influences protein (bovine serum albumin, lysozyme) adsorption and 3T3 fibroblast cell adhesion. For proteins, we observe the presence of thresholds for surface wettability and electrostatic driving forces necessary to affect adhesion. Beyond these thresholds, the individual effects of electrostatic forces and wettability are observed. For fibroblast, both surface charge and wettability have an effect on its adhesion. The combined effects of positive charge and hydrophilicity lead to the highest cell adhesion, whereas negative charge and hydrophobicity lead to the lowest cell adhesion. Our design strategy can potentially form the basis for studying the distinct behaviors of electrostatic force or wettability driven interfacial phenomena and serve as a reference in future studies assessing protein adsorption and cell adhesion to surfaces with known charge and wettability within the property range studied here.

  7. Amyloid precursor-like protein 1 (APLP1) exhibits stronger zinc-dependent neuronal adhesion than amyloid precursor protein and APLP2.

    PubMed

    Mayer, Magnus C; Schauenburg, Linda; Thompson-Steckel, Greta; Dunsing, Valentin; Kaden, Daniela; Voigt, Philipp; Schaefer, Michael; Chiantia, Salvatore; Kennedy, Timothy E; Multhaup, Gerhard

    2016-04-01

    The amyloid precursor protein (APP) and its paralogs, amyloid precursor-like protein 1 (APLP1) and APLP2, are metalloproteins with a putative role both in synaptogenesis and in maintaining synapse structure. Here, we studied the effect of zinc on membrane localization, adhesion, and secretase cleavage of APP, APLP1, and APLP2 in cell culture and rat neurons. For this, we employed live-cell microscopy techniques, a microcontact printing adhesion assay and ELISA for protein detection in cell culture supernatants. We report that zinc induces the multimerization of proteins of the amyloid precursor protein family and enriches them at cellular adhesion sites. Thus, zinc facilitates the formation of de novo APP and APLP1 containing adhesion complexes, whereas it does not have such influence on APLP2. Furthermore, zinc-binding prevented cleavage of APP and APLPs by extracellular secretases. In conclusion, the complexation of zinc modulates neuronal functions of APP and APLPs by (i) regulating formation of adhesion complexes, most prominently for APLP1, and (ii) by reducing the concentrations of neurotrophic soluble APP/APLP ectodomains. Earlier studies suggest a function of the amyloid precursor protein (APP) family proteins in neuronal adhesion. We report here that adhesive function of these proteins is tightly regulated by zinc, most prominently for amyloid precursor-like protein 1 (APLP1). Zinc-mediated APLP1 multimerization, which induced formation of new neuronal contacts and decreased APLP1 shedding. This suggests that APLP1 could function as a zinc receptor processing zinc signals to stabilized or new neuronal contacts.

  8. In vivo post-translational modifications of recombinant mussel adhesive protein in insect cells.

    PubMed

    Lim, Seonghye; Kim, Kyoung Ro; Choi, Yoo Seong; Kim, Dae-Kyum; Hwang, Daehee; Cha, Hyung Joon

    2011-01-01

    Mussel adhesive proteins (MAPs) have been suggested as promising bioadhesives for diverse application fields, including medical uses. Previously, we successfully constructed and produced a new type of functional recombinant MAP, fp-151, in a prokaryotic Escherichia coli expression system. Even though the E. coli-derived MAP showed several excellent features, such as high production yield and efficient purification, in vitro enzymatic modification is required to convert tyrosine residues to l-3,4-dihydroxyphenyl alanine (dopa) molecules for its adhesive ability, due to the intrinsic inability of E. coli to undergo post-translational modification. In this work, we produced a soluble recombinant MAP in insect Sf9 cells, which are widely used as an effective and convenient eukaryotic expression system for eukaryotic foreign proteins. Importantly, we found that insect-derived MAP contained converted dopa residues by in vivo post-translational modification. In addition, insect-derived MAP also had other post-translational modifications including phosphorylation of serine and hydroxylation of proline that originally occurred in some natural MAPs. To our knowledge, this is the first report on in vivo post-translational modifications of MAP containing dopa and other modified amino acid residues.

  9. Adhesive surface proteins of Erysipelothrix rhusiopathiae bind to polystyrene, fibronectin, and type I and IV collagens.

    PubMed

    Shimoji, Yoshihiro; Ogawa, Yohsuke; Osaki, Makoto; Kabeya, Hidenori; Maruyama, Soichi; Mikami, Takeshi; Sekizaki, Tsutomu

    2003-05-01

    Erysipelothrix rhusiopathiae is a gram-positive bacterium that causes erysipelas in animals and erysipeloid in humans. We found two adhesive surface proteins of E. rhusiopathiae and determined the nucleotide sequences of the genes, which were colocalized and designated rspA and rspB. The two genes were present in all of the serovars of E. rhusiopathiae strains examined. The deduced RspA and RspB proteins contain the C-terminal anchoring motif, LPXTG, which is preceded by repeats of consensus amino acid sequences. The consensus sequences are composed of 78 to 92 amino acids and repeat 16 and 3 times in RspA and RspB, respectively. Adhesive surface proteins of other gram-positive bacteria, including Listeria monocytogenes adhesin-like protein, Streptococcus pyogenes protein F2 and F2-like protein, Streptococcus dysgalactiae FnBB, and Staphylococcus aureus Cna, share the same consensus repeats. Furthermore, the N-terminal regions of RspA and RspB showed characteristics of the collagen-binding domain that was described for Cna. RspA and RspB were expressed in Escherichia coli as histidine-tagged fusion proteins and purified. The recombinant proteins showed a high degree of capacity to bind to polystyrene and inhibited the binding of E. rhusiopathiae onto the abiotic surface in a dose dependent manner. In a solid-phase binding assay, both of the recombinant proteins bound to fibronectin, type I and IV collagens, indicating broad spectrum of their binding ability. It was suggested that both RspA and RspB were exposed on the cell surface of E. rhusiopathiae, as were the bacterial cells agglutinated by the anti-RspA immunoglobulin G (IgG) and anti-RspB IgG. RspA and RspB were present both in surface-antigen extracts and the culture supernatants of E. rhusiopathiae Fujisawa-SmR (serovar 1a) and SE-9 (serovar 2). The recombinant RspA, but not RspB, elicited protection in mice against experimental challenge. These results suggest that RspA and RspB participate in initiation

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

    PubMed Central

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

    2016-01-01

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

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

    NASA Astrophysics Data System (ADS)

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

    2016-06-01

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

  12. Tethered agonists: a new mechanism underlying adhesion G protein-coupled receptor activation.

    PubMed

    Schöneberg, Torsten; Liebscher, Ines; Luo, Rong; Monk, Kelly R; Piao, Xianhua

    2015-06-01

    The family of adhesion G protein-coupled receptors (aGPCRs) comprises 33 members in the human genome, which are subdivided into nine subclasses. Many aGPCRs undergo an autoproteolytic process via their GPCR Autoproteolysis-INducing (GAIN) domain during protein maturation to generate an N- and a C-terminal fragments, NTF and CTF, respectively. The NTF and CTF are non-covalently reassociated on the plasma membrane to form a single receptor unit. How aGPCRs are activated upon ligand binding remains one of the leading questions in the field of aGPCR research. Recent work from our labs and others shows that ligand binding can remove the NTF from the plasma membrane-bound CTF, exposing a tethered agonist which potently activates downstream signaling.

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

    PubMed Central

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

    2013-01-01

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

  14. Spatiotemporal segregation of endothelial cell integrin and nonintegrin extracellular matrix-binding proteins during adhesion events

    PubMed Central

    1990-01-01

    Bovine aortic endothelial cell (BAEC) attachments to laminin, fibronectin, and fibrinogen are inhibited by soluble arginine-glycine- aspartate (RGD)-containing peptides, and YGRGDSP activity is responsive to titration of either soluble peptide or matrix protein. To assess the presence of RGD-dependent receptors, immunoprecipitation and immunoblotting studies were conducted and demonstrated integrin beta 1, beta 3, and associated alpha subunits as well as a beta 1 precursor. Immunofluorescence of BAECs plated on laminin, fibronectin, and fibrinogen reveals different matrix-binding specificities of each of these integrin subclasses. By 1 h after plating, organization of beta 1 integrin into fibrillar streaks is influenced by laminin and fibronectin, whereas beta 3 integrin punctate organization is influenced by fibrinogen and the integrin spatial distribution changes with time in culture. In contrast, the nonintegrin laminin-binding protein LB69 only organizes after cell-substrate contact is well established several hours after plating. Migration of BAECs is also mediated by both integrin and nonintegrin matrix-binding proteins. Specifically, BAEC migration on laminin is remarkably sensitive to RGD peptide inhibition, and, in its presence, beta 1 integrin organization dissipates and reorganizes into perinuclear vesicles. However, RGD peptides do not alter LB69 linear organization during migration. Similarly, agents that block LB69--e.g., antibodies to LB69 as well as YIGSR-NH2 peptide--do not inhibit attachment of nonmotile BAECs to laminin. However, both anti-LB69 and YIGSR-NH2 inhibit late adhesive events such as spreading. Accordingly, we propose that integrin and nonintegrin extracellular matrix-binding protein organizations in BAECs are both temporally and spatially segregated during attachment processes. High affinity nonintegrin interaction with matrix may create necessary stable contacts for longterm attachment, while lower affinity integrins may be important

  15. 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.

  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. Major Membrane Protein TDE2508 Regulates Adhesive Potency in Treponema denticola

    PubMed Central

    Abiko, Yuki; Nagano, Keiji; Yoshida, Yasuo; Yoshimura, Fuminobu

    2014-01-01

    The cultivation and genetic manipulation of Treponema denticola, a Gram-negative oral spirochaeta associated with periodontal diseases, is still challenging. In this study, we formulated a simple medium based on a commercially available one, and established a transformation method with high efficiency. We then analyzed proteins in a membrane fraction in T. denticola and identified 16 major membrane-associated proteins, and characterized one of them, TDE2508, whose biological function was not yet known. Although this protein, which exhibited a complex conformation, was presumably localized in the outer membrane, we did not find conclusive evidence that it was exposed on the cell surface. Intriguingly, a TDE2508-deficient mutant exhibited significantly increased biofilm formation and adherent activity on human gingival epithelial cells. However, the protein deficiency did not alter autoaggregation, coaggregation with Porphyromonas gingivalis, hemagglutination, cell surface hydrophobicity, motility, or expression of Msp which was reported to be an adherent molecule in this bacteria. In conclusion, the major membrane protein TDE2508 regulates biofilm formation and the adhesive potency of T. denticola, although the underlying mechanism remains unclear. PMID:24586498

  18. Blood compatibility of a ferulic acid (FA)-eluting PHBHHx system for biodegradable magnesium stent application.

    PubMed

    Zhang, Erlin; Shen, Feng

    2015-01-01

    Magnesium stent has shown potential application as a new biodegradable stent. However, the fast degradation of magnesium stent limited its clinic application. Recently, a biodegradable and drug-eluting coating system was designed to prevent magnesium from fast degradation by adding ferulic acid (FA) in poly (3-hydroxybutyrate-co-3-hydroxyhexanoate) (PHBHHx) by a physical method. In vitro study has demonstrated that the FA-eluting system exhibited strong promotion to the endothelialization, which might be a choice for the stent application. In this paper, the hemolysis rate, the plasma recalcification time (PRT), the plasma prothrombin time (PT) and the kinetic clotting time of the FA-eluting films were investigated and the platelet adhesion was observed in order to assess the blood compatibility of the FA-eluting PHBHHx films in comparison with PHBHHx film. The results have shown that the addition of FA had no influence on the hemolysis, but prolonged PRT, PT and the clotting time and reduced the platelet adhesion and activation, displaying that the FA-eluting PHBHHx exhibited better blood compatibility than PHBHHx. In addition, the effect of alkali treatment on the blood compatibility of FA-eluting PHBHHx was also studied. It was indicated that alkali treatment had no effect on the hemolysis and the coagulation time, but enhanced slightly the platelet adhesion. All these demonstrated that FA-eluting PHBHHx film had good blood compatibility and might be a candidate surface coating for the biodegradable magnesium stent.

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

    PubMed

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

    2011-08-01

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

  20. Platelet adhesion and protein adsorption on silicone rubber surface by ozone-induced grafted polymerization with carboxybetaine monomer.

    PubMed

    Zhou, Jun; Yuan, Jiang; Zang, Xiaopeng; Shen, Jian; Lin, Sicong

    2005-03-10

    Platelet adhesion and protein adsorption on the silicone rubber film grafted with N,N'-dimethyl-N-methacryloyloxyethyl-N-(2-carboxyethyl) ammonium (DMMCA) was studied. The grafting was carried out by means of ozone-induced method and was confirmed by ATR-FTIR and XPS investigations. The grafted films possessed relatively hydrophilic surface revealed by contact angle measurement. The blood compatibility of the grafted film was evaluated in vitro by platelet adhesion in platelet-rich plasma (PRP) and protein absorption in bovine fibrinogen (BFG) using silicone film as the reference. No substantial platelet adhesion was observed for the grafted films incubated in PRP for 60 and 180 min. The protein absorption was also significantly reduced after incubated in bovine fibrinogen for 60 min. Both the results indicated that the blood compatibility of silicone rubber was greatly improved by ozone-induced grafting of carboxybetaine zwitterionic polymer onto its surface.

  1. Activation of cyclic amp/protein kinase: a signaling pathway enhances osteoblast cell adhesion on biomaterials for regenerative engineering.

    PubMed

    Lo, Kevin W-H; Ashe, Keshia M; Kan, Ho Man; Lee, Duron A; Laurencin, Cato T

    2011-04-01

    Osteoblast cell adhesion on biomaterials is an important goal for implants to be useful in bone regeneration technologies. The adhesion of osteoblastic cells to biomaterials has been investigated in the field of bone regenerative engineering. Previous work from our group demonstrated that osteoblastic cells adhering to biodegradable biomaterials require the expression of integrins on the cell surface. However, the underlying molecular signaling mechanism is still not fully clear. We report here that cyclic adenosine monophosphate (cAMP), a small signaling molecule, regulates osteoblast cell adhesion to biomaterial surfaces. We used an in vitro cell adhesion assay to demonstrate that at 0.1 mM, 8-Br-cAMP, a cell-permeable cAMP analog, significantly enhances osteoblast-like cells' (MC3T3-E1) adherence to biomaterials. Moreover, we demonstrate that a commonly used cAMP-elevating agent, forskolin, promotes cell adhesion similar to that of the cell permeable cAMP analog. By using different target-specific cAMP analogs: 8-CPT-2Me-cAMP which specifically activates exchange protein activated by cAMP (Epac), and 6-Bnz-cAMP which specifically activates protein kinase A (PKA), we observed that the PKA signaling pathway plays a dominant role in this process. Thus, this report suggests a new method to enhance osteoblast cell adhesion on biodegradable biomaterials for bone regenerative engineering applications.

  2. 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.

  3. Integrin adhesions suppress syncytium formation in the Drosophila larval epidermis

    PubMed Central

    Wang, Yan; Antunes, Marco; Anderson, Aimee E.; Kadrmas, Julie L.; Jacinto, Antonio; Galko, Michael J.

    2015-01-01

    Summary Integrins are critical for barrier epithelial architecture. Integrin loss in vertebrate skin leads to blistering and wound healing defects. However, how Integrins and associated proteins maintain the regular morphology of epithelia is not well understood. We found that targeted knockdown of the integrin focal adhesion (FA) complex components βIntegrin, PINCH, and Integrin-linked kinase (ILK), caused formation of multinucleate epidermal cells within the Drosophila larval epidermis. This phenotype was specific to the Integrin FA complex and not due to secondary effects on polarity or junctional structures. The multinucleate cells resembled the syncytia caused by physical wounding. Live imaging of wound-induced syncytium formation in the pupal epidermis suggested direct membrane breakdown leading to cell-cell fusion and consequent mixing of cytoplasmic contents. Activation of Jun N-terminal kinase (JNK) signaling, which occurs upon wounding, also correlated with syncytium formation induced by PINCH knockdown. Further, ectopic JNK activation directly caused epidermal syncytium formation. No mode of syncytium formation including that induced by wounding, genetic loss-of FA-proteins, or local JNK hyperactivation, involved misregulation of mitosis or apoptosis. Finally, the mechanism of epidermal syncytium formation following JNK hyperactivation and wounding appeared to be direct disassembly of FA complexes. In conclusion, the loss of function phenotype of Integrin FA components in the larval epidermis resembles a wound. Integrin FA loss in mouse and human skin also causes a wound-like appearance. Our results reveal a novel and unexpected role for proper Integrin-based adhesion in suppressing larval epidermal cell-cell fusion– a role that may be conserved in other epithelia. PMID:26255846

  4. 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

  5. Leukocyte trafficking-associated vascular adhesion protein 1 is expressed and functionally active in atherosclerotic plaques

    PubMed Central

    Silvola, Johanna M. U.; Virtanen, Helena; Siitonen, Riikka; Hellberg, Sanna; Liljenbäck, Heidi; Metsälä, Olli; Ståhle, Mia; Saanijoki, Tiina; Käkelä, Meeri; Hakovirta, Harri; Ylä-Herttuala, Seppo; Saukko, Pekka; Jauhiainen, Matti; Veres, Tibor Z.; Jalkanen, Sirpa; Knuuti, Juhani; Saraste, Antti; Roivainen, Anne

    2016-01-01

    Given the important role of inflammation and the potential association of the leukocyte trafficking-associated adhesion molecule vascular adhesion protein 1 (VAP-1) with atherosclerosis, this study examined whether functional VAP-1 is expressed in atherosclerotic lesions and, if so, whether it could be targeted by positron emission tomography (PET). First, immunohistochemistry revealed that VAP-1 localized to endothelial cells of intra-plaque neovessels in human carotid endarterectomy samples from patients with recent ischemic symptoms. In low-density lipoprotein receptor-deficient mice expressing only apolipoprotein B100 (LDLR−/−ApoB100/100), VAP-1 was expressed on endothelial cells lining inflamed atherosclerotic lesions; normal vessel walls in aortas of C57BL/6N control mice were VAP-1-negative. Second, we discovered that the focal uptake of VAP-1 targeting sialic acid-binding immunoglobulin-like lectin 9 based PET tracer [68Ga]DOTA-Siglec-9 in atherosclerotic plaques was associated with the density of activated macrophages (r = 0.58, P = 0.022). As a final point, we found that the inhibition of VAP-1 activity with small molecule LJP1586 decreased the density of macrophages in inflamed atherosclerotic plaques in mice. Our results suggest for the first time VAP-1 as a potential imaging target for inflamed atherosclerotic plaques, and corroborate VAP-1 inhibition as a therapeutic approach in the treatment of atherosclerosis. PMID:27731409

  6. Preliminary studies of the effects of vascular adhesion protein-1 inhibitors on experimental corneal neovascularization.

    PubMed

    Enzsöly, Anna; Dunkel, Petra; Récsán, Zsuzsa; Gyorffy, Hajnalka; Tóth, Jeanette; Marics, Gábor; Bori, Zoltán; Tóth, Miklós; Zelkó, Romána; Di Paolo, Maria Luisa; Mátyus, Péter; Németh, János

    2011-07-01

    Vascular adhesion protein-1 (VAP-1) controls the adhesion of lymphocytes to endothelial cells and is upregulated at sites of inflammation. Moreover, it expresses amine oxidase activity, due to the sequence identity with semicarbazide-sensitive amine oxidase. Recent studies indicate a significant role for VAP-1 in neovascularization, besides its contribution to inflammation. Pathological blood vessel development in severe ocular diseases (such as diabetes, age-related macula degeneration, trauma and infections) might lead to decreased visual acuity and finally to blindness, yet there is no clear consensus as to its appropriate treatment. In the present case study, the effects of two VAP-1 inhibitors on experimentally induced corneal neovascularization in rabbits were compared with the effects of a known inhibitor of angiogenesis, bevacizumab, an anti-vascular endothelial growth factor antibody. In accordance with recent literature data, the results of the preliminary study reported here indicate that the administration of VAP-1 inhibitors is a potentially valuable therapeutic option in the treatment of corneal neovascularization.

  7. 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

  8. 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.

  9. Adhesion of mussel foot protein-3 to TiO2 surfaces: the effect of pH.

    PubMed

    Yu, Jing; Wei, Wei; Menyo, Matthew S; Masic, Admir; Waite, J Herbert; Israelachvili, Jacob N

    2013-04-08

    The underwater adhesion of marine mussels relies on mussel foot proteins (mfps) rich in the catecholic amino acid 3,4-dihydroxyphenylalanine (Dopa). As a side chain, Dopa is capable of strong bidentate interactions with a variety of surfaces, including many minerals and metal oxides. Titanium is among the most widely used medical implant material and quickly forms a TiO2 passivation layer under physiological conditions. Understanding the binding mechanism of Dopa to TiO2 surfaces is therefore of considerable theoretical and practical interest. Using a surface forces apparatus, we explored the force-distance profiles and adhesion energies of mussel foot protein 3 (mfp-3) to TiO2 surfaces at three different pHs (pH 3, 5.5 and 7.5). At pH 3, mfp-3 showed the strongest adhesion force on TiO2, with an adhesion energy of ∼-7.0 mJ/m(2). Increasing the pH gives rise to two opposing effects: (1) increased oxidation of Dopa, thus, decreasing availability for the Dopa-mediated adhesion, and (2) increased bidentate Dopa-Ti coordination, leading to the further stabilization of the Dopa group and, thus, an increase in adhesion force. Both effects were reflected in the resonance-enhanced Raman spectra obtained at the three deposition pHs. The two competing effects give rise to a higher adhesion force of mfp-3 on the TiO2 surface at pH 7.5 than at pH 5.5. Our results suggest that Dopa-containing proteins and synthetic polymers have great potential as coating materials for medical implant materials, particularly if redox activity can be controlled.

  10. 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.

  11. Novel pyridazinone inhibitors for vascular adhesion protein-1 (VAP-1): old target-new inhibition mode.

    PubMed

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

    2013-12-27

    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. Although 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.

  12. Development of robust biocompatible silicone with high resistance to protein adsorption and bacterial adhesion.

    PubMed

    Lin, Weifeng; Zhang, Juan; Wang, Zhen; Chen, Shengfu

    2011-05-01

    A new biocompatible silicone comprising a carboxybetaine (CB) ester analogue, 3-methacryloxypropyltris(trimethylsiloxy)silane (TRIS) and an organic silicone macromer (bis-α,ω-(methacryloxypropyl) polydimethylsiloxane) has been developed using photo-polymerisation. Following interfacial hydrolysis of the CB ester, the resulting zwitterionic material became significantly more hydrophilic and exhibited high resistance to both non-specific protein adsorption and bacterial adhesion. Moreover, the stability of these non-fouling properties was dramatically improved by using a slow and controlled rate of ester hydrolysis of the original protective hydrophobic matrix. The subsequent ability to maintain the original optical and mechanical properties of the bare silicone following surface activation makes this material an ideal candidate for preparing contact lenses and other medical devices.

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

    PubMed Central

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

    2014-01-01

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

  14. Bioinspired Universal Monolayer Coatings by Combining Concepts from Blood Protein Adsorption and Mussel Adhesion.

    PubMed

    Yu, Leixiao; Cheng, Chong; Ran, Qidi; Schlaich, Christoph; Noeske, Paul-Ludwig Michael; Li, Wenzhong; Wei, Qiang; Haag, Rainer

    2017-02-22

    Despite the increasing need for universal polymer coating strategies, only a few approaches have been successfully developed, and most of them are suffering from color, high thickness, or high roughness. In this paper, we present for the first time a universal monolayer coating that is only a few nanometers thick and independent of the composition, size, shape, and structure of the substrate. The coating is based on a bioinspired synthetic amphiphilic block copolymer that combines two concepts from blood protein adsorption and mussel adhesion. This polymer can be rapidly tethered on various substrates including both planar surfaces and nanosystems with high grafting density. The resulting monolayer coatings are, on the one hand, inert to the adsorption of multiple polymer layers and prevent biofouling. On the other hand, they are chemically active for secondary functionalization and provide a new platform for selective material surface modification.

  15. A mutant form of the rho protein can restore stress fibers and adhesion plaques in v-src transformed fibroblasts.

    PubMed

    Mayer, T; Meyer, M; Janning, A; Schiedel, A C; Barnekow, A

    1999-03-25

    The organization of polymerized actin in the mammalian cell is regulated by several members of the rho family. Three rho proteins, cdc42, rac and rho act in a cascade to organize the intracellular actin cytoskeleton. Rho proteins are involved in the formation of actin stress fibers and adhesion plaques in fibroblasts. During transformation of mammalian cells by oncogenes the cytoskeleton is rearranged and stress fibers and adhesion plaques are disintegrated. In this paper we investigate the function of the rho protein in RR1022 rat fibroblasts transformed by the Rous sarcoma virus. Two activated mutants of the rho protein, rho G14V and rho Q63L, and a dominant negative mutant, rho N1171, were stably transfected into RR1022 cells. The resulting cell lines were analysed for the organization of polymerized actin and adhesion plaques. Cells expressing rho Q63L, but not rho wt, rho G14V or rho N1171, showed an altered morphology. These cells displayed a flat, fibroblast like shape when compared with the RR1022 ancestor cells. Immunofluorescence analyses revealed that actin stress fibers and adhesion plaques were rearranged in these cells. We conclude from these data that an active rho protein can restore elements of the actin cytoskeleton in transformed cells by overriding the tyrosine kinase phosphorylation induced by the pp60(v-src).

  16. Link protein hyaluronan-binding motif abrogates CD44-hyaluronan-mediated leukemia-liver cell adhesion.

    PubMed

    Chen, Jing; Li, Na; Li, Gongchu

    2013-05-01

    The liver is a frequent site for the metastasis of cancer cells originating from other sites. Leukemic liver metastasis is associated with poor prognosis. The ligation of CD44 with hyaluronan (HA) has been shown to contribute to the drug resistance of leukemic cells. In this study, a link protein HA-binding motif was genetically fused with enhanced green fluorescence protein (EGFP) to generate an EGFP-L fusion protein. Furthermore, a coculture system was established to investigate the interaction of leukemic cells with liver cells. CD44-positive Kasumi-1, but not CD44-negative HL-60 cells, were observed to adhere to the liver cell line L02. This cell-cell adhesion was significantly blocked by HA, indicating that Kasumi-L02 cell adhesion was mediated by the CD44-HA interaction. Compared to EGFP, EGFP-L fusion protein bound to L02 and BEL7404 liver cells. EGFP-L partially abrogated the Kasumi-L02 adhesion, suggesting that the link protein-binding motif is able to inhibit CD44-HA-mediated leukemia-liver adhesion. These results may help provide insight into novel therapeutic methods for leukemic patients diagnosed with liver metastasis.

  17. Exported proteins in probiotic bacteria: adhesion to intestinal surfaces, host immunomodulation and molecular cross-talking with the host.

    PubMed

    Sánchez, Borja; Bressollier, Philippe; Urdaci, María C

    2008-10-01

    The group of exported proteins of a bacterium are those proteins that are sorted from the cytoplasm to the bacterial surface or to the surroundings of the microorganism. In probiotic bacteria, these proteins are of special relevance because they might determine important traits such as adhesion to intestinal surfaces and molecular cross-talking with the host. Current knowledge about the presence and biological relevance of exported proteins produced by the main genera of probiotic bacteria in the gastrointestinal environment is reviewed in this minireview. As will be seen, some of these proteins are involved in host adhesion or are able to modify certain signalization pathways within host cells, whereas others are important for the physiology of probiotic bacteria in the gastrointestinal tract.

  18. 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

  19. Effect of anticoagulants on the protein corona-induced reduced drug carrier adhesion efficiency in human blood flow.

    PubMed

    Sobczynski, Daniel J; Eniola-Adefeso, Omolola

    2017-01-15

    Plasma proteins rapidly coat the surfaces of particulate drug carriers to form a protein corona upon their injection into the bloodstream. The high presence of immunoglobulins in the corona formed on poly(lactic-co-glycolic acid) (PLGA) vascular-targeted carrier (VTC) surfaces was recently shown to negatively impact their adhesion to activated endothelial cells (aECs) in vitro. Here, we characterized the influence of anticoagulants, or their absence, on the binding efficiency of VTCs of various materials via modulation of their protein corona. Specifically, we evaluated the adhesion of PLGA, poly(lactic acid) (PLA), polycaprolactone (PCL), silica, and polystyrene VTCs to aECs in heparinized, citrated, and non-anticoagulated (serum and whole) blood flows relative to buffer control. Particle adhesion is substantially reduced in non-anticoagulated blood flows regardless of the material type while only moderate to minimal reduction is observed for VTCs in anticoagulant-containing blood flow depending on the anticoagulant and material type. The substantial reduction in VTC adhesion in blood flows was linked to a high presence of immunoglobulin-sized proteins in the VTC corona via SDS-PAGE analysis. Of all the materials evaluated, PLGA was the most sensitive to plasma protein effects while PCL was the most resistant, suggesting particle hydrophobicity is a critical component of the observed negative plasma protein effects. Overall, this work demonstrates that anticoagulant positively alters the effect of plasma proteins in prescribing VTC adhesion to aECs in human blood flow, which has implication in the use of in vitro blood flow assays for functional evaluation of VTCs for in vivo use.

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

    NASA Astrophysics Data System (ADS)

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

    2016-09-01

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

  1. Nanoscale organization of synaptic adhesion proteins revealed by single-molecule localization microscopy.

    PubMed

    Chamma, Ingrid; Levet, Florian; Sibarita, Jean-Baptiste; Sainlos, Matthieu; Thoumine, Olivier

    2016-10-01

    The advent of superresolution imaging has created a strong need for both optimized labeling strategies and analysis methods to probe the nanoscale organization of complex biological structures. We present a thorough description of the distribution of synaptic adhesion proteins at the nanoscopic scale, namely presynaptic neurexin-[Formula: see text] ([Formula: see text]), and its two postsynaptic binding partners neuroligin-1 (Nlg1) and leucine-rich-repeat transmembrane protein 2 (LRRTM2). We monitored these proteins in the membrane of neurons by direct stochastic optical reconstruction microscopy, after live surface labeling with Alexa647-conjugated monomeric streptavidin. The small probe ([Formula: see text]) efficiently penetrates into crowded synaptic junctions and reduces the distance to target. We quantified the organization of the single-molecule localization data using a tesselation-based analysis technique. We show that Nlg1 exhibits a fairly disperse organization within dendritic spines, while LRRTM2 is organized in compact domains, and [Formula: see text] in presynaptic terminals displays a dual-organization pattern intermediate between that of Nlg1 and LRRTM2. These results suggest that part of [Formula: see text] interacts transsynaptically with Nlg1 and the other part with LRRTM2.

  2. Mispolarization of desmosomal proteins and altered intercellular adhesion in autosomal dominant polycystic kidney disease.

    PubMed

    Silberberg, Melina; Charron, Audra J; Bacallao, Robert; Wandinger-Ness, Angela

    2005-06-01

    Polycystin-1, the product of the major gene mutated in autosomal dominant polycystic kidney disease (ADPKD), has been shown to associate with multiple epithelial cell junctions. Our hypothesis is that polycystin-1 is an important protein for the initial establishment of cell-cell junctions and maturation of the cell and that polycystin-1 localization is dependent on the degree of cell polarization. Using laser-scanning confocal microscopy and two models of cell polarization, polycystin-1 and desmosomes were found to colocalize during the initial establishment of cell-cell contact when junctions were forming. However, colocalization was lost in confluent monolayers. Parallel morphological and biochemical evaluations revealed a profound mispolarization of desmosomal components to both the apical and basolateral domains in primary ADPKD cells and tissue. Studies of the intermediate filament network associated with desmosomes showed that there is a decrease in cytokeratin levels and an abnormal expression of the mesenchymal protein vimentin in the disease. Moreover, we show for the first time that the structural alterations seen in adherens and desmosomal junctions have a functional impact, leaving the ADPKD cells with weakened cell-cell adhesion. In conclusion, in this paper we show that polycystin-1 transiently colocalizes with desmosomes and that desmosomal proteins are mislocalized as a consequence of polycystin-1 mutation.

  3. 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.

  4. Catechol-functionalized adhesive polymer nanoparticles for controlled local release of bone morphogenetic protein-2 from titanium surface.

    PubMed

    Lee, Hong Jae; Koo, Ahn Na; Lee, Suk Won; Lee, Myung Hyun; Lee, Sang Cheon

    2013-09-10

    We report on a novel surface functionalization approach to equip the titanium (Ti) surfaces with osteogenic properties. A key feature of the approach is the treatment of the Ti surfaces with Ti-adhesive nanoparticles that can stably load and controllably release bone morphogenetic protein-2 (BMP-2). Ti-adhesive nanoparticles were prepared by self-assembly of a catechol-functionalized poly(amino acid) diblock copolymer, catechol-poly(L-aspartic acid)-b-poly(L-phenylalanine) (Cat-PAsp-PPhe). The nanoparticles consist of Ti-adhesive peripheral catechol groups, anionic PAsp shells, and PPhe inner cores. Field-emission scanning electron microscopy (Fe-SEM) images showed that the Ti-adhesive nanoparticles could be uniformly immobilized on Ti surfaces. X-ray photoelectron spectroscopy (XPS) confirmed the successful anchoring of nanoparticles onto Ti surfaces. After surface immobilization of the nanoparticles, the static water contact angle of the Ti substrate decreased from 75.3° to 50.0° or 36.4°, depending on the surface nanoparticle. Fluorescence microscopic analysis showed that BMP-2 could be effectively incorporated onto the Ti surface with adhesive nanoparticles. BMP-2 was controllably released for up to 40 days. The Ti substrate functionalized with BMP-2-incorporated nanoparticles significantly promoted attachment, proliferation, spreading, and alkaline phosphatase (ALP) activity of human adipose-derived stem cell (hADSC). The catechol-functionalized adhesive nanoparticles may be applied to various medical devices to create surfaces for improved performance.

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

    PubMed Central

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

    2016-01-01

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

  6. Cytoplasmic Domain of Zebrafish Myelin Protein Zero: Adhesive Role Depends on β-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 β-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 β→α structural transition at lipid/protein (L/P) molar ratios >50, and adopted a β-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 β-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 β-conformation. PMID:17693467

  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

    , 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. B-cell receptor-associated protein 31 regulates human embryonic stem cell adhesion, stemness, and survival via control of epithelial cell adhesion molecule.

    PubMed

    Kim, Won-Tae; Seo Choi, Hong; Min Lee, Hyun; Jang, Young-Joo; Ryu, Chun Jeih

    2014-10-01

    B-Cell receptor-associated protein 31 (BAP31) regulates the export of secreted membrane proteins from the endoplasmic reticulum (ER) to the downstream secretory pathway. Previously, we generated a monoclonal antibody 297-D4 against the surface molecule on undifferentiated human embryonic stem cells (hESCs). Here, we found that 297-D4 antigen was localized to pluripotent hESCs and downregulated during early differentiation of hESCs and identified that the antigen target of 297-D4 was BAP31 on the hESC-surface. To investigate the functional role of BAP31 in hESCs, BAP31 expression was knocked down by small interfering RNA. BAP31 depletion impaired hESC self-renewal and pluripotency and drove hESC differentiation into multicell lineages. BAP31 depletion hindered hESC proliferation by arresting cell cycle at G0/G1 phase and inducing caspase-independent cell death. Interestingly, BAP31 depletion reduced hESC adhesion to extracellular matrix (ECM). Analysis of cell surface molecules showed decreased expression of epithelial cell adhesion molecule (EpCAM) in BAP31-depleted hESCs, while ectopic expression of BAP31 elevated the expression of EpCAM. EpCAM depletion also reduced hESC adhesion to ECM, arrested cell cycle at G0/G1 phase and induced cell death, producing similar effects to those of BAP31 depletion. BAP31 and EpCAM were physically associated and colocalized at the ER and cell surface. Both BAP31 and EpCAM depletion decreased cyclin D1 and E expression and suppressed PI3K/Akt signaling, suggesting that BAP31 regulates hESC stemness and survival via control of EpCAM expression. These findings provide, for the first time, mechanistic insights into how BAP31 regulates hESC stemness and survival via control of EpCAM expression.

  9. 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.

  10. 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

  11. Multifunctional and Redundant Roles of Borrelia burgdorferi Outer Surface Proteins in Tissue Adhesion, Colonization, and Complement Evasion

    PubMed Central

    Caine, Jennifer A.; Coburn, Jenifer

    2016-01-01

    Borrelia burgdorferi is the causative agent of Lyme disease in the U.S., with at least 25,000 cases reported to the CDC each year. B. burgdorferi is thought to enter and exit the bloodstream to achieve rapid dissemination to distal tissue sites during infection. Travel through the bloodstream requires evasion of immune surveillance and pathogen clearance in the host, a process at which B. burgdorferi is adept. B. burgdorferi encodes greater than 19 adhesive outer surface proteins many of which have been found to bind to host cells or components of the extracellular matrix. Several others bind to host complement regulatory factors, in vitro. Production of many of these adhesive proteins is tightly regulated by environmental cues, and some have been shown to aid in vascular interactions and tissue colonization, as well as survival in the blood, in vivo. Recent work has described multifaceted and redundant roles of B. burgdorferi outer surface proteins in complement component interactions and tissue targeted adhesion and colonization, distinct from their previously identified in vitro binding capabilities. Recent insights into the multifunctional roles of previously well-characterized outer surface proteins such as BBK32, DbpA, CspA, and OspC have changed the way we think about the surface proteome of these organisms during the tick–mammal life cycle. With the combination of new and old in vivo models and in vitro techniques, the field has identified distinct ligand binding domains on BBK32 and DbpA that afford tissue colonization or blood survival to B. burgdorferi. In this review, we describe the multifunctional and redundant roles of many adhesive outer surface proteins of B. burgdorferi in tissue adhesion, colonization, and bloodstream survival that, together, promote the survival of Borrelia spp. throughout maintenance in their multi-host lifestyle. PMID:27818662

  12. 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.

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

    PubMed

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

    2009-03-27

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

  14. Myelin Basic Protein Cleaves Cell Adhesion Molecule L1 and Promotes Neuritogenesis and Cell Survival*

    PubMed Central

    Lutz, David; Loers, Gabriele; Kleene, Ralf; Oezen, Iris; Kataria, Hardeep; Katagihallimath, Nainesh; Braren, Ingke; Harauz, George; Schachner, Melitta

    2014-01-01

    The cell adhesion molecule L1 is a Lewisx-carrying glycoprotein that plays important roles in the developing and adult nervous system. Here we show that myelin basic protein (MBP) binds to L1 in a Lewisx-dependent manner. Furthermore, we demonstrate that MBP is released by murine cerebellar neurons as a sumoylated dynamin-containing protein upon L1 stimulation and that this MBP cleaves L1 as a serine protease in the L1 extracellular domain at Arg687 yielding a transmembrane fragment that promotes neurite outgrowth and neuronal survival in cell culture. L1-induced neurite outgrowth and neuronal survival are reduced in MBP-deficient cerebellar neurons and in wild-type cerebellar neurons in the presence of an MBP antibody or L1 peptide containing the MBP cleavage site. Genetic ablation of MBP in shiverer mice and mutagenesis of the proteolytically active site in MBP or of the MBP cleavage site within L1 as well as serine protease inhibitors and an L1 peptide containing the MBP cleavage site abolish generation of the L1 fragment. Our findings provide evidence for novel functions of MBP in the nervous system. PMID:24671420

  15. Universal method for protein bioconjugation with nanocellulose scaffolds for increased cell adhesion.

    PubMed

    Kuzmenko, Volodymyr; Sämfors, Sanna; Hägg, Daniel; Gatenholm, Paul

    2013-12-01

    Bacterial nanocellulose (BNC) is an emerging biomaterial since it is biocompatible, integrates well with host tissue and can be biosynthesized in desired architecture. However, being a hydrogel, it exhibits low affinity for cell attachment, which is crucial for the cellular fate process. To increase cell attachment, the surface of BNC scaffolds was modified with two proteins, fibronectin and collagen type I, using an effective bioconjugation method applying 1-cyano-4-dimethylaminopyridinium (CDAP) tetrafluoroborate as the intermediate catalytic agent. The effect of CDAP treatment on cell adhesion to the BNC surface is shown for human umbilical vein endothelial cells and the mouse mesenchymal stem cell line C3H10T1/2. In both cases, the surface modification increased the number of cells attached to the surfaces. In addition, the morphology of the cells indicated more healthy and viable cells. CDAP activation of bacterial nanocellulose is shown to be a convenient method to conjugate extracellular proteins to the scaffold surfaces. CDAP treatment can be performed in a short period of time in an aqueous environment under heterogeneous and mild conditions preserving the nanofibrillar network of cellulose.

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

    PubMed

    Liebscher, Ines; Ackley, Brian; Araç, Demet; Ariestanti, Donna M; Aust, Gabriela; Bae, Byoung-il; Bista, Bigyan R; Bridges, James P; Duman, Joseph G; Engel, Felix B; Giera, Stefanie; Goffinet, André M; Hall, Randy A; Hamann, Jörg; Hartmann, Nicole; Lin, Hsi-Hsien; Liu, Mingyao; Luo, Rong; Mogha, Amit; Monk, Kelly R; Peeters, Miriam C; Prömel, Simone; Ressl, Susanne; Schiöth, Helgi B; Sigoillot, Séverine M; Song, Helen; Talbot, William S; Tall, Gregory G; White, James P; Wolfrum, Uwe; Xu, Lei; Piao, Xianhua

    2014-12-01

    The class of adhesion G protein-coupled receptors (aGPCRs), with 33 human homologs, is the second largest family of GPCRs. In addition to a seven-transmembrane α-helix-a structural feature of all GPCRs-the class of aGPCRs is characterized by the presence of a large N-terminal extracellular region. In addition, all aGPCRs but one (GPR123) contain a GPCR autoproteolysis-inducing (GAIN) domain that mediates autoproteolytic cleavage at the GPCR autoproteolysis site motif to generate N- and a C-terminal fragments (NTF and CTF, respectively) during protein maturation. Subsequently, the NTF and CTF are associated noncovalently as a heterodimer at the plasma membrane. While the biological function of the GAIN domain-mediated autocleavage is not fully understood, mounting evidence suggests that the NTF and CTF possess distinct biological activities in addition to their function as a receptor unit. We discuss recent advances in understanding the biological functions, signaling mechanisms, and disease associations of the aGPCRs.

  17. Orphan G protein-coupled receptor GPRC5A modulates integrin β1-mediated epithelial cell adhesion.

    PubMed

    Bulanova, Daria R; Akimov, Yevhen A; Rokka, Anne; Laajala, Teemu D; Aittokallio, Tero; Kouvonen, Petri; Pellinen, Teijo; Kuznetsov, Sergey G

    2016-10-07

    G-Protein Coupled Receptor (GPCR), Class C, Group 5, Member A (GPRC5A) has been implicated in several malignancies. The underlying mechanisms, however, remain poorly understood. Using a panel of human cell lines, we demonstrate that CRISPR/Cas9-mediated knockout and RNAi-mediated depletion of GPRC5A impairs cell adhesion to integrin substrates: collagens I and IV, fibronectin, as well as to extracellular matrix proteins derived from the Engelbreth-Holm-Swarm (EHS) mouse sarcoma (Matrigel). Consistent with the phenotype, knock-out of GPRC5A correlated with a reduced integrin β1 (ITGB1) protein expression, impaired phosphorylation of the focal adhesion kinase (FAK), and lower activity of small GTPases RhoA and Rac1. Furthermore, we provide the first evidence for a direct interaction between GPRC5A and a receptor tyrosine kinase EphA2, an upstream regulator of FAK, although its contribution to the observed adhesion phenotype is unclear. Our findings reveal an unprecedented role for GPRC5A in regulation of the ITGB1-mediated cell adhesion and it's downstream signaling, thus indicating a potential novel role for GPRC5A in human epithelial cancers.

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

    NASA Astrophysics Data System (ADS)

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

    2015-08-01

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

  19. Regulation of the L-type calcium channel by alpha 5beta 1 integrin requires signaling between focal adhesion proteins.

    PubMed

    Wu, X; Davis, G E; Meininger, G A; Wilson, E; Davis, M J

    2001-08-10

    The L-type calcium channel is the major calcium influx pathway in vascular smooth muscle and is regulated by integrin ligands, suggesting an important link between extracellular matrix and vascular tone regulation in tissue injury and remodeling. We examined the role of integrin-linked tyrosine kinases and focal adhesion proteins in regulation of L-type calcium current in single vascular myocytes. Soluble tyrosine kinase inhibitors blocked the increase in current produced by alpha(5) integrin antibody or fibronectin, whereas tyrosine phosphatase inhibition enhanced the effect. Cell dialysis with an antibody to focal adhesion kinase or with FRNK, the C-terminal noncatalytic domain of focal adhesion kinase, produced moderate (24 or 18%, respectively) inhibition of basal current but much greater inhibition (63 or 68%, respectively) of integrin-enhanced current. A c-Src antibody and peptide inhibitors of the Src homology-2 domain or a putative Src tyrosine phosphorylation site on the channel produced similar inhibition. Antibodies to the cytoskeletal proteins paxillin and vinculin, but not alpha-actinin, inhibited integrin-dependent current by 65-80%. Therefore, alpha(5)beta(1) integrin appears to regulate a tyrosine phosphorylation cascade involving Src and various focal adhesion proteins that control the function of the L-type calcium channel. This interaction may represent a novel mechanism for control of calcium influx in vascular smooth muscle and other cell types.

  20. Cell adhesion to proteins separated by lithium dodecyl sulfate-polyacrylamide gel electrophoresis and blotted onto a polyvinylidene difluoride membrane: a new cell-blotting technique.

    PubMed

    Seshi, B

    1994-12-02

    Cell blotting, although conceptually simple, has failed to achieve wide practical application. Described here is a new cell-blotting technique which involves cell adhesion to protein bands after separation by lithium dodecyl sulfate-polyacrylamide gel electrophoresis (LDS-PAGE) and blotting onto polyvinylidene difluoride (PVDF) membrane at 4 degrees C. Cell bands adherent on PVDF are detected using hematoxylin, or propidium iodide (PI) staining followed by viewing under ultraviolet (UV) light. The technique allows quick microscopic visualization of adherent cells composing the bands, without requiring clearing of the membrane. Representative cell adhesion proteins from different sources, i.e., plant lectins (e.g., phytohemagglutinin, PHA; concanavalin A, ConA; and wheat germ agglutinin, WGA); extracellular matrix (ECM) proteins; and integral membrane proteins (e.g., recombinant soluble vascular cell adhesion molecule-1, rs VCAM-1) were tested for cell binding by the new cell-blotting technique using human lymphoid progenitor (NALM-6) and myeloid progenitor (KG1a) cell lines. Cell adhesion proteins retained their adhesion function in all cases tested. Specificity of cell binding on PVDF blot was demonstrated by inhibition of cell adhesion to WGA protein bands using an appropriate sugar, i.e., N-acetyl D-glucosamine. The cell blotting assay was comparable in sensitivity to Coomassie blue staining of protein bands. The ability to conduct protein extraction, separation and blotting at low temperature avoids thermal denaturation, thereby preserving the adhesion properties of the proteins. The electrophoretic/blotting system has unique detergent removal/protein renaturation properties and the ability to preserve functionally active adhesion protein complexes. The cell-blotting technique described is sufficiently robust for routine application in the investigation of novel cell adhesion proteins.

  1. 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

  2. Effects of protein tyrosine kinase inhibitors on cytokine-induced adhesion molecule expression by human umbilical vein endothelial cells.

    PubMed Central

    May, M. J.; Wheeler-Jones, C. P.; Pearson, J. D.

    1996-01-01

    1. Endothelial cells can be stimulated by the pro-inflammatory cytokines interleukin (IL)-1 alpha and tumour necrosis factor (TNF) alpha to express the leukocyte adhesion molecules E-selectin, vascular cell adhesion molecule (VCAM)-1 and intercellular adhesion molecule (ICAM)-1 but the intracellular signalling mechanisms leading to this expression are incompletely understood. We have investigated the role of protein tyrosine kinases (PTK) in adhesion molecule expression by cytokine-activated human umbilical vein endothelial cells (HUVEC) using the PTK inhibitors genistein and herbimycin A, and the protein tyrosine phosphatase (PTP) inhibitor sodium orthovanadate. 2. Maximal E-selectin expression induced by incubation of HUVEC for 4 h with IL-1 alpha (100 u ml-1) and TNF alpha (100 u ml-1) was dose-dependently inhibited by genistein and herbimycin A. Although similar effects were seen on phorbol 12-myristate, 13-acetate (PMA)-induced expression, this was not due to inhibition of protein kinase C (PKC) activity as the selective inhibitors of PKC, bisindolylmaleimide (BIM), Ro31-7549 or Ro31-8220 did not affect IL-1 alpha- or TNF alpha-induced E-selectin expression at concentrations which maximally inhibited PMA-induced expression. 3. Genistein inhibited VCAM-1 expression induced by incubation of HUVEC for 24 h with TNF alpha or IL-1 alpha whereas it did not affect ICAM-1 expression induced by 24 h incubation with either of these cytokines. Herbimycin A inhibited both VCAM-1 and ICAM-1 expression induced by TNF alpha. 4. Basal expression of E-selectin, VCAM-1 and ICAM-1 was dose-dependently enhanced by sodium orthovanadate. In contrast, vanadate differentially affected TNF alpha-induced expression of these molecules with maximal E-selectin and ICAM-1 expression being slightly enhanced and VCAM-1 expression dose-dependently reduced. 5. We also studied the effects of PTK and PTP inhibitors on adhesion of the human pre-myeloid cell line U937 to TNF alpha-stimulated HUVEC

  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

  4. Redox control of surface protein sulphhydryls in bovine spermatozoa reversibly modulates sperm adhesion to the oviductal epithelium and capacitation.

    PubMed

    Gualtieri, Roberto; Mollo, Valentina; Duma, Gennaro; Talevi, Riccardo

    2009-07-01

    Oviductal fluid molecules, such as sulphated glycosaminoglycans and disulphide-reductants, may represent periovulatory signals for the release of spermatozoa from the oviductal reservoir in the bovine species. Disulphide-reductants release spermatozoa through the reduction of sperm-surface disulphides to sulphhydryls (SH). Herein, we studied sperm-surface protein SH through labelling with maleimidylpropionyl biocytin in the initial sperm suspension, in the subpopulations able and unable to adhere to the in vitro cultured oviductal epithelium, and in spermatozoa released either through the disulphide-reductant penicillamine (PEN) or the sulphated glycosaminoglycan heparin (HEP). Adhesion assays were performed to study the ability of released spermatozoa to readhere to the oviductal epithelium. Results showed that the level of SH in sperm-surface proteins was: 1) low in adhering spermatozoa; 2) high in spermatozoa unable to adhere; and 3) markedly increased in released spermatozoa. Adhesion assays showed that: 1) PEN-released spermatozoa promptly recovered adhesion after removal of the disulphide-reductant and could be released again in response to PEN; 2) conversely, a limited number of HEP-released spermatozoa was able to readhere to the oviductal epithelium and this ability was not affected by HEP removal. Recovery of adhesion was associated to reoxidation of sperm-surface protein SH and to the reversal of capacitation. In conclusion, redox modulation of sperm-surface protein SH is involved in the release of spermatozoa adhering to the oviduct in vitro; the reversible action of disulphide-reductants might be responsible for intermittent phases of adhesions and releases; and the irreversible action of HEP indicates that it may represent a terminal releasing signal.

  5. Human DCXR - another 'moonlighting protein' involved in sugar metabolism, carbonyl detoxification, cell adhesion and male fertility?

    PubMed

    Ebert, Bettina; Kisiela, Michael; Maser, Edmund

    2015-02-01

    Dicarbonyl/L-xylulose reductase (DCXR; SDR20C1), a member of the short-chain dehydrogenase/reductase (SDR) superfamily catalyzes the reduction of α-dicarbonyl compounds and monosaccharides. Its role in the metabolism of L-xylulose has been known since 1970, when essential pentosuria was found to be associated with DCXR deficiency. Despite its early discovery, our knowledge about the role of human DCXR in normal physiology and pathophysiology is still incomplete. Sporadic studies have demonstrated aberrant expression in several cancers, but their physiological significance is unknown. In reproductive medicine, where DCXR is commonly referred to as 'sperm surface protein P34H', it serves as marker for epididymal sperm maturation and is essential for gamete interaction and successful fertilization. DCXR exhibits a multifunctional nature, both acting as a carbonyl reductase and also performing non-catalytic functions, possibly resulting from interactions with other proteins. Recent observations associate DCXR with a role in cell adhesion, pointing to a novel function involving tumour progression and possibly metastasis. This review summarizes the current knowledge about human DCXR and its orthologs from mouse and Caenorhabditis elegans (DHS-21) with an emphasis on its multifunctional characteristics. Due to its close structural relationship with DCXR, carbonyl reductase 2 (Cbr2), a tetrameric enzyme found in several non-primate species is also discussed. Similar to human DCXR, Cbr2 from golden hamster (P26h) and cow (P25b) is essential for sperm-zona pellucida interaction and fertilization. Because of the apparent similarity of these two proteins and the inconsistent use of alternative names previously, we provide an overview of the systematic classification of DCXR and Cbr2 and a phylogenetic analysis to illustrate their ancestry.

  6. Silk protein as a new optically transparent adhesion layer for an ultra-smooth sub-10 nm gold layer

    NASA Astrophysics Data System (ADS)

    Min, Kyungtaek; Umar, Muhammad; Ryu, Shinyoung; Lee, Soonil; Kim, Sunghwan

    2017-03-01

    Ultra-thin and ultra-smooth gold (Au) films are appealing for photonic applications including surface plasmon resonances and transparent contacts. However, poor adhesion at the Au–dielectric interface prohibits the formation of a mechanically stable, ultra-thin, and ultra-smooth Au film. A conventional solution is to use a metallic adhesion layer, such as titanium and chromium, however such layers cause the optical properties of pure Au to deteriorate. Here we report the use of silk protein to enhance the adhesion at the Au–dielectric interface, thus obtaining ultra-smooth sub-10 nm Au films. The Au films that were deposited onto the silk layer exhibited superior surface roughness to those deposited on SiO2, Si, and poly(methyl methacrylate), along with improved adhesion, electrical conductivity, and optical transparency. Additionally, we confirm that a metal–insulator–metal optical resonator can be successfully generated using a silk insulating layer without the use of a metallic adhesion layer.

  7. Low density lipoprotein receptor-related protein 1 mediated endocytosis of β1-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 β1-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 β1-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 β1-integrin and decreased β1-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 β1-integrin mediating integrin internalization and thus correlates with downstream signaling of β1-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.

  8. Roles for the tubulin- and PTP-PEST-binding paxillin LIM domains in cell adhesion and motility.

    PubMed

    Brown, Michael C; Turner, Christopher E

    2002-07-01

    Cell dynamics mediated through cell-extracellular matrix contacts, such as adhesion and motility involve the precise regulation of large complexes of structural and signaling molecules called focal adhesions (FAs). Paxillin is a multi-domain FA adaptor protein containing five amino-terminal paxillin leucine-aspartate repeat (LD) motifs and four carboxyl-terminal Lin-11 Isl-1 and Mec-3 (LIM) domains. The LD motifs support paxillin binding to actopaxin, integrin linked kinase (ILK), FA kinase (FAK), paxillin kinase linker (PKL) and vinculin. Of the LIM domains, LIM2 and 3 comprise the paxillin FA-targeting motif, with phosphorylation of these domains modulating paxillin targeting and cell adhesion to fibronectin (Fn). The identity of the paxillin FA targeting partner remains to be determined; however, the LIM domains mediate interactions with tubulin and the protein-tyrosine phosphatase (PTP)-PEST. PTP-PEST binding requires both LIM3 and 4, whereas, the precise LIM target of tubulin binding is not known. In this report, we demonstrate that the individual paxillin LIM2 and 3 domains support specific binding to tubulin and suggest a potential role for this interaction in the regulation of paxillin sub-cellular compartmentalization. In addition, expression of paxillin molecules with mutations in the tubulin- and PTP-PEST-binding LIM domains differentially impaired Chinese hamster ovary K1 (CHO.K1) cell adhesion and migration to Fn. Perturbation of LIM3 or 4 inhibited adhesion while mutation of LIM2 or 4 decreased cell motility. Interestingly, expression of tandem LIM2-3 inhibited cell adhesion and spreading while LIM3-4 stimulated a well-spread polarized phenotype. These data offer further support for a critical role for paxillin in cell adhesion and motility.

  9. Transforming growth factor beta-induced (TGFBI) is an anti-adhesive protein regulating the invasive growth of melanoma cells.

    PubMed

    Nummela, Pirjo; Lammi, Johanna; Soikkeli, Johanna; Saksela, Olli; Laakkonen, Pirjo; Hölttä, Erkki

    2012-04-01

    Melanoma is a malignancy characterized by high invasive/metastatic potential, with no efficient therapy after metastasis. Understanding the molecular mechanisms underlying the invasive/metastatic tendency is therefore important. Our genome-wide gene expression analyses revealed that human melanoma cell lines WM793 and especially WM239 (vertical growth phase and metastatic cells, respectively) overexpress the extracellular matrix (ECM) protein transforming growth factor β induced (TGFBI). In adhesion assays, recombinant TGFBI was strongly anti-adhesive for both melanoma cells and skin fibroblasts. TGFBI further impaired the adhesion of melanoma cells to the adhesive ECM proteins fibronectin, collagen-I, and laminin, known to interact with it. Unexpectedly, WM239 cells migrated/invaded more effectively in three-dimensional collagen-I and Matrigel cultures after knockdown of TGFBI by shRNA expression. However, in the physiological subcutaneous microenvironment in nude mice, after TGFBI knockdown, these cells showed markedly impaired tumor growth and invasive capability; the initially formed small tumors later underwent myxoid degeneration and completely regressed. By contrast, the expanding control tumors showed intense TGFBI staining at the tumor edges, co-localizing with the fibrillar fibronectin/tenascin-C/periostin structures that characteristically surround melanoma cells at invasion fronts. Furthermore, TGFBI was found in similar fibrillar structures in clinical human melanoma metastases as well, co-localizing with fibronectin. These data imply an important role for TGFBI in the ECM deposition and invasive growth of melanoma cells, rendering TGFBI a potential target for therapeutic interventions.

  10. Mammalian adenylyl cyclase-associated protein 1 (CAP1) regulates cofilin function, the actin cytoskeleton, and cell adhesion.

    PubMed

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

    2013-07-19

    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.

  11. 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.

  12. Modulation of endogenous Cysteine Protease Inhibitor (ICP) 1 expression in Entamoeba histolytica affects amoebic adhesion to Extracellular Matrix proteins.

    PubMed

    Lee, Young Ah; Saito-Nakano, Yumiko; Kim, Kyeong Ah; Min, Arim; Nozaki, Tomoyoshi; Shin, Myeong Heon

    2015-02-01

    Entamoeba histolytica is an enteric tissue-invading protozoan parasite that causes amoebic colitis and occasionally liver abscess in humans. During tissue invasion, amoebic adhesion to host components is an important event for host cell death leading to successful invasion and infection. Among amoebic virulence factors, Gal/GalNAc lectin is known to be major adhesion factor to host cells. In this study, we investigated the role of amoebic secreted CP (Cysteine Proteases) in amoebic adhesion to extracellular matrix (ECM) protein using CP inhibitor and E. histolytica strains in which the endogenous inhibitor of cysteine protease (ICP) 1 gene was overexpressed (ICP1(+)) or repressed by antisense small RNA-mediated gene silencing (ICP1(-)). We found that pretreatment of wild-type amoebae with CP inhibitor E64, or thiol-group modifiers such as diamide and N-Ethylmaleimide resulted in a significant decrease in adhesion to laminin and collagen ECM proteins. Furthermore, ICP1(+) strain, with a reduction of secreted CP activity, exhibited reduced ability by 40% to adhere to laminin. In contrast, ICP1(-) strain, with a 1.9-fold increase of secreted CP activity, showed a two-fold increase in amoebic adherence to laminin compared to the control strain. In addition, total amount of secreted CP5 was decreased in ICP1(+) amoeba. Conversely, total amount of secreted CP1 and mature-form CP5 were increased in ICP1(-) amoeba. We also found that ICP1 was secreted into extracellular milieu. These results suggest that secreted CP activity by E. histolytica may be an important factor affecting adhesion to host proteins, and regulation of CP secretion by ICP plays a major role in pathogenesis. This study provides insight into the CP-mediated tissue pathogenesis in amoeba-invaded lesions during human amoebiasis.

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

    PubMed Central

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

    2015-01-01

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

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

    PubMed

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

    2015-01-01

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

  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

    2016-01-05

    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-κB) 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-κB activation and the expression of NF-κB-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 (TNFα) was used as a representative proinflammatory cytokine. In an adhesion assay using THP-1 cells, CTRP9 reduced TNFα-induced adhesion of monocytes to HAECs. Treatment with CTRP9 significantly decreased TNFα-induced activation of NF-κB, 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 AMPKα1-specific siRNA. Our study is the first to demonstrate that CTRP9 attenuates cytokine-induced vascular inflammation in endothelial cells mediated by AMPK activation.

  16. 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; Dé, 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 81–176 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 81–176 ΔCadF 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

  17. Adhesion protein VSIG1 is required for the proper differentiation of glandular gastric epithelia.

    PubMed

    Oidovsambuu, Odgerel; Nyamsuren, Gunsmaa; Liu, Shuai; Göring, Wolfgang; Engel, Wolfgang; Adham, Ibrahim M

    2011-01-01

    VSIG1, a cell adhesion protein of the immunoglobulin superfamily, is preferentially expressed in stomach, testis, and certain gastric, esophageal and ovarian cancers. Here, we describe the expression patterns of three alternatively spliced isoforms of mouse Vsig1 during pre- and postnatal development of stomach and potential function of Vsig1 in differentiation of gastric epithelia. We show that isoforms Vsig1A and Vsig1B, which differ in the 3'untranslated region, are expressed in the early stages of stomach development. Immunohistochemical analysis revealed that VSIG1 is restricted to the adherens junction of the glandular epithelium. The shorter transcript Vsig1C is restricted to the testis, encodes an N-terminal truncated protein and is presumably regulated by an internal promoter, which is located upstream of exon 1b. To determine whether the 5' flanking region of exon 1a specifically targets the expression of Vsig1 to stomach epithelia, we generated and analyzed transgenic mice. The 4.8-kb fragment located upstream of exon 1a was sufficient to direct the expression of the reporter gene to the glandular epithelia of transgenic stomach. To determine the role of VSIG1 during the development of stomach epithelia, an X-linked Vsig1 was inactivated in embryonic stem cells (ESCs). Although Vsig1(-/Y) ESCs were only able to generate low coat color chimeric mice, no male chimeras transmitted the targeted allele to their progeny suggesting that the high contribution of Vsig1(-/Y) cells leads to the lethality of chimeric embryos. Analysis of chimeric stomachs revealed the differentiation of VSIG1-null cells into squamous epithelia inside the glandular region. These results suggest that VSIG1 is required for the establishment of glandular versus squamous epithelia in the stomach.

  18. Conserved roles of the prion protein domains on subcellular localization and cell-cell adhesion.

    PubMed

    Solis, Gonzalo P; Radon, Yvonne; Sempou, Emily; Jechow, Katharina; Stuermer, Claudia A O; Málaga-Trillo, Edward

    2013-01-01

    Analyses of cultured cells and transgenic mice expressing prion protein (PrP) deletion mutants have revealed that some properties of PrP -such as its ability to misfold, aggregate and trigger neurotoxicity- are controlled by discrete molecular determinants within its protein domains. Although the contributions of these determinants to PrP biosynthesis and turnover are relatively well characterized, it is still unclear how they modulate cellular functions of PrP. To address this question, we used two defined activities of PrP as functional readouts: 1) the recruitment of PrP to cell-cell contacts in Drosophila S2 and human MCF-7 epithelial cells, and 2) the induction of PrP embryonic loss- and gain-of-function phenotypes in zebrafish. Our results show that homologous mutations in mouse and zebrafish PrPs similarly affect their subcellular localization patterns as well as their in vitro and in vivo activities. Among PrP's essential features, the N-terminal leader peptide was sufficient to drive targeting of our constructs to cell contact sites, whereas lack of GPI-anchoring and N-glycosylation rendered them inactive by blocking their cell surface expression. Importantly, our data suggest that the ability of PrP to homophilically trans-interact and elicit intracellular signaling is primarily encoded in its globular domain, and modulated by its repetitive domain. Thus, while the latter induces the local accumulation of PrPs at discrete punctae along cell contacts, the former counteracts this effect by promoting the continuous distribution of PrP. In early zebrafish embryos, deletion of either domain significantly impaired PrP's ability to modulate E-cadherin cell adhesion. Altogether, these experiments relate structural features of PrP to its subcellular distribution and in vivo activity. Furthermore, they show that despite their large evolutionary history, the roles of PrP domains and posttranslational modifications are conserved between mouse and zebrafish.

  19. Internalization of adhesion junction proteins and their association with recycling endosome marker proteins in rat seminiferous epithelium.

    PubMed

    Young, J'Nelle S; Takai, Yoshimi; Kojic, Katarina L; Vogl, A Wayne

    2012-03-01

    Tubulobulbar complexes (TBCs) are elaborate cytoskeleton-related structures that are formed in association with intercellular junctions in the seminiferous epithelium. They consist of a cylindrical double-membrane core composed of the plasma membranes of the two attached cells, cuffed by a dendritic network of actin filaments. TBCs are proposed to be subcellular machines that internalize intercellular junctions during the extensive junction remodeling that occurs during spermatogenesis. At the apical sites of attachment between Sertoli cells and spermatids, junction disassembly is part of the sperm release mechanism. In this study, we used immunological probes to explore junction internalization and recycling at apical TBCs in the rat seminiferous epithelium. We demonstrate that β1-integrin and nectin 2 were concentrated at the ends of TBCs and for the first time show that the early endosome marker RAB5A was also distinctly localized at the ends of TBCs that appear to be the 'bulbar' regions of the complexes. Significantly, we also demonstrate that the 'long-loop' recycling endosome marker RAB11A was co-distributed with nectin 2 at junctions with early spermatids deeper in the epithelium. Our results are consistent with the hypothesis that TBCs associated with late spermatids internalize adhesion junctions and also indicate that some of the internalized junction proteins may be recycled to form junctions with the next generation of spermatids.

  20. SDF-1-induced adhesion of monocytes to vascular endothelium is modulated by azelnidipine via protein kinase C inhibition.

    PubMed

    Takahashi, Keiko; Shimokado, Kentaro; Yoshida, Masayuki

    2006-12-15

    Monocyte-endothelial interaction and its modulation by chemokines play a key role in atherogenesis and inflammation. We examined the potential effects of stromal cell-derived factor (SDF-1) and azelnidipine, a novel dihydropyridine derivative, toward monocyte-endothelial interaction. Human monocytes were prepared from peripheral blood mononuclear cells obtained from healthy volunteers and pretreated with azelnidipine (1 microM) for 48 h, after which their adhesion to interleukin-1beta (IL-1beta)-activated human umbilical vein endothelial cells (HUVECs) was analyzed using an in vitro flow apparatus with a shear stress of 1 dyn/cm(2). In some experiments, monocytes were incubated in the presence of stromal cell-derived factor (SDF-1), a chemokine, just prior to the assay. Pre-incubation of monocytes with SDF-1 enhanced their adhesion to activated HUVECs. When monocytes were pre-incubated in the presence of azelnidipine, baseline levels as well as SDF-1-induced monocyte adhesion levels were reduced. Interestingly, the surface expressions of the adhesion molecules CD11a, CD11b, and CD36, were not changed by azelnidipine treatment. Western blotting analysis revealed that activation of protein kinase C (PKC)alpha was inhibited by azelnidipine treatment, while it also reduced the SDF-1-induced increase in intracellular calcium concentration ([Ca(2+)](i)). Further, pre-incubation of monocytes with Go6976, a potent inhibitor of PKCalpha, significantly reduced monocyte adhesion to HUVECs. Our results demonstrated an inhibitory action of azelnidipine toward adhesive interactions of monocytes to HUVECs, which involves inhibition of PKCalpha and a reduction in [Ca(2+)](i). These findings imply a protective role of azelnidipine against inflammation in atherosclerosis.

  1. KDM4B histone demethylase and G9a regulate expression of vascular adhesion proteins in cerebral microvessels

    PubMed Central

    Choi, Ji-Young; Yoon, Sang-Sun; Kim, Sang-Eun; Ahn Jo, Sangmee

    2017-01-01

    Intercellular adhesion molecule 1 (ICAM1) mediates the adhesion and transmigration of leukocytes across the endothelium, promoting inflammation. We investigated the epigenetic mechanism regulating ICAM1 expression. The pro-inflammatory cytokine TNF-α dramatically increased ICAM1 mRNA and protein levels in human brain microvascular endothelial cells and mouse brain microvessels. Chromatin immunoprecipitation revealed that TNF-α reduced methylation of histone H3 at lysines 9 and 27 (H3K9 and H3K27), well-known residues involved in gene suppression. Inhibition of G9a and EZH2, histone methyltransferases responsible for methylation at H3K9 and H3K27, respectively as well as G9a overexpression demonstrated the involvement of G9a in TNF-α-induced ICAM1 expression and leukocyte adhesion and transmigration. A specific role for KDM4B, a histone demethylase targeting H3K9me2, in TNF-α-induced ICAM1 upregulation was validated with siRNA. Moreover, treating mice with a KDM4 inhibitor ML324 blocked TNF-α-mediated neutrophil adhesion. Similarly, TNF-α-induced VCAM1 expression was suppressed by G9a overexpression and KDM4B knockdown. Collectively, we demonstrated that modification of H3K9me2 by G9a and KDM4B regulates expression of vascular adhesion molecules, and that depletion of these proteins or KDM4B reduces inflammation-induced leukocyte extravasation. Thus, blocking ICAM1 or KDM4B could offer a novel therapeutic opportunity treating brain diseases. PMID:28327608

  2. A Role for the Protein Tyrosine Phosphatase CD45 in Macrophage Adhesion through the Regulation of Paxillin Degradation

    PubMed Central

    St-Pierre, Joëlle; Ostergaard, Hanne L.

    2013-01-01

    CD45 is a protein tyrosine phosphatase expressed on all cells of hematopoietic origin that is known to regulate Src family kinases. In macrophages, the absence of CD45 has been linked to defects in adhesion, however the molecular mechanisms involved remain poorly defined. In this study, we show that bone marrow derived macrophages from CD45-deficient mice exhibit abnormal cell morphology and defective motility. These defects are accompanied by substantially decreased levels of the cytoskeletal-associated protein paxillin, without affecting the levels of other proteins. Degradation of paxillin in CD45-deficient macrophages is calpain-mediated, as treatment with a calpain inhibitor restores paxillin levels in these cells and enhances cell spreading. Inhibition of the tyrosine kinases proline-rich tyrosine kinase (Pyk2) and focal adhesion kinase (FAK), kinases that are capable of mediating tyrosine phosphorylation of paxillin, also restored paxillin levels, indicating a role for these kinases in the CD45-dependent regulation of paxillin. These data demonstrate that CD45 functions to regulate Pyk2/FAK activity, likely through the activity of Src family kinases, which in turn regulates the levels of paxillin to modulate macrophage adhesion and migration. PMID:23936270

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

    PubMed

    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.

  4. A possible role of cellulose-binding protein A (CBPA) in the adhesion of Eubacterium cellulosolvens 5 to cellulose.

    PubMed

    Toyoda, Atsushi; Takano, Kazunori; Minato, Hajime

    2003-08-01

    The cellulose-binding protein A (CBPA) of Eubacterium cellulosolvens 5 is a modular enzyme comprised of a catalytic domain, a cellulose-binding domain and a cell wall-binding domain. Cellobiose-grown cells changed their adhesion ability to cellulose depending on the growth phase. On the other hand, carboxymethyl cellulose (CMC)-grown cells bound to cellulose regardless of their growth phase. The distribution of CBPA in the culture supernatant and cell fractions changed depending on the carbon source contained in the medium and growth phase. The cellobiose-grown cells harvested from the culture of the late stationary growth phase did not bind to cellulose, but their adhesion ability was recovered by treatment with recombinant CBPA. Moreover, cellobiose-grown cells harvested from the culture of an early exponential growth phase bound to cellulose, but their adhesion ability was inhibited by treatment with anti-CBPA antiserum. CBPA rapidly decreased the viscosity of CMC, indicating that CBPA was endoglucanase. The results obtained in this study indicate that CBPA plays an important role in the adhesion of E. cellulosolvens 5 cells to cellulose.

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

    SciTech Connect

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

    2007-07-13

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

  6. Localization of vascular adhesion protein-1 (VAP-1) in the human eye.

    PubMed

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

    2010-01-01

    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 therefore investigated by immunohistochemistry. Five micrometer thick sections were generated from human ocular tissues embedded in paraffin. Sections were incubated overnight with primary mAbs against VAP-1 (5 microg/ml), smooth muscle actin (1 microg/ml), CD31 or isotype-matched IgG at 4 degrees C. Subsequently, a secondary mAb was used for 30 min 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 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 colocalized 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 nearly 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.

  7. GPR133 (ADGRD1), an adhesion G-protein-coupled receptor, is necessary for glioblastoma growth

    PubMed Central

    Bayin, N S; Frenster, J D; Kane, J R; Rubenstein, J; Modrek, A S; Baitalmal, R; Dolgalev, I; Rudzenski, K; Scarabottolo, L; Crespi, D; Redaelli, L; Snuderl, M; Golfinos, J G; Doyle, W; Pacione, D; Parker, E C; Chi, A S; Heguy, A; MacNeil, D J; Shohdy, N; Zagzag, D; Placantonakis, D G

    2016-01-01

    Glioblastoma (GBM) is a deadly primary brain malignancy with extensive intratumoral hypoxia. Hypoxic regions of GBM contain stem-like cells and are associated with tumor growth and angiogenesis. The molecular mechanisms that regulate tumor growth in hypoxic conditions are incompletely understood. Here, we use primary human tumor biospecimens and cultures to identify GPR133 (ADGRD1), an orphan member of the adhesion family of G-protein-coupled receptors, as a critical regulator of the response to hypoxia and tumor growth in GBM. GPR133 is selectively expressed in CD133+ GBM stem cells (GSCs) and within the hypoxic areas of PPN in human biospecimens. GPR133 mRNA is transcriptionally upregulated by hypoxia in hypoxia-inducible factor 1α (Hif1α)-dependent manner. Genetic inhibition of GPR133 with short hairpin RNA reduces the prevalence of CD133+ GSCs, tumor cell proliferation and tumorsphere formation in vitro. Forskolin rescues the GPR133 knockdown phenotype, suggesting that GPR133 signaling is mediated by cAMP. Implantation of GBM cells with short hairpin RNA-mediated knockdown of GPR133 in the mouse brain markedly reduces tumor xenograft formation and increases host survival. Analysis of the TCGA data shows that GPR133 expression levels are inversely correlated with patient survival. These findings indicate that GPR133 is an important mediator of the hypoxic response in GBM and has significant protumorigenic functions. We propose that GPR133 represents a novel molecular target in GBM and possibly other malignancies where hypoxia is fundamental to pathogenesis. PMID:27775701

  8. 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 μm thick sections were generated from human ocular tissues embedded in paraffin. Sections were incubated overnight with primary mAbs against VAP-1 (5μg/ml), smooth muscle actin (1μg/ml), CD31 or isotype-matched IgG at 4°C. 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

  9. Optical measurements of dynamic adhesive forces between bacteria and protein-coated surfaces

    NASA Astrophysics Data System (ADS)

    Simpson, Kathryn H.; Bowden, Gabriela; Hook, Magnus; Anvari, Bahman

    2003-06-01

    Bacterial adhesion to host tissue is an initial step in the infectious process. Staphylococcus aureus, a major human pathogen, has covalently anchored cell surface adhesins called microbial surface components recognizing adhesive matrix molecules (MSCRAMMs) which mediate specific adhesion to extracellular matrix (ECM) molecules. Understanding MSCRAMM binding is potentially useful in developing effective antibacterial drugs. In this study, optical tweezers were used in conjunction with a quadrant photodetector to measure adhesive forces between MSCRAMMs and surfaces coated with the ECM molecule fibronectin. Using a piezoelectrically driven stage, a fibronectin-coated microsphere adherent to a coverslip was brought into contact with a cell optically trapped at 830 nm. The microsphere was subsequently moved away from the cell, and a quadrant photodiode monitored the cell displacement from the trap center during the detachment process. The photodetector voltage signals were subsequently converted into the adhesive forces between MSCRAMMs and fibronectin based on a calibration using Stoke"s law for viscous drag. Optical detection of the trapped bead displacement allowed us to study both the dynamics of the detachment process and observe the effects of various loading rates. This technique can be extended to identify the contributions of various MSCRAMM domains to adhesion in order to develop new methods of treating infections.

  10. 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, João; Voabil, Paula; Liberal, Joana; Chiavaroli, Carlo; Bauer, Jacques; Cunha-Vaz, José; Ambrósio, António 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 blood–retinal 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-κB 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-κB 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-κB activation, possibly through the inhibition of oxidative

  11. PDK1 regulates focal adhesion disassembly by modulating endocytosis of αvβ3 integrin.

    PubMed

    di Blasio, Laura; Gagliardi, Paolo Armando; Puliafito, Alberto; Sessa, Roberto; Seano, Giorgio; Bussolino, Federico; Primo, Luca

    2015-03-01

    Non-amoeboid cell migration is characterised by dynamic competition among multiple protrusions to establish new adhesion sites at the cell's leading edge. However, the mechanisms that regulate the decision to disassemble or to grow nascent adhesions are not fully understood. Here we show that, in endothelial cells, 3-phosphoinositide-dependent protein kinase 1 (PDK1) promotes focal adhesion (FA) turnover by controlling endocytosis of integrin αvβ3 in a PI3K-dependent manner. We demonstrate that PDK1 binds and phosphorylates integrin αvβ3. Downregulation of PDK1 increases FA size and slows down their disassembly. This process requires both PDK1 kinase activity and PI3K activation but does not involve Akt. Moreover, PDK1 silencing stabilises FA in membrane protrusions decreasing migration of endothelial cells on vitronectin. These results indicate that modulation of integrin endocytosis by PDK1 hampers endothelial cell adhesion and migration on extracellular matrix, thus unveiling a novel role for this kinase.

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

    PubMed

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

    2014-04-01

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

  13. Dualistic nature of adhesive protein function: fibronectin and its biologically active peptide fragments can autoinhibit fibronectin function

    PubMed Central

    1984-01-01

    Fibronectin and certain polypeptide regions of this adhesive glycoprotein mediate cell attachment and spreading on various substrates. We explored the theoretical prediction that this adhesive protein could become a competitive inhibitor of fibronectin-mediated processes if present in solution at appropriately high concentrations. Fibronectin function was inhibited by purified plasma fibronectin at 5- 10 mg/ml, by a 75,000-dalton cell-interaction fragment of the protein at 0.5-1 mg/ml, and even by two synthetic peptides containing a conserved, hydrophilic amino acid sequence at 0.1-0.5 mg/ml. Inhibition of fibronectin-dependent cell spreading was dose dependent, noncytotoxic, and reversible. It was competitive in nature, since increased quantities of substrate-adsorbed fibronectin or longer incubation periods decreased the inhibition. A peptide inhibitory for fibronectin-mediated cell spreading also inhibited fibronectin-mediated attachment of cells to type I collagen, but it did not affect concanavalin A-mediated spreading. These results demonstrate the potential of a cell adhesion molecule and its biologically active peptide fragments to act as competitive inhibitors, and they suggest that fibronectin may act by binding to a saturable cell surface receptor. PMID:6736130

  14. Exploring the Molecular Origins of Bio(in)compatibility: Adhesion Between Proteins and Individual Chains of Poly(ethylene oxide)

    NASA Astrophysics Data System (ADS)

    Rixman, Monica A.; Ortiz, Christine

    2002-03-01

    A critical determinant of the biocompatibility of implanted blood-contacting devices is the initial noncovalent adsorption of blood plasma proteins onto the biomaterial surface. Using high-resolution force spectroscopy, we have measured the complex intermolecular interaction forces between individual end-grafted PEO chains and a probe tip covalently bound with human serum albumin, the most abundant blood plasma protein in the human body. On approach, a long-range, nonlinear repulsive force is observed. Upon retraction, however, adhesion between the HSA probe tip and PEO chain occurs, which in many cases is strong enough to allow long-range adhesion and stretching of the individual PEO chains. The known PEO strain-induced conformational transition from the helical (ttg) to the planar (ttt) conformation is clearly observed and seen to shift to lower force values. Statistical analysis of adhesion data, comparison to a variety of control experiments, and theoretical modeling enable us to interpret these experimental results in terms of electrostatic interactions, hydrogen bonding, and steric forces.

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

    PubMed

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

    2012-02-01

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

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

    PubMed

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

    2016-02-01

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

  17. Construction of multifunctional proteins for tissue engineering: epidermal growth factor with collagen binding and cell adhesive activities.

    PubMed

    Hannachi Imen, Elloumi; Nakamura, Makiko; Mie, Masayasu; Kobatake, Eiry

    2009-01-01

    The development of different techniques based on natural and polymeric scaffolds are useful for the design of different biomimetic materials. These approaches, however, require supplementary steps for the chemical or physical modification of the biomaterial. To avoid such steps, in the present study, we constructed a new multifunctional protein that can be easily immobilized onto hydrophobic surfaces, and at the same time helps enhance specific cell adhesion and proliferation onto collagen substrates. A collagen binding domain was fused to a previously constructed protein, which had an epidermal growth factor fused to a hydrophobic peptide that allows for cell adhesion. The new fusion protein, designated fnCBD-ERE-EGF is produced in Escherichia coli, and its abilities to bind to collagen and promote cell proliferation were investigated. fnCBD-ERE-EGF was shown to keep both collagen binding and cell growth-promoting activities comparable to those of the corresponding unfused proteins. The results obtained in this study also suggest the use of a fnCBD-ERE-EGF as an alternative for the design of multifunctional ECM-bound growth factor based materials.

  18. Congeneric bio-adhesive mussel foot proteins designed by modified prolines revealed a chiral bias in unnatural translation.

    PubMed

    Larregola, Maud; Moore, Shannon; Budisa, Nediljko

    2012-05-18

    Chiral bias in the unnatural translation and 'sticky' mussel proteins. The residue-specific in vivo incorporation of hydroxylated amino acids as well as other synthetic analogs, such as fluoroprolines, emerges as the method of choice for recombinant synthesis of Pro-rich mussel adhesive protein congeners. Chemical diversifications introduced in this way provide a general route towards bio-adhesive congeners endowed with properties not developed by natural evolution. Most importantly, we have found that the co-translational incorporation of (4R)-, and (4S)-hyroxylated and fluorinated analogs into mussel proteins presented a chiral bias: the expressed protein was only detectable in samples incubated with analogs with (4R)-substituents. Possible relationship of these stereochemical preferences for (4R)-stereoisomers in the translation to intracellular tRNA concentrations, ribosomal editing and proofreading or structural effects such as preorganization remains to be addressed in future studies. These studies will generally provide a mechanistic framework for the flexibility of the translational machinery and establish the boundaries of the unnatural translation.

  19. The Adhesion of Lactobacillus salivarius REN to a Human Intestinal Epithelial Cell Line Requires S-layer Proteins

    PubMed Central

    Wang, Ran; Jiang, Lun; Zhang, Ming; Zhao, Liang; Hao, Yanling; Guo, Huiyuan; Sang, Yue; Zhang, Hao; Ren, Fazheng

    2017-01-01

    Lactobacillus salivarius REN, a novel probiotic isolated from Chinese centenarians, can adhere to intestinal epithelial cells and subsequently colonize the host. We show here that the surface-layer protein choline-binding protein A (CbpA) of L. salivarius REN was involved in adherence to the human colorectal adenocarcinoma cell line HT-29. Adhesion of a cbpA deletion mutant was significantly reduced compared with that of wild-type, suggesting that CbpA acts as an adhesin that mediates the interaction between the bacterium and its host. To identify the molecular mechanism of adhesion, we determined the crystal structure of a truncated form of CbpA that is likely involved in binding to its cell-surface receptor. The crystal structure identified CbpA as a peptidase of the M23 family whose members harbor a zinc-dependent catalytic site. Therefore, we propose that CbpA acts as a multifunctional surface protein that cleaves the host extracellular matrix and participates in adherence. Moreover, we identified enolase as the CbpA receptor on the surface of HT-29 cells. The present study reveals a new class of surface-layer proteins as well as the molecular mechanism that may contribute to the ability of L. salivarius REN to colonize the human gut. PMID:28281568

  20. 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

    2015-09-15

    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.

  1. The influence of cross-linking on protein-protein interactions in a marine adhesive: the case of two byssus plaque proteins from the blue mussel.

    PubMed

    Fant, Camilla; Elwing, Hans; Höök, Fredrik

    2002-01-01

    The interaction between two proteins, Mefp-1 and Mefp-2, from the byssal plaque of the blue mussel, Mytilus edulis, was investigated using a quartz crystal microbalance with dissipation monitoring (QCM-D) technique. The challenge in using a surface-sensitive technique to investigate the interaction between two strongly adhesive proteins was met by coupling a biotinylated version of one of the proteins (b-Mefp-1) to an inert two-dimensional arrangement of streptavidin (SA) formed on top of a biotin-doped supported phospholipid bilayer. The interaction between Mefp-1 and Mefp-2 was further investigated by addition of Mefp-2 to SA-coupled b-Mefp-1, where the latter was either in the native state or cross-linked using sodium periodate (NaIO(4)), Cu(2+), or mushroom tyrosinase. With this coupling strategy it is shown that a requirement for attraction between the two proteins is that tyrosinase is used as the cross-linking agent of b-Mefp-1. By inhibiting the enzymatic activity of tyrosinase it is also shown that enzymatic activity is required for both efficient binding of tyrosinase to SA-coupled b-Mefp-1 as well as for the subsequent binding of Mefp-2. In contrast, spontaneous adsorption of Mefp-1 to a methyl-terminated (thiolated) gold surface followed by addition of Mefp-2 results in binding of Mefp-2 for all cross-linking agents. This suggests that cross-linking of Mefp-1 adsorbed on a solid surface induces structural changes in the adsorbed protein layer, resulting in exposure of free surface patches on which Mefp-2 binds.

  2. Regulation of cell-matrix adhesion by OLA1, the Obg-like ATPase 1

    PubMed Central

    Jeyabal, Prince VS; Rubio, Valentina; Chen, Huarong; Zhang, Jiawei; Shi, Zheng-Zheng

    2014-01-01

    Attachment of cells to the extracellular matrix induces clustering of membrane receptor integrins which in turn triggers the formation of focal adhesions (FAs). The adaptor/scaffold proteins in FAs provide linkage to actin cytoskeleton, whereas focal adhesion kinase (FAK) and other FA-associated kinases and phosphatases transduce integrin-mediated signaling cascades, promoting actin polymerization and progression of cell spreading. In this study, we explored the role of OLA1, a newly identified member of Obg-like ATPases, in regulating cell adhesion processes. We showed that in multiple human cell lines RNAi-mediated downregulation of OLA1 significantly accelerated cell adhesion and spreading, and conversely overexpression of OLA1 by gene transfection resulted in delayed cell adhesion and spreading. We further found that OLA1-deficient cells had elevated levels of FAK protein and decreased Ser3 phosphorylation of cofilin, an actin-binding protein and key regulator of actin filament dynamics, while OLA1-overexpressing cells exhibited the opposite molecular alterations in FAK and cofilin. These findings suggest that OLA1 plays an important negative role in cell adhesion and spreading, in part through the regulation of FAK expression and cofilin phosphorylation, and manipulation of OLA1 may lead to significant changes in cell adhesion and the associated phenotypes. PMID:24486488

  3. 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.

  4. Differential impact of plasma proteins on the adhesion efficiency of vascular-targeted carriers (VTCs) in blood of common laboratory animals

    PubMed Central

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

    2016-01-01

    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 micro-sized, but not nano-sized, VTCs in porcine blood. Overall, the results suggest that porcine models, as opposed to mouse, can serve as a better model 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

  5. Mechanism for adhesion G protein-coupled receptor GPR56-mediated RhoA activation induced by collagen III stimulation.

    PubMed

    Luo, Rong; Jeong, Sung-Jin; Yang, Annie; Wen, Miaoyun; Saslowsky, David E; Lencer, Wayne I; Araç, 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.

  6. A Chemomechanical Model of Matrix and Nuclear Rigidity Regulation of Focal Adhesion Size

    PubMed Central

    Cao, Xuan; Lin, Yuan; Driscoll, Tristian P.; Franco-Barraza, Janusz; Cukierman, Edna; Mauck, Robert L.; Shenoy, Vivek B.

    2015-01-01

    In this work, a chemomechanical model describing the growth dynamics of cell-matrix adhesion structures (i.e., focal adhesions (FAs)) is developed. We show that there are three regimes for FA evolution depending on their size. Specifically, nascent adhesions with initial lengths below a critical value that are yet to engage in actin fibers will dissolve, whereas bigger ones will grow into mature FAs with a steady state size. In adhesions where growth surpasses the steady state size, disassembly will occur until their sizes are reduced to the equilibrium state. This finding arises from the fact that polymerization of adhesion proteins is force-dependent. Under actomyosin contraction, individual integrin bonds within small FAs (i.e., nascent adhesions or focal complexes) must transmit higher loads while the phenomenon of stress concentration occurs at the edge of large adhesion patches. As such, an effective stiffness of the FA-extracellular matrix complex that is either too small or too large will be relatively low, resulting in a limited actomyosin pulling force developed at the edge that is insufficient to prevent disassembly. Furthermore, it is found that a stiffer extracellular matrix and/or nucleus, as well as a stronger chemomechanical feedback, will induce larger adhesions along with a higher level of contraction force. Interestingly, switching the extracellular side from an elastic half-space, corresponding to some widely used in vitro gel substrates, to a one-dimensional fiber (as in the case of cells anchoring to a fibrous scaffold in vivo) does not qualitative change these conclusions. Our model predictions are in good agreement with a variety of experimental observations obtained in this study as well as those reported in the literature. Furthermore, this new model, to our knowledge, provides a framework with which to understand how both intracellular and extracellular perturbations lead to changes in adhesion structure number and size. PMID:26536258

  7. 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.

  8. 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.

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

    PubMed

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

    2000-06-15

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

  10. Small Heat Shock Protein αB-Crystallin Controls Shape and Adhesion of Glioma and Myoblast Cells in the Absence of Stress

    PubMed Central

    2016-01-01

    Cell shape and adhesion and their proper controls are fundamental for all biological systems. Mesenchymal cells migrate at an average rate of 6 to 60 μm/hr, depending on the extracellular matrix environment and cell signaling. Myotubes, fully differentiated muscle cells, are specialized for power-generation and therefore lose motility. Cell spreading and stabilities of focal adhesion are regulated by the critical protein vinculin from immature myoblast to mature costamere of differentiated myotubes where myofibril Z-band linked to sarcolemma. The Z-band is constituted from microtubules, intermediate filaments, cell adhesion molecules and other adapter proteins that communicate with the outer environment. Mesenchymal cells, including myoblast cells, convert actomyosin contraction forces to tension through mechano-responsive adhesion assembly complexes as Z-band equivalents. There is growing evidence that microtubule dynamics are involved in the generation of contractile forces; however, the roles of microtubules in cell adhesion dynamics are not well determined. Here, we show for the first time that αB-crystallin, a molecular chaperon for tubulin/microtubules, is involved in cell shape determination. Moreover, knockdown of this molecule caused myoblasts and glioma cells to lose their ability for adhesion as they tended to behave like migratory cells. Surprisingly, αB-crystallin knockdown in both C6 glial cells and L6 myoblast permitted cells to migrate more rapidly (2.7 times faster for C6 and 1.3 times faster for L6 cells) than dermal fibroblast. On the other hand, overexpression of αB-crystallin in cells led to an immortal phenotype because of persistent adhesion. Position of matured focal adhesion as visualized by vinculin immuno-staining, stress fiber direction, length, and density were clearly αB-crystallin dependent. These results indicate that the small HSP αB-crystallin has important roles for cell adhesion, and thus microtubule dynamics are necessary

  11. SpyAD, a moonlighting protein of group A Streptococcus contributing to bacterial division and host cell adhesion.

    PubMed

    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; Grandi, Guido; Margarit, Immaculada; Bensi, Giuliano

    2014-07-01

    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).

  12. Isolation of a laminin-binding protein from the protozoan parasite Leishmania donovani that may mediate cell adhesion.

    PubMed Central

    Ghosh, A; Bandyopadhyay, K; Kole, L; Das, P K

    1999-01-01

    Extracellular matrix (ECM)-binding proteins on the surface of Leishmania are thought to play a crucial role in the onset of leishmaniasis, as these parasites invade mononuclear phagocytes in various organs after migrating through the ECM. In a previous report, we presented several lines of evidence suggesting that Leishmania has a specific receptor for laminin, a major ECM protein, with a Kd in the nanomolar range. Here we describe the identification, purification and biochemical characterization of the Leishmania laminin receptor. When the outer membrane proteins of L. donovani were blotted on to nitrocellulose paper and probed with laminin, a prominent laminin-binding protein of 67 kDa was identified. The purified protein was isolated by a three-step process involving DEAE-cellulose, Con A (concanavalin A)-Sepharose and laminin-Sepharose affinity chromatography and was used to raise a monospecific antibody. The same protein was obtained when parasite membrane extracts were adsorbed to antibody affinity matrix and eluted with glycine. The affinity-purified protein bound to laminin in a detergent-solubilized form as well as after integration into artificial bilayers, and was subsequently characterized as an integral membrane protein. Metaperiodate oxidation and metabolic inhibition of glycosylation studies indicate the binding protein to be glycoprotein in nature and that N-linked oligosaccharides play a part in the interaction of laminin with the binding protein. Surface-labelled parasites attached to microtitre wells coated with laminin and the 67 kDa protein blocked the adhesion to laminin substrate. We propose that the 67 kDa protein is an adhesin involved in the attachment of Leishmania to host tissues. PMID:9895301

  13. The Role of TSC Proteins in Regulating Cell Adhesion and Motility

    DTIC Science & Technology

    2006-09-01

    regulate cell adhesion and motility as it relates to the genetic disorder tuberous sclerosis complex (TSC). The pathogenesis of TSC that develops due to the...from seizures, mental retardation, and autism . Thus, TSC represents a major cause of developmental disorders and epilepsy in the pediatric...insights on TSC1 and TSC2, and the pathogenesis of tuberous sclerosis. Cancer Biol. Ther. 2:471–476. Kwiatkowski, D.J., H. Zhang, J.L. Bandura, K.M

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

    SciTech Connect

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

    2012-03-09

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

  15. An immediate-early protein of white spot syndrome virus modulates the phosphorylation of focal adhesion kinase of shrimp.

    PubMed

    Lu, Huasong; Ruan, Lingwei; Xu, Xun

    2011-10-25

    WSSV interacts with integrin during infection of shrimps and modulate the focal adhesion kinase which is known as a regulator of several downstream signaling pathways. Viral protein kinases are thought to be important for virus infection by regulating the host signaling pathways. WSV083 is an immediate-early gene of white spot syndrome virus that contains a Ser/Thr protein kinase domain. So, does WSSV modulate FAK phosphorylation via the WSV083 molecule? In this study, co-transfection of WSV083 and MjFAK genes proceeded in insect cells revealed that the MjFAK phosphorylation and cell adhesion activity could be inhibited by the expression of WSV083. Kinase domain mutants of WSV083 lost its ability of inhibiting FAK phosphorylation. Moreover, silencing of FAK gene through RNAi accelerated the shrimp death rate upon WSSV challenge. These results demonstrate for the first time that modulation of FAK phosphorylation by WSV083 plays a critical role in the pathogenesis of WSSV infection.

  16. Adhesive properties, extracellular protein production, and metabolism in the Lactobacillus rhamnosus GG strain when grown in the presence of mucin.

    PubMed

    Sanchez, Borja; Saad, Naima; Schmitter, Jean-Marie; Bressollier, Philippe; Urdaci, Maria C

    2010-06-01

    This paper examines the probiotic bacterium Lactobacillus rhamnosus GG, and how it reacts to the presence of mucin in its extracellular milieu. Parameters studied included cell clustering, adhesion to mucin, extracellular protein production, and formation of final metabolites. L. rhamnosus GG was found to grow efficiently in the presence of glucose, N-acetylglucosamine, or mucin (partially purified or purified) as sole carbon sources. However, it was unable to grow using other mucin constituents, such as fucose or glucuronic acid. Mucin induced noticeable changes in all the parameters studied when compared with growth using glucose, including in the formation of cell clusters, which were easily disorganized with trypsin. Mucin increased adhesion of the bacterium, and modulated the production of extracellular proteins. SDS-PAGE revealed that mucin was not degraded during L. rhamnosus GG growth, suggesting that this bacterium is able to partially use the glucidic moiety of glycoprotein. This study goes some way towards developing an understanding of the metabolic and physiological changes that L. rhamnosus GG undergoes within the human gastrointestinal tract.

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

    PubMed

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

    2015-11-01

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

  18. GPM Video of In-fa

    NASA Video Gallery

    On Nov. 19 GPM saw a few towering storms in In-fa's eye wall were reaching heights of up to 17.3 km (10.7 miles). The most intense precipitation was measured in In-fa's eye wall by DPR where it was...

  19. FANCI Regulates Recruitment of the FA Core Complex at Sites of DNA Damage Independently of FANCD2.

    PubMed

    Castella, Maria; Jacquemont, Celine; Thompson, Elizabeth L; Yeo, Jung Eun; Cheung, Ronald S; Huang, Jen-Wei; Sobeck, Alexandra; Hendrickson, Eric A; Taniguchi, Toshiyasu

    2015-10-01

    The Fanconi anemia (FA)-BRCA pathway mediates repair of DNA interstrand crosslinks. The FA core complex, a multi-subunit ubiquitin ligase, participates in the detection of DNA lesions and monoubiquitinates two downstream FA proteins, FANCD2 and FANCI (or the ID complex). However, the regulation of the FA core complex itself is poorly understood. Here we show that the FA core complex proteins are recruited to sites of DNA damage and form nuclear foci in S and G2 phases of the cell cycle. ATR kinase activity, an intact FA core complex and FANCM-FAAP24 were crucial for this recruitment. Surprisingly, FANCI, but not its partner FANCD2, was needed for efficient FA core complex foci formation. Monoubiquitination or ATR-dependent phosphorylation of FANCI were not required for the FA core complex recruitment, but FANCI deubiquitination by USP1 was. Additionally, BRCA1 was required for efficient FA core complex foci formation. These findings indicate that FANCI functions upstream of FA core complex recruitment independently of FANCD2, and alter the current view of the FA-BRCA pathway.

  20. GPR116, an adhesion G-protein-coupled receptor, promotes breast cancer metastasis via the Gαq-p63RhoGEF-Rho GTPase pathway.

    PubMed

    Tang, Xiaolong; Jin, Rongrong; Qu, Guojun; Wang, Xiu; Li, Zhenxi; Yuan, Zengjin; Zhao, Chen; Siwko, Stefan; Shi, Tieliu; Wang, Ping; Xiao, Jianru; Liu, Mingyao; Luo, Jian

    2013-10-15

    Adhesion G-protein-coupled receptors (GPCR), which contain adhesion domains in their extracellular region, have been found to play important roles in cell adhesion, motility, embryonic development, and immune response. Because most adhesion molecules with adhesion domains have vital roles in cancer metastasis, we speculated that adhesion GPCRs are potentially involved in cancer metastasis. In this study, we identified GPR116 as a novel regulator of breast cancer metastasis through expression and functional screening of the adhesion GPCR family. We found that knockdown of GPR116 in highly metastatic (MDA-MB-231) breast cancer cells suppressed cell migration and invasion. Conversely, ectopic GPR116 expression in poorly metastatic (MCF-7 and Hs578T) cells promoted cell invasion. We further showed that knockdown of GPR116 inhibited breast cancer cell metastasis in two mammary tumor metastasis mouse models. Moreover, GPR116 modulated the formation of lamellipodia and actin stress fibers in cells in a RhoA- and Rac1-dependent manner. At a molecular level, GPR116 regulated cell motility and morphology through the Gαq-p63RhoGEF-RhoA/Rac1 pathway. The biologic significance of GPR116 in breast cancer is substantiated in human patient samples, where GPR116 expression is significantly correlated with breast tumor progression, recurrence, and poor prognosis. These findings show that GPR116 is crucial for the metastasis of breast cancer and support GPR116 as a potential prognostic marker and drug target against metastatic human breast cancer.

  1. 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, Adéla; Claes, Aurélie; 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

  2. Enantiopure chiral poly(glycerol methacrylate) self-assembled monolayers knock down protein adsorption and cell adhesion.

    PubMed

    Li, Zheng; Köwitsch, Alexander; Zhou, Guoying; Groth, Thomas; Fuhrmann, Bodo; Niepel, Marcus; Amado, Elkin; Kressler, Jörg

    2013-10-01

    Chirality plays a fundamental role not only in biological systems, but also in synthetic materials intended for bio-applications. Self-assembled monolayers (SAMs) are prepared on gold surfaces through a "grafting to" method from racemic or enantiopure chiral poly(glycerol methacrylate)s (PGMA(rac), PGMA(R), and PGMA(S)), having a thiol endgroup. Such SAMs constitute a chemically and structurally well-defined model substrate for studying protein adsorption and cell adhesion as a function of the polymer chirality. Surface plasmon resonance measurements reveal that PGMA SAMs greatly reduce the adsorption of bovine serum albumin (BSA) compared to bare gold surfaces. Interestingly, enantiopure SAMs based on PGMA(R) or PGMA(S) show a significantly larger reduction in BSA adsorption than PGMA(rac)-covered surfaces. Studies with the monocytic cell line THP-1 show a similar relationship between enantiopurity of PGMA SAMs and the extent of cell adhesion. Ellipsometry and Raman spectroscopy measurements indicate that SAMs formed by PGMA(rac) have a higher grafting density compared to SAMs of PGMA(R) and PGMA(S). This seems to be due to the ability of PGMA(rac) to form more intermolecular hydrogen bonds among polymer chains compared to the enantiopure PGMAs. Circular dichroism spectroscopy provide evidence that enantiopure polymers adopt a chiral ordered conformation, most likely helical, in aqueous solutions. It is concluded that a higher water content of SAMs formed by enantiopure PGMA(S) and PGMA(R) SAMs arises from the macromolecular chiral conformation adopted by their enantiopure PGMA chains, and it is the decisive reason for the reduced BSA adsorption and cell adhesion as compared to PGMA(rac) SAMs.

  3. Nicotine stimulates adhesion molecular expression via calcium influx and mitogen-activated protein kinases in human endothelial cells.

    PubMed

    Wang, Yajing; Wang, Zhaoxia; Zhou, Ying; Liu, Liming; Zhao, Yangxing; Yao, Chenjiang; Wang, Lianyun; Qiao, Zhongdong

    2006-02-01

    To evaluate the effect of nicotine on endothelium dysfunction and development of vascular diseases, we investigated the influence on adhesion molecular expression mediated by nicotine and the mechanism of this effect in human umbilical vein endothelial cells (HUVECs). The result showed that nicotine could induce surface/soluble vascular cell adhesion molecule (VCAM-1) and endothelial selectin (E-selectin) expression in a time-response decline manner and the peak appeared at 15 min. This action could be mediated by mitogen-activated protein kinase/extracellular signal regulated kinase 1/2 (MAPK/ERK1/2) and MAPK/p38 because their activation could be distinctly blocked by MAPK inhibitors, PD098059 or SB203580. Mecamylamine (non-selective nicotinic receptor inhibitor), alpha-bungarotoxin (alpha7 nicotinic receptor inhibitor) could block Ca2+ accumulation, and then, prevented the phosphorylation on ERK1/2 and p38. They also inhibited the surface/soluble VCAM-1, E-selectin production of HUVECs modulated by nicotine. Therefore, we concluded that: (i) nicotine obviously up-regulates VCAM-1 and E-selectin expression at 15 min in HUVECs, (ii) nicotine activates HUVECs triggered by the ERK1/2 and p38 phosphorylation with an involvement of intracellular calcium mobilization chiefly mediated by alpha7 nicotinic receptor, (iii) intracellular Ca2+ activates a sequential pathway from alpha7 nicotinic receptor to the phosphorylation of ERK1/2, p38. These elucidate that nicotine activates HUVECs through fast signal transduction pathway and arguments their capacity of adhesion molecular production. Further more nicotine may contribute its influence to the progression of vascular disease such as atherosclerotic lesion.

  4. Parathyroid hormone-related protein (PTHrP) modulates adhesion, migration and invasion in bone tumor cells.

    PubMed

    Mak, Isabella W Y; Turcotte, Robert E; Ghert, Michelle

    2013-07-01

    Parathyroid-hormone-related protein (PTHrP) has been shown to be an important factor in osteolysis in the setting of metastatic carcinoma to the bone. However, PTHrP may also be central in the setting of primary bone tumors. Giant cell tumor of bone (GCT) is an aggressive osteolytic bone tumor characterized by osteoclast-like giant cells that are recruited by osteoblast-like stromal cells. The stromal cells of GCT are well established as the only neoplastic element of the tumor, and we have previously shown that PTHrP is highly expressed by these cells both in vitro and in vivo. We have also found that the stromal cells exposed to a monoclonal antibody to PTHrP exhibited rapid plate detachment and quickly died in vitro. Therefore, PTHrP may serve in an autocrine manner to increase cell proliferation and promote invasive properties in GCT. The purpose of this study was to use transcriptomic microarrays and functional assays to examine the effects of PTHrP neutralization on cell adhesion, migration and invasion. Microarray and proteomics data identified genes that were differentially expressed in GCT stromal cells under various PTHrP treatment conditions. Treatment of GCT stromal cells with anti-PTHrP antibodies showed a change in the expression of 13 genes from the integrin family relative to the IgG control. Neutralization of PTHrP reduced cell migration and invasion as evidenced by functional assays. Adhesion and anoikis assays demonstrated that although PTHrP neutralization inhibits cell adhesion properties, cell detachment related to PTHrP neutralization did not result in associated cell death, as expected in mesenchymal stromal cells. Based on the data presented herein, we conclude that PTHrP excreted by GCT stromal cells increases bone tumor cell local invasiveness and migration.

  5. The oxidase activity of vascular adhesion protein-1 (VAP-1) induces endothelial E- and P-selectins and leukocyte binding.

    PubMed

    Jalkanen, Sirpa; Karikoski, Marika; Mercier, Nathalie; Koskinen, Kaisa; Henttinen, Tiina; Elima, Kati; Salmivirta, Katriina; Salmi, Marko

    2007-09-15

    Leukocyte migration from the blood into tissues is pivotal in immune homeostasis and in inflammation. During the multistep extravasation cascade, endothelial selectins (P- and E-selectin) and vascular adhesion protein-1 (VAP-1), a cell-surface-expressed oxidase, are important in tethering and rolling. Here, we studied the signaling functions of the catalytic activity of VAP-1. Using human endothelial cells transfected with wild-type VAP-1 and an enzymatically inactive VAP-1 point mutant, we show that transcription and translation of E- and P-selectins are induced through the enzymatic activity of VAP-1. Moreover, use of VAP-1-deficient animals and VAP-1-deficient animals carrying the human VAP-1 as a transgene show a VAP-enzyme activity-dependent induction of P-selectin in vivo. Up-regulation of P-selectin was found both in high endothelial venules in lymphoid tissues and in flat-walled vessels in noninflamed tissues. VAP-1 activity in vivo led to increased P-selectin-dependent binding of lymphocytes to endothelial cells. These data show that the oxidase reaction catalyzed by VAP-1 alters the expression of other molecules involved in the leukocyte extravasation cascade. Our findings indicate cross-talk between adhesion molecules involved in the tethering and rolling of leukocytes and show that VAP-1-dependent signaling can prime the vessels for an enhanced inflammatory response.

  6. Structural analysis of the synaptic protein neuroligin and its beta-neurexin complex: determinants for folding and cell adhesion.

    PubMed

    Fabrichny, Igor P; Leone, Philippe; Sulzenbacher, Gerlind; Comoletti, Davide; Miller, Meghan T; Taylor, Palmer; Bourne, Yves; Marchot, Pascale

    2007-12-20

    The neuroligins are postsynaptic cell adhesion proteins whose associations with presynaptic neurexins participate in synaptogenesis. Mutations in the neuroligin and neurexin genes appear to be associated with autism and mental retardation. The crystal structure of a neuroligin reveals features not found in its catalytically active relatives, such as the fully hydrophobic interface forming the functional neuroligin dimer; the conformations of surface loops surrounding the vestigial active center; the location of determinants that are critical for folding and processing; and the absence of a macromolecular dipole and presence of an electronegative, hydrophilic surface for neurexin binding. The structure of a beta-neurexin-neuroligin complex reveals the precise orientation of the bound neurexin and, despite a limited resolution, provides substantial information on the Ca2+-dependent interactions network involved in trans-synaptic neurexin-neuroligin association. These structures exemplify how an alpha/beta-hydrolase fold varies in surface topography to confer adhesion properties and provide templates for analyzing abnormal processing or recognition events associated with autism.

  7. Taking Orders from Light: Photo-Switchable Working/Inactive Smart Surfaces for Protein and Cell Adhesion.

    PubMed

    Zhang, Junji; Ma, Wenjing; He, Xiao-Peng; Tian, He

    2017-03-15

    Photoresponsive smart surfaces are promising candidates for a variety of applications in optoelectronics and sensing devices. The use of light as an order signal provides advantages of remote and noninvasive control with high temporal and spatial resolutions. Modification of the photoswitches with target biomacromolecules, such as peptides, DNA, and small molecules including folic acid derivatives and sugars, has recently become a popular strategy to empower the smart surfaces with an improved detection efficiency and specificity. Herein, we report the construction of photoswitchable self-assembled monolayers (SAMs) based on sugar (galactose/mannose)-decorated azobenzene derivatives and determine their photoswitchable, selective protein/cell adhesion performances via electrochemistry. Under alternate UV/vis irradiation, interconvertible high/low recognition and binding affinity toward selective lectins (proteins that recognize sugars) and cells that highly express sugar receptors are achieved. Furthermore, the cis-SAMs with a low binding affinity toward selective proteins and cells also exhibit minimal response toward unselective protein and cell samples, which offers the possibility in avoiding unwanted contamination and consumption of probes prior to functioning for practical applications. Besides, the electrochemical technique used facilitates the development of portable devices based on the smart surfaces for on-demand disease diagnosis.

  8. Specific degradation of the mucus adhesion-promoting protein (MapA) of Lactobacillus reuteri to an antimicrobial peptide.

    PubMed

    Bøhle, Liv Anette; Brede, Dag Anders; Diep, Dzung B; Holo, Helge; Nes, Ingolf F

    2010-11-01

    The intestinal flora of mammals contains lactic acid bacteria (LAB) that may provide positive health effects for the host. Such bacteria are referred to as probiotic bacteria. From a pig, we have isolated a Lactobacillus reuteri strain that produces an antimicrobial peptide (AMP). The peptide was purified and characterized, and it was unequivocally shown that the AMP was a well-defined degradation product obtained from the mucus adhesion-promoting protein (MapA); it was therefore termed AP48-MapA. This finding demonstrates how large proteins might inherit unexpected pleiotropic functions by conferring antimicrobial capacities on the producer. The MapA/AP48-MapA system is the first example where a large protein of an intestinal LAB is shown to give rise to such an AMP. It is also of particular interest that the protein that provides this AMP is associated with the binding of the bacterium producing it to the surface/lining of the gut. This finding gives us new perspective on how some probiotic bacteria may successfully compete in this environment and thereby contribute to a healthy microbiota.

  9. The GPS motif is a molecular switch for bimodal activities of adhesion class G protein-coupled receptors.

    PubMed

    Prömel, Simone; Frickenhaus, Marie; Hughes, Samantha; Mestek, Lamia; Staunton, David; Woollard, Alison; Vakonakis, Ioannis; Schöneberg, Torsten; Schnabel, Ralf; Russ, Andreas P; Langenhan, Tobias

    2012-08-30

    Adhesion class G protein-coupled receptors (aGPCR) form the second largest group of seven-transmembrane-spanning (7TM) receptors whose molecular layout and function differ from canonical 7TM receptors. Despite their essential roles in immunity, tumorigenesis, and development, the mechanisms of aGPCR activation and signal transduction have remained obscure to date. Here, we use a transgenic assay to define the protein domains required in vivo for the activity of the prototypical aGPCR LAT-1/Latrophilin in Caenorhabditis elegans. We show that the GPCR proteolytic site (GPS) motif, the molecular hallmark feature of the entire aGPCR class, is essential for LAT-1 signaling serving in two different activity modes of the receptor. Surprisingly, neither mode requires cleavage but presence of the GPS, which relays interactions with at least two different partners. Our work thus uncovers the versatile nature of aGPCR activity in molecular detail and places the GPS motif in a central position for diverse protein-protein interactions.

  10. 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.

  11. Biocompatible Adhesives

    DTIC Science & Technology

    1991-03-01

    pressure sensitive elastomer, polyisobutylene. with water soluble adhesives such as carboxy methyl ceiiulose, pectin and gelatin for adhesion to... cellulose and nylon films, were most often used in 180 peel adhesion tests on the adhesives. Films were cast on one substrate and the other was moistened...irritation. 4. Peel adhesion to hydrated cellulose , nylon and cotton cloth substrates was satisfactory. So too was the peel adhesion as a function of

  12. Downregulation of human platelet reactivity by neutrophils. Participation of lipoxygenase derivatives and adhesive proteins.

    PubMed Central

    Valles, J; Santos, M T; Marcus, A J; Safier, L B; Broekman, M J; Islam, N; Ullman, H L; Aznar, J

    1993-01-01

    Unstimulated neutrophils inhibited activation and recruitment of thrombin- or collagen-stimulated platelets in an agonist-specific manner. This occurred under conditions of close physical cell-cell contact, although biochemical adhesion between the cells as mediated by P-selectin was not required. Moreover, in the presence of monoclonal P-selectin antibodies that blocked biochemical platelet-neutrophil adhesion, thrombin-stimulated platelets were more efficiently downregulated by neutrophils. This suggested a prothrombotic role for P-selectin under these circumstances. The neutrophil downregulatory effect on thrombin-stimulated platelets was amplified by lipoxygenase inhibition with 5,8,11,14-eicosatetraynoic acid. In contrast, the neutrophil inhibitory effect on platelets was markedly reduced by platelet-derived 12S-hydroxy-5,8-cis, 10-trans, 14-cis-eicosatetraenoic acid (12S-HETE), as well as by the platelet-neutrophil transcellular product, 12S,20-dihydroxy-5,8,10,14-eicosatetraenoic acid (12S,20-DiHETE), but not by another comparable metabolite, 5S,12S-dihydroxy-6-trans, 8-cis, 10-trans, 14-cis-eicosatetraenoic acid (5S,12S-DiHETE), or the neutrophil-derived hydroxy acid leukotriene B4. The neutrophil downregulatory effect on thrombin-induced platelet reactivity was enhanced by aspirin treatment. This may represent a novel action of aspirin as an inhibitor of platelet function. These results provide in vitro biochemical and functional evidence for the thromboregulatory role of neutrophils and emphasize the multicellular aspect of hemostasis and thrombosis. Images PMID:7690778

  13. Adhesion-Linked Protein Tyrosine Phosphatases, Morphogenesis and Breast Cancer Progression

    DTIC Science & Technology

    2006-07-01

    baculoviral expression system to prepare a full length recombinant protein in Sf9 insect cells. We reasoned that expression of such a large protein...baculovirus expressing PTPN4 6 wild type and active site mutants as N terminally six histidine tagged proteins in Sf9 cells to facilitate rapid one step...from infected Sf9 cells from both wild type and active site mutant (band at ~116kDa present only in cells infected with PTPN4 recombinant baculovirus

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

    PubMed

    Mongiardini, Elías J; Parisi, Gustavo D; Quelas, Juan I; Lodeiro, Aníbal R

    2016-01-01

    Adhesion of symbiotic bacteria to host plants is an essential early step of the infection process that leads to the beneficial interaction. In the Bradyrhizobium diazoefficiens-soybean symbiosis few molecular determinants of adhesion are known. Here we identified the tight-adhesion gene products TadGEF in the open-reading frames blr3941-blr3943 of the B. diazoefficiens USDA 110 complete genomic sequence. Predicted structure of TadG indicates a transmembrane domain and two extracytosolic domains, from which the C-terminal has an integrin fold. TadE and TadF are also predicted as bearing transmembrane segments. Mutants in tadG or the small cluster tadGEF were impaired in adhesion to soybean roots, and the root infection was delayed. However, nodule histology was not compromised by the mutations, indicating that these effects were restricted to the earliest contact of the B. diazoefficiens and root surfaces. Knowledge of preinfection determinants is important for development of inoculants that are applied to soybean crops worldwide.

  15. Hepatocyte nuclear factor 4alpha orchestrates expression of cell adhesion proteins during the epithelial transformation of the developing liver.

    PubMed

    Battle, Michele A; Konopka, Genevieve; Parviz, Fereshteh; Gaggl, Alexandra Lerch; Yang, Chuhu; Sladek, Frances M; Duncan, Stephen A

    2006-05-30

    Epithelial formation is a central facet of organogenesis that relies on intercellular junction assembly to create functionally distinct apical and basal cell surfaces. How this process is regulated during embryonic development remains obscure. Previous studies using conditional knockout mice have shown that loss of hepatocyte nuclear factor 4alpha (HNF4alpha) blocks the epithelial transformation of the fetal liver, suggesting that HNF4alpha is a central regulator of epithelial morphogenesis. Although HNF4alpha-null hepatocytes do not express E-cadherin (also called CDH1), we show here that E-cadherin is dispensable for liver development, implying that HNF4alpha regulates additional aspects of epithelial formation. Microarray and molecular analyses reveal that HNF4alpha regulates the developmental expression of a myriad of proteins required for cell junction assembly and adhesion. Our findings define a fundamental mechanism through which generation of tissue epithelia during development is coordinated with the onset of organ function.

  16. The N Terminus of the Prion Protein Mediates Functional Interactions with the Neuronal Cell Adhesion Molecule (NCAM) Fibronectin Domain.

    PubMed

    Slapšak, Urška; Salzano, Giulia; Amin, Ladan; Abskharon, Romany N N; Ilc, Gregor; Zupančič, Blaž; Biljan, Ivana; Plavec, Janez; Giachin, Gabriele; Legname, Giuseppe

    2016-10-14

    The cellular form of the prion protein (PrP(C)) is a highly conserved glycoprotein mostly expressed in the central and peripheral nervous systems by different cell types in mammals. A misfolded, pathogenic isoform, denoted as prion, is related to a class of neurodegenerative diseases known as transmissible spongiform encephalopathy. PrP(C) function has not been unequivocally clarified, and it is rather defined as a pleiotropic protein likely acting as a dynamic cell surface scaffolding protein for the assembly of different signaling modules. Among the variety of PrP(C) protein interactors, the neuronal cell adhesion molecule (NCAM) has been studied in vivo, but the structural basis of this functional interaction is still a matter of debate. Here we focused on the structural determinants responsible for human PrP(C) (HuPrP) and NCAM interaction using stimulated emission depletion (STED) nanoscopy, SPR, and NMR spectroscopy approaches. PrP(C) co-localizes with NCAM in mouse hippocampal neurons, and this interaction is mainly mediated by the intrinsically disordered PrP(C) N-terminal tail, which binds with high affinity to the NCAM fibronectin type-3 domain. NMR structural investigations revealed surface-interacting epitopes governing the interaction between HuPrP N terminus and the second module of the NCAM fibronectin type-3 domain. Our data provided molecular details about the interaction between HuPrP and the NCAM fibronectin domain, and revealed a new role of PrP(C) N terminus as a dynamic and functional element responsible for protein-protein interaction.

  17. Molecular Characterization of a Strawberry FaASR Gene in Relation to Fruit Ripening

    PubMed Central

    Jiang, Yue-ming; Zhao, Ming-lei; Shan, Wei; Kuang, Jian-fei; Lu, Wang-jin

    2011-01-01

    Background ABA-, stress- and ripening-induced (ASR) proteins have been reported to act as a downstream component involved in ABA signal transduction. Although much attention has been paid to the roles of ASR in plant development and stress responses, the mechanisms by which ABA regulate fruit ripening at the molecular level are not fully understood. In the present work, a strawberry ASR gene was isolated and characterized (FaASR), and a polyclonal antibody against FaASR protein was prepared. Furthermore, the effects of ABA, applied to two different developmental stages of strawberry, on fruit ripening and the expression of FaASR at transcriptional and translational levels were investigated. Methodology/Principal Findings FaASR, localized in the cytoplasm and nucleus, contained 193 amino acids and shared common features with other plant ASRs. It also functioned as a transcriptional activator in yeast with trans-activation activity in the N-terminus. During strawberry fruit development, endogenous ABA content, levels of FaASR mRNA and protein increased significantly at the initiation of ripening at a white (W) fruit developmental stage. More importantly, application of exogenous ABA to large green (LG) fruit and W fruit markedly increased endogenous ABA content, accelerated fruit ripening, and greatly enhanced the expression of FaASR transcripts and the accumulation of FaASR protein simultaneously. Conclusions These results indicate that FaASR may be involved in strawberry fruit ripening. The observed increase in endogenous ABA content, and enhanced FaASR expression at transcriptional and translational levels in response to ABA treatment might partially contribute to the acceleration of strawberry fruit ripening. PMID:21915355

  18. TRIM.FaTE Evaluation Report

    EPA Pesticide Factsheets

    The TRIM.FaTE Evaluation Report is composed of three volumes. Volume I presents conceptual, mechanistic, and structural complexity evaluations of various aspects of the model. Volumes II and III present performance evaluation.

  19. Guiding Cell Attachment in 3D Microscaffolds Selectively Functionalized with Two Distinct Adhesion Proteins.

    PubMed

    Richter, Benjamin; Hahn, Vincent; Bertels, Sarah; Claus, Tanja K; Wegener, Martin; Delaittre, Guillaume; Barner-Kowollik, Christopher; Bastmeyer, Martin

    2017-02-01

    The combination of three different photoresists into a single direct laser written 3D microscaffold permits functionalization with two bioactive full-length proteins. The cell-instructive microscaffolds consist of a passivating framework equipped with light activatable constituents featuring distinct protein-binding properties. This allows directed cell attachment of epithelial or fibroblast cells in 3D.

  20. Regulation of extracellular matrix proteins and integrin cell substratum adhesion receptors on epithelium during cutaneous human wound healing in vivo.

    PubMed Central

    Juhasz, I.; Murphy, G. F.; Yan, H. C.; Herlyn, M.; Albelda, S. M.

    1993-01-01

    Although changes in extracellular matrix proteins during wound healing have been well documented, little is known about the regulation of corresponding extracellular matrix adhesion receptors (integrins). To study this process in a human in vivo model, full thickness human skin grafts were transplanted onto severe combined immunodeficient mice and deep excisional wounds involving both the epidermal and dermal layers were then made. The changes in the expression of cell matrix proteins and epithelial integrins over time were analyzed with specific antibodies using immunohistochemistry. Wounding was associated with alterations in extracellular matrix proteins, namely, loss of laminin and type IV collagen in the region of the wound and expression of tenascin and fibronectin. Changes were also noted in the integrins on the migrating keratinocytes. There was marked up-regulation of the alpha v subunit and de novo expression of the fibronectin receptor (alpha 5 beta 1) during the stage of active migration (days 1 to 3 after wounding). In the later stages of wound healing, after epithelial integrity had been established, redistribution of the alpha 2, alpha 3, alpha 6, and beta 4 collagen/laminin-binding integrin subunits to suprabasal epidermal layers was noted. Thus, during cutaneous wound healing, keratinocytes up-regulate fibronectin/fibrinogen-binding integrins and redistribute collagen/laminin-binding integrins. This study demonstrates that the human skin/severe combined immunodeficient chimera provides a useful model to study events during human wound repair. Images Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 PMID:7694470

  1. Human pathogens utilize host extracellular matrix proteins laminin and collagen for adhesion and invasion of the host.

    PubMed

    Singh, Birendra; Fleury, Christophe; Jalalvand, Farshid; Riesbeck, Kristian

    2012-11-01

    Laminin (Ln) and collagen are multifunctional glycoproteins that play an important role in cellular morphogenesis, cell signalling, tissue repair and cell migration. These proteins are ubiquitously present in tissues as a part of the basement membrane (BM), constitute a protective layer around blood capillaries and are included in the extracellular matrix (ECM). As a component of BMs, both Lns and collagen(s), thus function as major mechanical containment molecules that protect tissues from pathogens. Invasive pathogens breach the basal lamina and degrade ECM proteins of interstitial spaces and connective tissues using various ECM-degrading proteases or surface-bound plasminogen and matrix metalloproteinases recruited from the host. Most pathogens associated with the respiratory, gastrointestinal, or urogenital tracts, as well as with the central nervous system or the skin, have the capacity to bind and degrade Lns and collagen(s) in order to adhere to and invade host tissues. In this review, we focus on the adaptability of various pathogens to utilize these ECM proteins as enhancers for adhesion to host tissues or as a targets for degradation in order to breach the cellular barriers. The major pathogens discussed are Streptococcus, Staphylococcus, Pseudomonas, Salmonella, Yersinia, Treponema, Mycobacterium, Clostridium, Listeria, Porphyromonas and Haemophilus; Candida, Aspergillus, Pneumocystis, Cryptococcus and Coccidioides; Acanthamoeba, Trypanosoma and Trichomonas; retrovirus and papilloma virus.

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

    PubMed

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

    2015-02-14

    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.

  3. Comparison of adhesive properties of water- and phosphate-buffer-washed cottonseed meals with cottonseed protein isolate on 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...

  4. Comparative studies on the effect of growth conditions on adhesion, hydrophobicity, and extracellular protein profile of Streptococcus sanguis G9B.

    PubMed Central

    Knox, K W; Hardy, L N; Markevics, L J; Evans, J D; Wicken, A J

    1985-01-01

    Streptococcus sanguis G9B was grown in continuous culture at different generation times and pH values in media containing either glucose or fructose and differing in the concentrations of Na+ and K+. The growth pH, carbohydrate, and cation concentration each affected the yield of organisms, their ability to adhere to saliva-coated hydroxyapatite beads, and their hydrophobicity, as measured by adhesion to hexadecane. There was no correlation between adhesion to saliva-coated hydroxyapatite beads and hydrophobicity, the values for hydrophobicity varying between 44 and 83% for organisms that adhered poorly and between 24 and 75% for those that adhered effectively. For organisms grown in batch culture at pH 6.0 or 7.0 there was similarly no correlation between adhesion and hydrophobicity. The growth conditions also had a considerable influence on the production of extracellular protein. The total amount was greater at pH 7.5 than at other pH values, and there were also differences in the individual components in response to changes in generation time, pH, carbohydrate source, and cation concentration. Two protein bands were identified, namely, glucosyltransferase and protein P1 (also called antigen B or I/II). However, there was no correlation between a particular protein component and adhesion. Images PMID:4055033

  5. Laminin-511: a multi-functional adhesion protein regulating cell migration, tumor invasion and metastasis.

    PubMed

    Pouliot, Normand; Kusuma, Nicole

    2013-01-01

    Laminins are major constituents of basement membranes. At least 16 isoforms have now been described, each with distinct spatio-temporal expression patterns and functions. The laminin-511 heterotrimer (α5β1γ1) is one of the more recent isoforms to be identified and a potent adhesive and pro-migratory substrate for a variety of normal and tumor cell lines in vitro. As our understanding of its precise function in normal tissues and in pathologies is rapidly unraveling, current evidence suggests an important regulatory role in cancer. This review describes published data on laminin-511 expression in several malignancies and experimental evidence from both in vitro and in vivo studies supporting its functional role during tumor progression. A particular emphasis is put on more recent studies from our laboratory and that of others indicating that laminin-511 contributes to tumor dissemination and metastasis in advanced breast carcinomas and other tumor types. Collectively, the experimental evidence suggests that high expression of laminin-511 has prognostic significance and that targeting tumor-laminin-511 interactions may have therapeutic potential in advanced cancer patients.

  6. A novel domain cassette identifies Plasmodium falciparum PfEMP1 proteins binding ICAM-1 and is a target of cross-reactive, adhesion-inhibitory antibodies.

    PubMed

    Bengtsson, Anja; Joergensen, Louise; Rask, Thomas S; Olsen, Rebecca W; Andersen, Marianne A; Turner, Louise; Theander, Thor G; Hviid, Lars; Higgins, Matthew K; Craig, Alister; Brown, Alan; Jensen, Anja T R

    2013-01-01

    Cerebral Plasmodium falciparum malaria is characterized by adhesion of infected erythrocytes (IEs) to the cerebral microvasculature. This has been linked to parasites expressing the structurally related group A subset of the P. falciparum erythrocyte membrane protein 1 (PfEMP1) family of IE adhesion ligands and to IEs with affinity for ICAM-1. However, recent evidence has cast doubt on both these associations, tempering hopes of the feasibility of developing a vaccine based on ICAM-1-binding PfEMP1. In this study, we report the identification of a domain cassette (DC) present in group A var genes from six genetically distinct P. falciparum parasites. The three domains in the cassette, which we call DC4, had a high level of sequence identity and cluster together phylogenetically. Erythrocytes infected by these parasites and selected in vitro for expression of DC4 adhered specifically to ICAM-1. The ICAM-1-binding capacity of DC4 was mapped to the C-terminal third of its Duffy-binding-like β3 domain. DC4 was the target of broadly cross-reactive and adhesion-inhibitory IgG Abs, and levels of DC4-specific and adhesion-inhibitory IgG increased with age among P. falciparum-exposed children. Our study challenges earlier conclusions that group A PfEMP1 proteins are not central to ICAM-1-specific IE adhesion and support the feasibility of developing a vaccine preventing cerebral malaria by inhibiting cerebral IE sequestration.

  7. 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.

  8. Expression, purification, crystallization and preliminary X-ray analysis of the olfactomedin domain from the sea urchin cell-adhesion protein amassin

    SciTech Connect

    Hillier, Brian J.; Sundaresan, Vidyasankar; Stout, C. David; Vacquier, Victor D.

    2006-01-01

    The olfactomedin (OLF) domain from the sea urchin cell-adhesion protein amassin has been crystallized. A native data set extending to 2.7 Å has been collected using an in-house X-ray source. A family of animal proteins is emerging which contain a conserved protein motif known as an olfactomedin (OLF) domain. Novel extracellular protein–protein interactions occur through this domain. The OLF-family member amassin, from the sea urchin Strongylocentrotus purpuratus, has previously been identified to mediate a rapid cell-adhesion event resulting in a large aggregation of coelomocytes, the circulating immune cells. In this work, heterologous expression and purification of the OLF domain from amassin was carried out and initial crystallization trials were performed. A native data set has been collected, extending to 2.7 Å under preliminary cryoconditions, using an in-house generator. This work leads the way to the determination of the first structure of an OLF domain.

  9. Xanthohumol lowers body weight and fasting plasma glucose in obese male Zucker fa/fa rats.

    PubMed

    Legette, Leecole L; Luna, Arlyn Y Moreno; Reed, Ralph L; Miranda, Cristobal L; Bobe, Gerd; Proteau, Rosita R; Stevens, Jan F

    2013-07-01

    Obesity contributes to increased risk for several chronic diseases including cardiovascular disease and type 2 diabetes. Xanthohumol, a prenylated flavonoid from hops (Humulus lupulus), was tested for efficacy on biomarkers of metabolic syndrome in 4 week old Zucker fa/fa rats, a rodent model of obesity. Rats received daily oral doses of xanthohumol at 0, 1.86, 5.64, and 16.9 mg/kg BW for 6 weeks. All rats were maintained on a high fat (60% kcal) AIN-93G diet for 3 weeks to induce severe obesity followed by a normal AIN-93G (15% kcal fat) diet for the last 3 weeks of the study. Weekly food intake and body weight were recorded. Plasma cholesterol, glucose, insulin, triglyceride, and monocyte chemoattractant protein-1 (MCP-1) levels were assessed using commercial assay kits. Plasma and liver tissue levels of XN and its metabolites were determined by liquid-chromatography tandem mass spectrometry. Plasma and liver tissue levels of xanthohumol were similar between low and medium dose groups and significantly (p<0.05) elevated in the highest dose group. There was a dose-dependent effect on body weight and plasma glucose levels. The highest dose group (n=6) had significantly lower plasma glucose levels compared to the control group (n=6) in male but not female rats. There was also a significant decrease in body weight for male rats in the highest dose group (16.9 mg/kg BW) compared to rats that received no xanthohumol, which was also not seen for female rats. Plasma cholesterol, insulin, triglycerides, and MCP-1 as well as food intake were not affected by treatment. The findings suggest that xanthohumol has beneficial effects on markers of metabolic syndrome.

  10. Atmospheric pressure plasma polymers for tuned QCM detection of protein adhesion.

    PubMed

    Rusu, G B; Asandulesa, M; Topala, I; Pohoata, V; Dumitrascu, N; Barboiu, M

    2014-03-15

    Our efforts have been concentrated in preparing plasma polymeric thin layers at atmospheric pressure grown on Quartz Crystal Microbalance-QCM electrodes for which the non-specific absorption of proteins can be efficiently modulated, tuned and used for QCM biosensing and quantification. Plasma polymerization reaction at atmospheric pressure has been used as a simple and viable method for the preparation of QCM bioactive surfaces, featuring variable protein binding properties. Polyethyleneglycol (ppEG), polystyrene (ppST) and poly(ethyleneglycol-styrene) (ppST-EG) thin-layers have been grown on QCM electrodes. These layers were characterized by Atomic Force Microscopy (AFM), Contact angle measurements, Fourier transform infrared (FTIR) and X-ray photoelectron spectroscopy (XPS). The plasma ppST QCM electrodes present a higher adsorption of Concanavalin A (ConA) and Bovine Serum Albumin (BSA) proteins when compared with the commercial coated polystyrene (ppST) ones. The minimum adsorption was found for ppEG, surface, known by their protein anti-fouling properties. The amount of adsorbed proteins can be tuned by the introduction of PEG precursors in the plasma discharge during the preparation of ppST polymers.

  11. Discovery of novel hematopoietic cell adhesion molecules from human bone marrow stromal cell membrane protein extracts by a new cell-blotting technique.

    PubMed

    Seshi, B

    1994-05-01

    In an attempt to define the role of cell adhesion molecules (CAMs) within the bone marrow (BM) microenvironment in normal hematopoiesis and in leukemia development, a novel cell-blotting technique that involved cell adhesion to protein bands after separation by lithium dodecyl sulfate-polyacrylamide gel electrophoresis (LDS-PAGE) and blotting onto polyvinylidene difluoride (PVDF) membrane has been developed. Human BM stromal cell membrane fractions have been prepared from Dexter-type cultures after cell lysis by sonification and differential centrifugations of the sonification contents. The 20,000 g pellets representing membrane fractions have been solubilized by 2% Triton X-100, 0.575% LDS, and 8 mol/L urea in sequential order. The protein extracts are fractionated by LDS-PAGE and screened for CAMs by the new cell-blotting technique. This led to identification of nine protein bands in lanes containing LDS extracts showing adhesion of KG1a (CD34+ progenitor myeloid) cells. Evidence that the BM proteins exhibiting KG1a cell adhesion are novel CAMs is based on the observations that these proteins, in comparison with known CAMs, specifically VCAM-1, CD54, and CD44, show (1) contrasting detergent-solubility properties, (2) different temperature requirement for mediating cell adhesion function, and (3) markedly distinct electrophoretic mobilities. The various cell types tested, notably KG1a, NALM-6, WIL-2, Ramos, HS-Sultan, K562, JY B lymphoblastoid cells, and T lymphoblasts, showed distinctive patterns of binding to different subsets of BM CAMs. These results demonstrate a new approach to studies of molecular mechanisms that may determine specificity of hematopoietic cellular localization within BM microenvironment and may play an important role in controlling hematopoiesis.

  12. Induction of Fibronectin-Binding Proteins and Increased Adhesion of Quinolone-Resistant Staphylococcus aureus by Subinhibitory Levels of Ciprofloxacin

    PubMed Central

    Bisognano, Carmelo; Vaudaux, Pierre; Rohner, Peter; Lew, Daniel P.; Hooper, David C.

    2000-01-01

    We recently reported that strain EN1252a, a fluoroquinolone-resistant derivative of Staphylococcus aureus NCTC8325 with mutations in grlA and gyrA, expressed increased levels of fibronectin-binding proteins (FnBPs) and showed a significantly higher attachment to fibronectin-coated polymer surfaces after growth in the presence of subinhibitory concentrations of ciprofloxacin. The present study evaluated the occurrence and frequency of fluoroquinolone-induced FnBP-mediated adhesion in clinical isolates of fluoroquinolone-resistant methicillin-resistant S. aureus (MRSA) and methicillin-susceptible S. aureus (MSSA). Eight of ten MRSA isolates and four of six MSSA isolates with grlA and gyrA mutations exhibited significant increases in attachment to fibronectin-coated surfaces after growth in the presence of one-quarter the MIC of ciprofloxacin. Fluoroquinolone-induced FnBP-mediated adhesion of one clinical MRSA strain and the double mutant strain EN1252a also occurred on coverslips removed from the subcutaneous space of guinea pigs. For strain EN1252a, the regulation of fnb transcription by sub-MICs of ciprofloxacin was studied on reporter plasmids carrying fnb-luxAB fusions. One-quarter of the MIC of ciprofloxacin significantly increased fnbB, but not fnbA, promoter activity of the fluoroquinolone-resistant mutant but not its fluoroquinolone-susceptible parent ISP794. This response was abolished by pretreatment with rifampin, indicating an effect at the level of transcription. Activation of the fnbB promoter was not due to an indirect effect of ciprofloxacin on growth rate and still occurred in an agr mutant of strain EN1252a. These data suggest that sub-MIC levels of ciprofloxacin activate the fnbB promoter of some laboratory and clinical isolates, thus contributing to increased production of FnBP(s) and leading to higher levels of bacterial attachment to fibronectin-coated or subcutaneously implanted coverslips. PMID:10817688

  13. In vivo selection for Neisseria gonorrhoeae opacity protein expression in the absence of human carcinoembryonic antigen cell adhesion molecules.

    PubMed

    Simms, Amy N; Jerse, Ann E

    2006-05-01

    The neisserial opacity (Opa) proteins are phase-variable, antigenically distinct outer membrane proteins that mediate adherence to and invasion of human cells. We previously reported that Neisseria gonorrhoeae Opa protein expression appeared to be selected for or induced during experimental murine genital tract infection. Here we further defined the kinetics of recovery of Opa variants from the lower genital tracts of female mice and investigated the basis for this initial observation. We found that the recovery of different Opa phenotypes from mice appears cyclical. Three phases of infection were defined. Following intravaginal inoculation with primarily Opa- gonococci, the majority of isolates recovered were Opa+ (early phase). A subsequent decline in the percentage of Opa+ isolates occurred in a majority of mice (middle phase) and was followed by a reemergence of Opa+ variants in mice that were infected for longer than 8 days (late phase). We showed the early phase was due to selection for preexisting Opa+ variants in the inoculum by constructing a chloramphenicol-resistant (Cm(r)) strain and following Cm(r) Opa+ populations mixed with a higher percentage of Opa- variants of the wild-type (Cm(s)) strain. Reciprocal experiments (Opa- Cm(r) gonococci spiked with Opa+ Cm(s) bacteria) were consistent with selection of Opa+ variants. Based on the absence in mice of human carcinoembryonic antigen cell adhesion molecules, the major class of Opa protein adherence receptors, we conclude the observed selection for Opa+ variants early in infection is not likely due to a specific adherence advantage and may be due to Opa-mediated evasion of innate defenses.

  14. Silk-fibronectin protein alloy fibres support cell adhesion and viability as a high strength, matrix fibre analogue

    PubMed Central

    Jacobsen, Matthew M.; Li, David; Gyune Rim, Nae; Backman, Daniel; Smith, Michael L.; Wong, Joyce Y.

    2017-01-01

    Silk is a natural polymer with broad utility in biomedical applications because it exhibits general biocompatibility and high tensile material properties. While mechanical integrity is important for most biomaterial applications, proper function and integration also requires biomaterial incorporation into complex surrounding tissues for many physiologically relevant processes such as wound healing. In this study, we spin silk fibroin into a protein alloy fibre with whole fibronectin using wet spinning approaches in order to synergize their respective strength and cell interaction capabilities. Results demonstrate that silk fibroin alone is a poor adhesive surface for fibroblasts, endothelial cells, and vascular smooth muscle cells in the absence of serum. However, significantly improved cell attachment is observed to silk-fibronectin alloy fibres without serum present while not compromising the fibres’ mechanical integrity. Additionally, cell viability is improved up to six fold on alloy fibres when serum is present while migration and spreading generally increase as well. These findings demonstrate the utility of composite protein alloys as inexpensive and effective means to create durable, biologically active biomaterials. PMID:28378749

  15. 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

  16. Silk-fibronectin protein alloy fibres support cell adhesion and viability as a high strength, matrix fibre analogue.

    PubMed

    Jacobsen, Matthew M; Li, David; Gyune Rim, Nae; Backman, Daniel; Smith, Michael L; Wong, Joyce Y

    2017-04-05

    Silk is a natural polymer with broad utility in biomedical applications because it exhibits general biocompatibility and high tensile material properties. While mechanical integrity is important for most biomaterial applications, proper function and integration also requires biomaterial incorporation into complex surrounding tissues for many physiologically relevant processes such as wound healing. In this study, we spin silk fibroin into a protein alloy fibre with whole fibronectin using wet spinning approaches in order to synergize their respective strength and cell interaction capabilities. Results demonstrate that silk fibroin alone is a poor adhesive surface for fibroblasts, endothelial cells, and vascular smooth muscle cells in the absence of serum. However, significantly improved cell attachment is observed to silk-fibronectin alloy fibres without serum present while not compromising the fibres' mechanical integrity. Additionally, cell viability is improved up to six fold on alloy fibres when serum is present while migration and spreading generally increase as well. These findings demonstrate the utility of composite protein alloys as inexpensive and effective means to create durable, biologically active biomaterials.

  17. Cyclic di-GMP contributes to adaption and virulence of Bacillus thuringiensis through a riboswitch-regulated collagen adhesion protein

    PubMed Central

    Tang, Qing; Yin, Kang; Qian, Hongliang; Zhao, Youwen; Wang, Wen; Chou, Shan-Ho; Fu, Yang; He, Jin

    2016-01-01

    Cyclic di-GMP is a ubiquitous second messenger that regulates diverse cellular processes in bacteria by binding to various protein or riboswitch effectors. In Bacillus thuringiensis BMB171, a c-di-GMP riboswitch termed Bc2 RNA resides in the 5′-untranslated region (5′-UTR) of an mRNA that encodes a collagen adhesion protein (Cap). The expression of cap was strongly repressed in parent strain BMB171 because of the presence of Bc2 RNA but was significantly promoted in the Bc2 RNA markerless deletion mutant. Bc2 RNA acts as a genetic “on” switch, which forms an anti-terminator structure to promote cap read-through transcription upon c-di-GMP binding. As a result, cap transcription was de-repressed under high c-di-GMP levels. Therefore, Bc2 RNA regulates cap expression using a repression/de-repression model. Bc2 RNA-regulated Cap was also found to be tightly associated with motility, aggregation, exopolysaccharide secretion, biofilm formation, and virulence of B. thuringiensis BMB171 against its host insect Helicoverpa armigera. PMID:27381437

  18. 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.

  19. Endothelial cell adhesion and growth within a bioassay chamber using microstamped ECM proteins

    NASA Astrophysics Data System (ADS)

    Rubenstein, David A.; Frame, Mary D.

    2011-06-01

    Our goal was to evaluate microvascular endothelial cell growth on microstamped patterns of extracellular matrix proteins (ECM). A combination of photo- and soft-lithography was used to make features ˜100 μm deep and 150μm wide. Polydimethylsiloxane imprints of features produced positive molds used to stamp collagen I, IV, laminin and fibronectin onto cleaned hydrophilic or hydrophobic glass coverslips. Human dermal microvascular endothelial cells were seeded at an initial density of 800 cells cm-2, and cultured for three days. Explanted murine aortas, serving as an initial source for autologous endothelial cells, were perfused at 240 μL min-1 for 1 day. Cell morphology was also quantified on both the non-patterned glass and within the microstamped patterns. Viability was high (>90%) on all microstamped proteins, regardless of glass hydrophobicity. Viability was reduced on bare hydrophobic glass. Cell density was 4 or 8 fold higher on microstamped ECM proteins compared with hydrophilic or hydrophobic glass, respectively. Confluence was approached more rapidly on microstamped proteins. Thus, rapid concentrated growth of endothelial cells was markedly enhanced within microstamped ECM patterns on hydrophilic and hydrophobic glass.

  20. Opuntia ficus indica (nopal) attenuates hepatic steatosis and oxidative stress in obese Zucker (fa/fa) rats.

    PubMed

    Morán-Ramos, Sofía; Avila-Nava, Azalia; Tovar, Armando R; Pedraza-Chaverri, José; López-Romero, Patricia; Torres, Nimbe

    2012-11-01

    Nonalcoholic fatty liver disease (NAFLD) is associated with multiple factors such as obesity, insulin resistance, and oxidative stress. Nopal, a cactus plant widely consumed in the Mexican diet, is considered a functional food because of its antioxidant activity and ability to improve biomarkers of metabolic syndrome. The aim of this study was to assess the effect of nopal consumption on the development of hepatic steatosis and hepatic oxidative stress and on the regulation of genes involved in hepatic lipid metabolism. Obese Zucker (fa/fa) rats were fed a control diet or a diet containing 4% nopal for 7 wk. Rats fed the nopal-containing diet had ∼50% lower hepatic TG than the control group as well as a reduction in hepatomegaly and biomarkers of hepatocyte injury such as alanine and aspartate aminotransferases. Attenuation of hepatic steatosis by nopal consumption was accompanied by a higher serum concentration of adiponectin and a greater abundance of mRNA for genes involved in lipid oxidation and lipid export and production of carnitine palmitoyltransferase-1 and microsomal TG transfer proteins in liver. Hepatic reactive oxygen species and lipid peroxidation biomarkers were significantly lower in rats fed nopal compared with the control rats. Furthermore, rats fed the nopal diet had a lower postprandial serum insulin concentration and a greater liver phosphorylated protein kinase B (pAKT):AKT ratio in the postprandial state. This study suggests that nopal consumption attenuates hepatic steatosis by increasing fatty acid oxidation and VLDL synthesis, decreasing oxidative stress, and improving liver insulin signaling in obese Zucker (fa/fa) rats.

  1. Structure and function of ameloblastin as an extracellular matrix protein: adhesion, calcium binding, and CD63 interaction in human and mouse.

    PubMed

    Zhang, Xu; Diekwisch, Thomas G H; Luan, Xianghong

    2011-12-01

    The functional significance of extracellular matrix proteins in the life of vertebrates is underscored by a high level of sequence variability in tandem with a substantial degree of conservation in terms of cell-cell and cell-matrix adhesion interactions. Many extracellular matrix proteins feature multiple adhesion domains for successful attachment to substrates, such as integrin, CD63, and heparin. Here we have used homology and ab initio modeling algorithms to compare mouse ameloblastin (mAMBN) and human ameloblastin (hABMN) isoforms and to analyze their potential for cell adhesion and interaction with other matrix molecules as well as calcium binding. Sequence comparison between mAMBN and hAMBN revealed a 26-amino-acid deletion in mAMBN, corresponding to a helix-loop-helix frameshift. The human AMBN domain (174Q-201G), homologous to the mAMBN 157E-178I helix-loop-helix region, formed a helix-loop motif with an extended loop, suggesting a higher degree of flexibility of hAMBN compared with mAMBN, as confirmed by molecular dynamics simulation. Heparin-binding domains, CD63-interaction domains, and calcium-binding sites in both hAMBN and mAMBN support the concept of AMBN as an extracellular matrix protein. The high level of conservation between AMBN functional domains related to adhesion and differentiation was remarkable when compared with only 61% amino acid sequence homology.

  2. Interleukin-6 enhances whereas tumor necrosis factor alpha and interferons inhibit integrin expression and adhesion of human mast cells to extracellular matrix proteins.

    PubMed

    Schoeler, Dagmar; Grützkau, Andreas; Henz, Beate M; Küchler, Jens; Krüger-Krasagakis, Sabine

    2003-05-01

    Integrins are expressed on mast cells and constitute an essential prerequisite for the accumulation of the cells at sites of inflammation. In order to clarify a potential contribution of inflammatory cytokines to this process, we have studied the modulation of integrin expression and adhesion of immature human mast cells (HMC-1) to extracellular matrix proteins by interleukin-6, tumor necrosis factor alpha, interferon-alpha and interferon-gamma. Corticosteroids were used for comparison. On fluorescence-activated cell sorter analysis, preincubation of cells for 48 h with different concentrations of interleukin-6 induced a significant, up to 40%, increase of alpha v alpha 5, CD49b (alpha 2), CD49e (alpha 5), CD49f (alpha 6), and CD51 (alpha v). In contrast, different concentrations of tumor necrosis factor alpha, interferon-alpha, interferon-gamma, and dexamethasone (10-8-10-10 M) inhibited expression of adhesion receptors by up to 60%, reaching significance for some but not all integrins. On semiquantitative polymerase chain reaction analysis, interleukin-6, the other cytokines, and corticosteroids significantly modulated expression of alpha1, alpha v and alpha 5 integrin chains at mRNA level. Functional significance of these findings was proven in adhesion assays using fibronectin, laminin, and vitronectin, with interleukin-6 causing significant enhancement of adhesion in all cases, tumor necrosis factor alpha and dexamethasone inducing significant reduction of adhesion to fibronectin and laminin, and interferon-gamma significantly inhibiting adhesion to fibronectin only. Specificity of interleukin-6-induced changes was demonstrated using antibodies against alpha1 and alpha 5 integrins in unstimulated and interleukin-6-prestimulated cells. These data show that interleukin-6 stimulates mast cell adhesion to extracellular matrix and thus allows for the accumulation of the cells at tissue sites by enhancing integrin expression, whereas tumor necrosis factor alpha

  3. The carboxyl terminus of FANCE recruits FANCD2 to the Fanconi Anemia (FA) E3 ligase complex to promote the FA DNA repair pathway.

    PubMed

    Polito, David; Cukras, Scott; Wang, Xiaozhe; Spence, Paige; Moreau, Lisa; D'Andrea, Alan D; Kee, Younghoon

    2014-03-07

    Fanconi anemia (FA) is a genome instability syndrome characterized by bone marrow failure and cellular hypersensitivity to DNA cross-linking agents. In response to DNA damage, the FA pathway is activated through the cooperation of 16 FA proteins. A central player in the pathway is a multisubunit E3 ubiquitin ligase complex or the FA core complex, which monoubiquitinates its substrates FANCD2 and FANCI. FANCE, a subunit of the FA core complex, plays an essential role by promoting the integrity of the complex and by directly recognizing FANCD2. To delineate its role in substrate ubiquitination from the core complex assembly, we analyzed a series of mutations within FANCE. We report that a phenylalanine located at the highly conserved extreme C terminus, referred to as Phe-522, is a critical residue for mediating the monoubiquitination of the FANCD2-FANCI complex. Using the FANCE mutant that specifically disrupts the FANCE-FANCD2 interaction as a tool, we found that the interaction-deficient mutant conferred cellular sensitivity in reconstituted FANCE-deficient cells to a similar degree as FANCE null cells, suggesting the significance of the FANCE-FANCD2 interaction in promoting cisplatin resistance. Intriguingly, ectopic expression of the FANCE C terminus fragment alone in FA normal cells disrupts DNA repair, consolidating the importance of the FANCE-FANCD2 interaction in the DNA cross-link repair.

  4. MPP2 is a postsynaptic MAGUK scaffold protein that links SynCAM1 cell adhesion molecules to core components of the postsynaptic density

    PubMed Central

    Rademacher, Nils; Schmerl, Bettina; Lardong, Jennifer A.; Wahl, Markus C.; Shoichet, Sarah A.

    2016-01-01

    At neuronal synapses, multiprotein complexes of trans-synaptic adhesion molecules, scaffold proteins and neurotransmitter receptors assemble to essential building blocks required for synapse formation and maintenance. Here we describe a novel role for the membrane-associated guanylate kinase (MAGUK) protein MPP2 (MAGUK p55 subfamily member 2) at synapses of rat central neurons. Through interactions mediated by its C-terminal SH3-GK domain module, MPP2 binds to the abundant postsynaptic scaffold proteins PSD-95 and GKAP and localises to postsynaptic sites in hippocampal neurons. MPP2 also colocalises with the synaptic adhesion molecule SynCAM1. We demonstrate that the SynCAM1 C-terminus interacts directly with the MPP2 PDZ domain and that MPP2 does not interact in this manner with other highly abundant postsynaptic transmembrane proteins. Our results highlight a previously unexplored role for MPP2 at postsynaptic sites as a scaffold that links SynCAM1 cell adhesion molecules to core proteins of the postsynaptic density. PMID:27756895

  5. Bactrian camel nanobody-based immunoassay for specific and sensitive detection of Cry1Fa toxin.

    PubMed

    Wang, Pingyan; Li, Guanghui; Yan, Junrong; Hu, Yonghong; Zhang, Cunzheng; Liu, Xianjin; Wan, Yakun

    2014-12-15

    The variable domain of the heavy-chain-only antibody (VHH) or nanobody (Nb), derived from camelids, begins to play an important role on the detection of protein markers. In this study, we constructed a phage-displayed library of VHHs against Cry1Fa by immunizing a healthy Bactrian camel with Cry1Fa toxin. After a series of bio-panning and screening by phage display technology, three anti-Cry1Fa nanobodies (Nbs) with great difference in complementarity determining region 3 (CDR3) were obtained and they were highly specific to Cry1Fa as well as showed full of activity when exposed to 70 °C for 3 h. Through modifying Nbs with Horseradish Peroxidase (HRP) and biotin, two Nbs which can recognize the different epitopes of Cry1Fa were determined and they were used to establish a novel sandwich immune ELISA based on biotin-SA interaction for Cry1Fa detection. The immunoassay exhibited a linear range from 1 to 100 ng/mL with a detection limit of 0.88 ng/mL. The recoveries from spiked corn and soybean samples were ranged from 83.33 to 117.17%, with a coefficient of variation (C.V) less than 6.0%. All together, the proposed immunoassay will be a promising way for sensitive and accurate determination of Cry1Fa toxin.

  6. 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.

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

    PubMed

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

    2014-07-01

    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.

  8. 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.

  9. Intrauterine Adhesions

    MedlinePlus

    ... adhesion formation are infections of the uterine lining (endometritis), removal of fibroids in the cavity of the ... to prevent adhesions from reforming. Hormonal treatment with estrogen and NSAIDs are frequently prescribed after surgery to ...

  10. 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.

  11. 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.

  12. The C. elegans F-spondin family protein SPON-1 maintains cell adhesion in neural and non-neural tissues

    PubMed Central

    Hudson, Martin L.; Swale, Ryann E.; Goncharov, Alexandr

    2008-01-01

    Summary The F-spondin family of extracellular matrix proteins has been implicated in axon outgrowth, fasciculation, and neuronal cell migration, as well as differentiation and proliferation of non-neuronal cells. In screens for mutants defective in C. elegans embryonic morphogenesis we identified SPON-1, the only C. elegans member of the spondin family. SPON-1 is synthesized in body muscles and localizes to integrin-containing structures on body muscles and to other basement membranes. SPON-1 maintains strong attachments of muscles to epidermis; in the absence of SPON-1, muscles progressively detach from the epidermis, causing defective epidermal elongation. In animals with reduced integrin function SPON-1 becomes dose-dependent, suggesting SPON-1 and integrins function in concert to promote attachment of muscles to the basement membrane. Although spon-1 mutants display largely normal neurite outgrowth, spon-1 synergizes with outgrowth defective mutants, revealing a cryptic role for SPON-1 in axon extension. In motor neurons SPON-1 acts in axon guidance and fasciculation, whereas in interneurons SPON-1 maintains process position. Our results show that a spondin maintains cell-matrix adhesion in multiple tissues. PMID:18614580

  13. Adhesion Protein ApfA of Actinobacillus pleuropneumoniae Is Required for Pathogenesis and Is a Potential Target for Vaccine Development

    PubMed Central

    Zhou, Yang; Li, Lu; Chen, Zhaohui; Yuan, Hong; Chen, Huanchun

    2013-01-01

    Actinobacillus pleuropneumoniae is the etiologic agent of porcine pleuropneumonia, which causes serious economic losses in the pig farming industry worldwide. Due to a lack of knowledge of its virulence factors and a lack of effective vaccines able to confer cross-serotype protection, it is difficult to place this disease under control. By analyzing its genome sequences, we found that type IV fimbrial subunit protein ApfA is highly conserved among different serotypes of A. pleuropneumoniae. Our study shows that ApfA is an adhesin since its expression was greatly upregulated (135-fold) upon contact with host cells, while its deletion mutant attenuated its capability of adhesion. The inactivation of apfA dramatically reduced the ability of A. pleuropneumoniae to colonize mouse lung, suggesting that apfA is a virulence factor. Purified recombinant ApfA elicited an elevated humoral immune response and conferred robust protection against challenges with A. pleuropneumoniae serovar 1 strain 4074 and serovar 7 strain WF83 in mice. Importantly, the anti-ApfA serum conferred significant protection against both serovar 1 and serovar 7 in mice. These studies indicate that ApfA promotes virulence through attachment to host cells, and its immunogenicity renders it a promising novel subunit vaccine candidate against infection with A. pleuropneumoniae. PMID:23269417

  14. Focal Adhesion Kinase-mediated Phosphorylation of Beclin1 Protein Suppresses Cardiomyocyte Autophagy and Initiates Hypertrophic Growth*♦

    PubMed Central

    Cheng, Zhaokang; Zhu, Qiang; Dee, Rachel; Opheim, Zachary; Mack, Christopher P.; Cyr, Douglas M.; Taylor, Joan M.

    2017-01-01

    Autophagy is an evolutionarily conserved intracellular degradation/recycling system that is essential for cellular homeostasis but is dysregulated in a number of diseases, including myocardial hypertrophy. Although it is clear that limiting or accelerating autophagic flux can result in pathological cardiac remodeling, the physiological signaling pathways that fine-tune cardiac autophagy are poorly understood. Herein, we demonstrated that stimulation of cardiomyocytes with phenylephrine (PE), a well known hypertrophic agonist, suppresses autophagy and that activation of focal adhesion kinase (FAK) is necessary for PE-stimulated autophagy suppression and subsequent initiation of hypertrophic growth. Mechanistically, we showed that FAK phosphorylates Beclin1, a core autophagy protein, on Tyr-233 and that this post-translational modification limits Beclin1 association with Atg14L and reduces Beclin1-dependent autophagosome formation. Remarkably, although ectopic expression of wild-type Beclin1 promoted cardiomyocyte atrophy, expression of a Y233E phosphomimetic variant of Beclin1 failed to affect cardiomyocyte size. Moreover, genetic depletion of Beclin1 attenuated PE-mediated/FAK-dependent initiation of myocyte hypertrophy in vivo. Collectively, these findings identify FAK as a novel negative regulator of Beclin1-mediated autophagy and indicate that this pathway can facilitate the promotion of compensatory hypertrophic growth. This novel mechanism to limit Beclin1 activity has important implications for treating a variety of pathologies associated with altered autophagic flux. PMID:27994061

  15. Controllable degradation of medical magnesium by electrodeposited composite films of mussel adhesive protein (Mefp-1) and chitosan.

    PubMed

    Jiang, Ping-Li; Hou, Rui-Qing; Chen, Cheng-Dong; Sun, Lan; Dong, Shi-Gang; Pan, Jin-Shan; Lin, Chang-Jian

    2016-09-15

    To control the degradation rate of medical magnesium in body fluid environment, biocompatible films composed of Mussel Adhesive Protein (Mefp-1) and chitosan were electrodeposited on magnesium surface in cathodic constant current mode. The compositions and structures of the films were characterized by atomic force microscope (AFM), scanning electron microscope (SEM) and infrared reflection absorption spectroscopy (IRAS). And the corrosion protection performance was investigated using electrochemical measurements and immersion tests in simulated body fluid (Hanks' solution). The results revealed that Mefp-1 and chitosan successfully adhered on the magnesium surface and formed a protective film. Compared with either single Mefp-1 or single chitosan film, the composite film of chitosan/Mefp-1/chitosan (CPC (chitosan/Mefp-1/chitosan)) exhibited lower corrosion current density, higher polarization resistance and more homogenous corrosion morphology and thus was able to effectively control the degradation rate of magnesium in simulated body environment. In addition, the active attachment and spreading of MC3T3-E1 cells on the CPC film coated magnesium indicated that the CPC film was significantly able to improve the biocompatibility of the medical magnesium.

  16. 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.

  17. 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.

  18. The Focal Adhesion: A Regulated Component of Aortic Stiffness

    PubMed Central

    Saphirstein, Robert J.; Gao, Yuan Z.; Jensen, Mikkel H.; Gallant, Cynthia M.; Vetterkind, Susanne; Moore, Jeffrey R.; Morgan, Kathleen G.

    2013-01-01

    Increased aortic stiffness is an acknowledged predictor and cause of cardiovascular disease. The sources and mechanisms of vascular stiffness are not well understood, although the extracellular matrix (ECM) has been assumed to be a major component. We tested here the hypothesis that the focal adhesions (FAs) connecting the cortical cytoskeleton of vascular smooth muscle cells (VSMCs) to the matrix in the aortic wall are a component of aortic stiffness and that this component is dynamically regulated. First, we examined a model system in which magnetic tweezers could be used to monitor cellular cortical stiffness, serum-starved A7r5 aortic smooth muscle cells. Lysophosphatidic acid (LPA), an activator of myosin that increases cell contractility, increased cortical stiffness. A small molecule inhibitor of Src-dependent FA recycling, PP2, was found to significantly inhibit LPA-induced increases in cortical stiffness, as well as tension-induced increases in FA size. To directly test the applicability of these results to force and stiffness development at the level of vascular tissue, we monitored mouse aorta ring stiffness with small sinusoidal length oscillations during agonist-induced contraction. The alpha-agonist phenylephrine, which also increases myosin activation and contractility, increased tissue stress and stiffness in a PP2- and FAK inhibitor 14-attenuated manner. Subsequent phosphotyrosine screening and follow-up with phosphosite-specific antibodies confirmed that the effects of PP2 and FAK inhibitor 14 in vascular tissue involve FA proteins, including FAK, CAS, and paxillin. Thus, in the present study we identify, for the first time, the FA of the VSMC, in particular the FAK-Src signaling complex, as a significant subcellular regulator of aortic stiffness and stress. PMID:23626821

  19. 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.

  20. GPM Video of In-fa

    NASA Video Gallery

    On Nov. 23, GPM saw In-fa dropping rain at an extreme rate of over 266 mm (10.5 inches) per hour in storms just to the northwest of the typhoon's eye where thunderstorms reached altitudes of over 1...

  1. Transcriptional regulation of adhesive properties of Bacillus subtilis to extracellular matrix proteins through the fibronectin-binding protein YloA.

    PubMed

    Rodriguez Ayala, Facundo; Bauman, Carlos; Bartolini, Marco; Saball, Ester; Salvarrey, Marcela; Leñini, Cecilia; Cogliati, Sebastián; Strauch, Mark; Grau, Roberto

    2017-03-13

    Bacterial adherence to extracellular matrix proteins (ECMp) plays important roles during host-pathogen interaction, however its genetic regulation remains poorly understood. yloA of the model bacterium Bacillus subtilis shows high homology to genes encoding fibronectin-binding proteins of Gram-positive pathogens. Here, we characterized the regulatory network of YloA-dependent adhesive properties of the probiotic B. subtilis natto (Bsn). YloA-proficient, but not YloA-deficient, Bsn specifically bound to ECMp in a concentration-dependent manner and were proficient in biofilm formation. yloA expression showed a continuous increase in activity during the growth phase and decreased during the stationary phase. The transcription factors AbrB and DegU downregulated yloA expression during the logarithmic and stationary growth phases respectively. Analysis of the yloA promoter region revealed the presence of AT-rich direct and inverted repeats previously reported to function as DegU-recognized binding sites. In spo0A cells, yloA expression was completely turned off because of upregulation of AbrB throughout growth. Accordingly, DNase I footprinting analysis confirmed that AbrB bound to the promoter region of yloA. Interestingly, Bsn bound fibronectin with higher affinity, lower Kd, than several bacterial pathogens and competitively excluded them from binding to immobilized-fibronectin, a finding that might be important for the anti-infective properties of B. subtilis and its relatives.

  2. Reversible Conformational Change in the Plasmodium falciparum Circumsporozoite Protein Masks Its Adhesion Domains.

    PubMed

    Herrera, Raul; Anderson, Charles; Kumar, Krishan; Molina-Cruz, Alvaro; Nguyen, Vu; Burkhardt, Martin; Reiter, Karine; Shimp, Richard; Howard, Randall F; Srinivasan, Prakash; Nold, Michael J; Ragheb, Daniel; Shi, Lirong; DeCotiis, Mark; Aebig, Joan; Lambert, Lynn; Rausch, Kelly M; Muratova, Olga; Jin, Albert; Reed, Steven G; Sinnis, Photini; Barillas-Mury, Carolina; Duffy, Patrick E; MacDonald, Nicholas J; Narum, David L

    2015-10-01

    The extended rod-like Plasmodium falciparum circumsporozoite protein (CSP) is comprised of three primary domains: a charged N terminus that binds heparan sulfate proteoglycans, a central NANP repeat domain, and a C terminus containing a thrombospondin-like type I repeat (TSR) domain. Only the last two domains are incorporated in RTS,S, the leading malaria vaccine in phase 3 trials that, to date, protects about 50% of vaccinated children against clinical disease. A seroepidemiological study indicated that the N-terminal domain might improve the efficacy of a new CSP vaccine. Using a panel of CSP-specific monoclonal antibodies, well-characterized recombinant CSPs, label-free quantitative proteomics, and in vitro inhibition of sporozoite invasion, we show that native CSP is N-terminally processed in the mosquito host and undergoes a reversible conformational change to mask some epitopes in the N- and C-terminal domains until the sporozoite interacts with the liver hepatocyte. Our findings show the importance of understanding processing and the biophysical change in conformation, possibly due to a mechanical or molecular signal, and may aid in the development of a new CSP vaccine.

  3. 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.

  4. Cloning of SEZ-12 encoding seizure-related and membrane-bound adhesion protein.

    PubMed

    Kajiwara, K; Nagasawa, H; Shimizu-Nishikawa, K; Ookura, T; Kimura, M; Sugaya, E

    1996-05-06

    SEZ-12 is one of the seizure-related cDNAs which was isolated by differential hybridization from primary cultured neurons from the mouse cerebral cortex with or without pentylenetetrazol (PTZ). SEZ-12 expression is transiently down-regulated in the mouse brain by injection of PTZ. To characterize SEZ-12, isolation of full-length cDNA and nucleotide sequence analysis were performed. The deduced amino acid sequence of SEZ-12 revealed that it encodes membrane-bound C-type lectin and has a significant homology to that of human cDNA, DGCR2 and IDD, which were cloned from a balanced translocation breakpoint associated with the DiGeorge syndrome. The isolated cDNA was about 4 kb in length and the message was expressed ubiquitously in various organs with low-abundance. Previously, we also cloned a transmembrane protein which is probably involved in cell-cell interaction by the differential hybridization technique. These findings suggest that transmembrane signaling in neuronal cells may have an important role in PTZ-induced seizure.

  5. JPRS Report Science & Technology Japan Future Prospects of FA-From FA to IMS

    DTIC Science & Technology

    2007-11-02

    STATEMENT A Approved lor public release; . Distribution Unlimited aa "lbAoa iaod sszs £0T 5S3D0ad sNiib 51? inzz 19980701 126 DTIC QUALITY INSPECTED...5 4. Tasks and Future Prospects of FA 13 5. Proposals 21 6. "Status of FA" Survey and Analysis 24 7.FA-related Vocabulary 31...came to about ¥ 13 trillion. This plant investment included such things as investment in new operations and R&D investment; investment for the purpose

  6. Utility of cytokine, adhesion molecule and acute phase proteins in early diagnosis of neonatal sepsis

    PubMed Central

    Fattah, M. A.; Omer, Al Fadhil A.; Asaif, S.; Manlulu, R.; Karar, T.; Ahmed, A.; Aljada, A.; Saleh, Ayman M.; Qureshi, Shoeb; Nasr, A.

    2017-01-01

    Background and Aim: Neonatal infection, including bacterial sepsis, is a major health care issue with an annual global mortality in excess of one million lives. Therefore, this study aimed to evaluate the potential diagnostic value of C-reactive protein (CRP), E-selectin, procalcitonin (PCT), interleukins-6 (IL-6), and tumor necrosis factor-α (TNF-α) both independently and in combination for the diagnosis of neonatal sepsis in its earliest stages. Materials and Methods: A total of 320 subjects were included in this study. A prospective cross-sectional study was conducted among neonates admitted to Neonatal Intensive Care Unit at King Abdulaziz Medical City, Riyadh, KSA during January 2013 to August 2015, the study based on three study groups categorized according to clinical symptoms and blood culture result. Study groups include healthy control neonates (n = 80), clinical sepsis (CS) group (n = 80) with clinical signs of sepsis but their blood culture was negative, and sepsis group with clinical signs of sepsis and their blood culture was positive. Results: The study observed significant difference in plasma levels of CRP, IL-6, TNF-α, E-selectin, and PCT in patients group when compared with control group (P < 0.001). Furthermore, the levels are significantly different between patient groups including CS and neonatal sepsis group. Moreover, result observed significant difference in CRP and IL-6 in early onset sepsis (EOS) when compared with late onset sepsis (LOS) neonates (P < 0.001 and 0.01), respectively, while there were no significant difference in TNF-α, E-selectin, and PCT between EOS and LOS (P = 0.44, 0.27 and 0.24), respectively. Regarding biomarkers accuracy, the result showed that CRP has the best diagnostic accuracy with cutoff value of 3.6 ng/ml (sensitivity 78% and specificity of 70%). The best combination is shown with CRP and IL-6 in which sensitivity increased to 89% and specificity to 79%. Conclusion: It was concluded that infected new

  7. A Novel Domain Cassette Identifies Plasmodium falciparum PfEMP1 Proteins Binding ICAM-1 and Is a Target of Cross-Reactive, Adhesion-Inhibitory Antibodies

    PubMed Central

    Bengtsson, Anja; Joergensen, Louise; Rask, Thomas S.; Olsen, Rebecca W.; Andersen, Marianne A.; Turner, Louise; Theander, Thor G.; Higgins, Matthew K.; Craig, Alister; Brown, Alan

    2013-01-01

    Cerebral Plasmodium falciparum malaria is characterized by adhesion of infected erythrocytes (IEs) to the cerebral microvasculature. This has been linked to parasites expressing the structurally related group A subset of the P. falciparum erythrocyte membrane protein 1 (PfEMP1) family of IE adhesion ligands and to IEs with affinity for ICAM-1. However, recent evidence has cast doubt on both these associations, tempering hopes of the feasibility of developing a vaccine based on ICAM-1–binding PfEMP1. In this study, we report the identification of a domain cassette (DC) present in group A var genes from six genetically distinct P. falciparum parasites. The three domains in the cassette, which we call DC4, had a high level of sequence identity and cluster together phylogenetically. Erythrocytes infected by these parasites and selected in vitro for expression of DC4 adhered specifically to ICAM-1. The ICAM-1–binding capacity of DC4 was mapped to the C-terminal third of its Duffy-binding–like β3 domain. DC4 was the target of broadly cross-reactive and adhesion-inhibitory IgG Abs, and levels of DC4-specific and adhesion-inhibitory IgG increased with age among P. falciparum–exposed children. Our study challenges earlier conclusions that group A PfEMP1 proteins are not central to ICAM-1–specific IE adhesion and support the feasibility of developing a vaccine preventing cerebral malaria by inhibiting cerebral IE sequestration. PMID:23209327

  8. Arabidopsis NDR1 is an integrin-like protein with a role in fluid loss and plasma membrane-cell wall adhesion.

    PubMed

    Knepper, Caleb; Savory, Elizabeth A; Day, Brad

    2011-05-01

    Arabidopsis (Arabidopsis thaliana) NON-RACE-SPECIFIC DISEASE RESISTANCE1 (NDR1), a plasma membrane-localized protein, plays an essential role in resistance mediated by the coiled-coil-nucleotide-binding site-leucine-rich repeat class of resistance (R) proteins, which includes RESISTANCE TO PSEUDOMONAS SYRINGAE2 (RPS2), RESISTANCE TO PSEUDOMONAS SYRINGAE PV MACULICOLA1, and RPS5. Infection with Pseudomonas syringae pv tomato DC3000 expressing the bacterial effector proteins AvrRpt2, AvrB, and AvrPphB activates resistance by the aforementioned R proteins. Whereas the genetic requirement for NDR1 in plant disease resistance signaling has been detailed, our study focuses on determining a global, physiological role for NDR1. Through the use of homology modeling and structure threading, NDR1 was predicted to have a high degree of structural similarity to Arabidopsis LATE EMBRYOGENESIS ABUNDANT14, a protein implicated in abiotic stress responses. Specific protein motifs also point to a degree of homology with mammalian integrins, well-characterized proteins involved in adhesion and signaling. This structural homology led us to examine a physiological role for NDR1 in preventing fluid loss and maintaining cell integrity through plasma membrane-cell wall adhesions. Our results show a substantial alteration in induced (i.e. pathogen-inoculated) electrolyte leakage and a compromised pathogen-associated molecular pattern-triggered immune response in ndr1-1 mutant plants. As an extension of these analyses, using a combination of genetic and cell biology-based approaches, we have identified a role for NDR1 in mediating plasma membrane-cell wall adhesions. Taken together, our data point to a broad role for NDR1 both in mediating primary cellular functions in Arabidopsis through maintaining the integrity of the cell wall-plasma membrane connection and as a key signaling component of these responses during pathogen infection.

  9. Seafood delicacy makes great adhesive

    ScienceCinema

    Idaho National Laboratory - Frank Roberto, Heather Silverman

    2016-07-12

    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

  10. 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

  11. Role of the actin-binding protein profilin1 in radial migration and glial cell adhesion of granule neurons in the cerebellum.

    PubMed

    Rust, Marco B; Kullmann, Jan A; Witke, Walter

    2012-01-01

    Profilins are small G-actin-binding proteins essential for cytoskeletal dynamics. Of the four mammalian profilin isoforms, profilin1 shows a broad expression pattern, profilin2 is abundant in the brain, and profilin3 and profilin4 are restricted to the testis. In vitro studies on cancer and epithelial cell lines suggested a role for profilins in cell migration and cell-cell adhesion. Genetic studies in mice revealed the importance of profilin1 in neuronal migration, while profilin2 has apparently acquired a specific function in synaptic physiology. We recently reported a mouse mutant line lacking profilin1 in the brain; animals display morphological defects that are typical for impaired neuronal migration. We found that during cerebellar development, profilin1 is specifically required for radial migration and glial cell adhesion of granule neurons. Profilin1 mutants showed cerebellar hypoplasia and aberrant organization of cerebellar cortex layers, with ectopically arranged granule neurons. In this commentary, we briefly introduce the profilin family and summarize the current knowledge on profilin activity in cell migration and adhesion. Employing cerebellar granule cells as a model, we shed some light on the mechanisms by which profilin1 may control radial migration and glial cell adhesion. Finally, a potential implication of profilin1 in human developmental neuropathies is discussed.

  12. Involvement of caveolin-1 in low shear stress-induced breast cancer cell motility and adhesion: Roles of FAK/Src and ROCK/p-MLC pathways.

    PubMed

    Xiong, Niya; Li, Shun; Tang, Kai; Bai, Hongxia; Peng, Yueting; Yang, Hong; Wu, Chunhui; Liu, Yiyao

    2017-01-01

    Tumor cells translocating to distant sites are subjected to hemodynamic shear forces during their passage in the blood vessels. Low shear stress (LSS) plays a critical role in the regulation of various aspects of tumor cells functions, including motility and adhesion. Beyond its structural role, caveolin-1 (Cav-1), the important component of caveolae, represents a modulator of several cancer-associated functions as tumor progression and metastasis. However, the role of Cav-1 in regulating tumor cells response to shear stress remains poorly explored. Here, we characterized the role of LSS and Cav-1 in mediating cell motility and adhesion on human breast carcinoma MDA-MB-231 cells. We first showed that LSS exposure promoted cell polarity and focal adhesion (FA) dynamics, thus indicating elevated cell migration. Silencing of Cav-1 leaded to a significantly lower formation of stress fibers. However, LSS exposure was able to rescue it via the alteration of actin-associated proteins expression, including ROCK, p-MLC, cofilin and filamin A. Time-lapse migration assay indicated that Cav-1 expression fostered MDA-MB-231 cells motility and LSS triggered cells to rapidly generate new lamellipodia. Furthermore, Cav-1 and LSS significantly influenced cell adhesion. Taken together, our findings provide insights into mechanisms underlying LSS triggered events mediated by downstream Cav-1, including FAK/Src and ROCK/p-MLC pathways, involved in the reorganization of the cytoskeleton, cell motility, FA dynamics and breast cancer cell adhesion.

  13. Biophysically inspired model for functionalized nanocarrier adhesion to cell surface: roles of protein expression and mechanical factors

    NASA Astrophysics Data System (ADS)

    Ramakrishnan, N.; Tourdot, Richard W.; Eckmann, David M.; Ayyaswamy, Portonovo S.; Muzykantov, Vladimir R.; Radhakrishnan, Ravi

    2016-06-01

    In order to achieve selective targeting of affinity-ligand coated nanoparticles to the target tissue, it is essential to understand the key mechanisms that govern their capture by the target cell. Next-generation pharmacokinetic (PK) models that systematically account for proteomic and mechanical factors can accelerate the design, validation and translation of targeted nanocarriers (NCs) in the clinic. Towards this objective, we have developed a computational model to delineate the roles played by target protein expression and mechanical factors of the target cell membrane in determining the avidity of functionalized NCs to live cells. Model results show quantitative agreement with in vivo experiments when specific and non-specific contributions to NC binding are taken into account. The specific contributions are accounted for through extensive simulations of multivalent receptor-ligand interactions, membrane mechanics and entropic factors such as membrane undulations and receptor translation. The computed NC avidity is strongly dependent on ligand density, receptor expression, bending mechanics of the target cell membrane, as well as entropic factors associated with the membrane and the receptor motion. Our computational model can predict the in vivo targeting levels of the intracellular adhesion molecule-1 (ICAM1)-coated NCs targeted to the lung, heart, kidney, liver and spleen of mouse, when the contributions due to endothelial capture are accounted for. The effect of other cells (such as monocytes, etc.) do not improve the model predictions at steady state. We demonstrate the predictive utility of our model by predicting partitioning coefficients of functionalized NCs in mice and human tissues and report the statistical accuracy of our model predictions under different scenarios.

  14. Biophysically inspired model for functionalized nanocarrier adhesion to cell surface: roles of protein expression and mechanical factors

    PubMed Central

    Ramakrishnan, N.; Tourdot, Richard W.; Eckmann, David M.; Ayyaswamy, Portonovo S.; Muzykantov, Vladimir R.; Radhakrishnan, Ravi

    2016-01-01

    In order to achieve selective targeting of affinity–ligand coated nanoparticles to the target tissue, it is essential to understand the key mechanisms that govern their capture by the target cell. Next-generation pharmacokinetic (PK) models that systematically account for proteomic and mechanical factors can accelerate the design, validation and translation of targeted nanocarriers (NCs) in the clinic. Towards this objective, we have developed a computational model to delineate the roles played by target protein expression and mechanical factors of the target cell membrane in determining the avidity of functionalized NCs to live cells. Model results show quantitative agreement with in vivo experiments when specific and non-specific contributions to NC binding are taken into account. The specific contributions are accounted for through extensive simulations of multivalent receptor–ligand interactions, membrane mechanics and entropic factors such as membrane undulations and receptor translation. The computed NC avidity is strongly dependent on ligand density, receptor expression, bending mechanics of the target cell membrane, as well as entropic factors associated with the membrane and the receptor motion. Our computational model can predict the in vivo targeting levels of the intracellular adhesion molecule-1 (ICAM1)-coated NCs targeted to the lung, heart, kidney, liver and spleen of mouse, when the contributions due to endothelial capture are accounted for. The effect of other cells (such as monocytes, etc.) do not improve the model predictions at steady state. We demonstrate the predictive utility of our model by predicting partitioning coefficients of functionalized NCs in mice and human tissues and report the statistical accuracy of our model predictions under different scenarios. PMID:27429783

  15. 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

  16. Cryptococcus neoformans activates RhoGTPase proteins followed by protein kinase C, focal adhesion kinase, and ezrin to promote traversal across the blood-brain barrier.

    PubMed

    Kim, Jong-Chul; Crary, Benjamin; Chang, Yun C; Kwon-Chung, Kyung J; Kim, Kee J

    2012-10-19

    Cryptococcus neoformans is an opportunistic fungal pathogen that causes meningoencephalitis. Previous studies have demonstrated that Cryptococcus binding and invasion of human brain microvascular endothelial cells (HBMEC) is a prerequisite for transmigration across the blood-brain barrier. However, the molecular mechanism involved in the cryptococcal blood-brain barrier traversal is poorly understood. In this study we examined the signaling events in HBMEC during interaction with C. neoformans. Analysis with inhibitors revealed that cryptococcal association, invasion, and transmigration require host actin cytoskeleton rearrangement. Rho pulldown assays revealed that Cryptococcus induces activation of three members of RhoGTPases, e.g. RhoA, Rac1, and Cdc42, and their activations are required for cryptococcal transmigration across the HBMEC monolayer. Western blot analysis showed that Cryptococcus also induces phosphorylation of focal adhesion kinase (FAK), ezrin, and protein kinase C α (PKCα), all of which are involved in the rearrangement of host actin cytoskeleton. Down-regulation of FAK, ezrin, or PKCα by shRNA knockdown, dominant-negative transfection, or inhibitors significantly reduces cryptococcal ability to traverse the HBMEC monolayer, indicating their positive role in cryptococcal transmigration. In addition, activation of RhoGTPases is the upstream event for phosphorylation of FAK, ezrin, and PKCα during C. neoformans-HBMEC interaction. Taken together, our findings demonstrate that C. neoformans activates RhoGTPases and subsequently FAK, ezrin, and PKCα to promote their traversal across the HBMEC monolayer, which is the critical step for cryptococcal brain infection and development of meningitis.

  17. Type III Methyltransferase M.NgoAX from Neisseria gonorrhoeae FA1090 Regulates Biofilm Formation and Interactions with Human Cells

    PubMed Central

    Kwiatek, Agnieszka; Mrozek, Agnieszka; Bacal, Pawel; Piekarowicz, Andrzej; Adamczyk-Popławska, Monika

    2015-01-01

    Neisseria gonorrhoeae is the etiological factor of the sexually transmitted gonorrhea disease that may lead, under specific conditions, to systemic infections. The gonococcal genome encodes many restriction modification (RM) systems, which main biological role is to defend the pathogen from potentially harmful foreign DNA. However, RM systems seem also to be involved in several other functions. In this study, we examined the effect of inactivation the N. gonorrhoeae FA1090 ngoAXmod gene encoding M.NgoAX methyltransferase on the global gene expression, biofilm formation, interactions with human epithelial host cells and overall bacterial growth. Expression microarrays showed at least a twofold deregulation of a total of 121 genes in the NgoAX knock-out mutant compared to the wild-type (wt) strain under standard grow conditions. Genes with changed expression levels encoded mostly proteins involved in cell metabolism, DNA replication and repair or regulating cellular processes and signaling (such as cell wall/envelop biogenesis). As determined by the assay with crystal violet, the NgoAX knock-out strain formed a slightly larger biofilm biomass per cell than the wt strain. Live biofilm observations showed that the biofilm formed by the gonococcal ngoAXmod gene mutant is more relaxed, dispersed and thicker than the one formed by the wt strain. This more relaxed feature of the biofilm, in respect to adhesion and bacterial interactions, can be involved in pathogenesis. Moreover, the overall adhesion of mutant bacterial cells to human cells was lower than adhesion of the wt gonococci [adhesion index = 0.672 (±0.2) and 2.15 (±1.53), respectively]; yet, a higher number of mutant than wt bacteria were found inside the Hec-1-B epithelial cells [invasion index = 3.38 (±0.93) × 105 for mutant and 4.67 (±3.09) × 104 for the wt strain]. These results indicate that NgoAX knock-out cells have lower ability to attach to human cells, but more easily penetrate inside the host

  18. Cryptosporidium parvum: identification of a new surface adhesion protein on sporozoite and oocyst by screening of a phage-display cDNA library.

    PubMed

    Yao, Longquan; Yin, Jigang; Zhang, Xichen; Liu, Quan; Li, Jianhua; Chen, Lifeng; Zhao, Yueping; Gong, Pengtao; Liu, Chengwu

    2007-04-01

    Cryptosporidium parvum is a significant cause of diarrheal disease worldwide. The specific molecules that mediate C. parvum-host interaction and the molecular mechanisms involved in the pathogenesis are unknown. In this study we described a novel phage display method to identify surface adhesion proteins of C. parvum. A cDNA library of the sporozoite and oocyst stages of C. parvum expressed on the surface of T7 phage was screened with intestinal epithelial cells (IECs) from the newborn Cryptosporidium-free Holstein calves. Proteins that selectively and specifically bound to IECs were then enriched using a multi-step panning procedure. Two proteins of C. parvum were selected, one was previously reported (p23), which was an important surface adhesion protein; the other was a novel surface adherence protein (CP12). Sequence analysis showed that CP12 has a N-terminal signal peptide, a transmembrane region, a N-glycosylation site, a casein kinase II phosphorylation site and two N-myristoylation sites. Immunofluorescence assay (IFA) using antibody specific for rCP12 demonstrated that the antibody can specifically bind the surface of sporozoite and oocyst, especially apical region of sporozoite. The surface localization of CP12 and its involvement in the host-parasite interaction suggest that it may serve as an effective target for specific preventive and therapeutic measures for cryptosporidiosis.

  19. 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.

  20. DDB2 (damaged-DNA binding 2) protein: a new modulator of nanomechanical properties and cell adhesion of breast cancer cells.

    PubMed

    Barbieux, Claire; Bacharouche, Jalal; Soussen, Charles; Hupont, Sébastien; Razafitianamaharavo, Angélina; Klotz, Rémi; Pannequin, Rémi; Brie, David; Bécuwe, Philippe; Francius, Grégory; Grandemange, Stéphanie

    2016-03-07

    DDB2, known for its role in DNA repair, was recently shown to reduce mammary tumor invasiveness by inducing the transcription of IκBα, an inhibitor of NF-κB activity. Since cellular adhesion is a key event during the epithelial to mesenchymal transition (EMT) leading to the invasive capacities of breast tumor cells, the aim of this study was to investigate the role of DDB2 in this process. Thus, using low and high DDB2-expressing MDA-MB231 and MCF7 cells, respectively, in which DDB2 expression was modulated experimentally, we showed that DDB2 overexpression was associated with a decrease of adhesion abilities on glass and plastic areas of breast cancer cells. Then, we investigated cell nanomechanical properties by atomic force microscopy (AFM). Our results revealed significant changes in the Young's Modulus value and the adhesion force in MDA-MB231 and MCF7 cells, whether DDB2 was expressed or not. The cell stiffness decrease observed in MDA-MB231 and MCF7 expressing DDB2 was correlated with a loss of the cortical actin-cytoskeleton staining. To understand how DDB2 regulates these processes, an adhesion-related gene PCR-Array was performed. Several adhesion-related genes were differentially expressed according to DDB2 expression, indicating that important changes are occurring at the molecular level. Thus, this work demonstrates that AFM technology is an important tool to follow cellular changes during tumorigenesis. Moreover, our data revealed that DDB2 is involved in early events occurring during metastatic progression of breast cancer cells and will contribute to define this protein as a new marker of metastatic progression in this type of cancer.

  1. The Non-Equilibrium Thermodynamics and Kinetics of Focal Adhesion Dynamics

    PubMed Central

    Olberding, Joseph E.; Thouless, Michael D.; Arruda, Ellen M.; Garikipati, Krishna

    2010-01-01

    Background We consider a focal adhesion to be made up of molecular complexes, each consisting of a ligand, an integrin molecule, and associated plaque proteins. Free energy changes drive the binding and unbinding of these complexes and thereby controls the focal adhesion's dynamic modes of growth, treadmilling and resorption. Principal Findings We have identified a competition among four thermodynamic driving forces for focal adhesion dynamics: (i) the work done during the addition of a single molecular complex of a certain size, (ii) the chemical free energy change associated with the addition of a molecular complex, (iii) the elastic free energy change associated with deformation of focal adhesions and the cell membrane, and (iv) the work done on a molecular conformational change. We have developed a theoretical treatment of focal adhesion dynamics as a nonlinear rate process governed by a classical kinetic model. We also express the rates as being driven by out-of-equilibrium thermodynamic driving forces, and modulated by kinetics. The mechanisms governed by the above four effects allow focal adhesions to exhibit a rich variety of behavior without the need to introduce special constitutive assumptions for their response. For the reaction-limited case growth, treadmilling and resorption are all predicted by a very simple chemo-mechanical model. Treadmilling requires symmetry breaking between the ends of the focal adhesion, and is achieved by driving force (i) above. In contrast, depending on its numerical value (ii) causes symmetric growth, resorption or is neutral, (iii) causes symmetric resorption, and (iv) causes symmetric growth. These findings hold for a range of conditions: temporally-constant force or stress, and for spatially-uniform and non-uniform stress distribution over the FA. The symmetric growth mode dominates for temporally-constant stress, with a reduced treadmilling regime. Significance In addition to explaining focal adhesion dynamics, this

  2. Sensing substrate rigidity by mechanosensitive ion channels with stress fibers and focal adhesions.

    PubMed

    Kobayashi, Takeshi; Sokabe, Masahiro

    2010-10-01

    Cell motility, spreading, proliferation and differentiation are critically influenced by substrate rigidity. To sense substrate rigidity, cells apply traction forces to cell-substrate adhesions via actin stress fibers (SFs) and measure mechanical responses of the substrate. Besides mechanosensitive adaptor proteins, mechanosensitive (MS) channels are involved in the substrate rigidity sensing. MS channels located at or near focal adhesions (FAs) convert the rigidity-dependent stress generated in SF/FA system into the level of cytoplasmic Ca(2+) concentration ([Ca(2+)]cyt) by locally altering their Ca(2+) permeability. Besides by external forces, cells spontaneously generate rigidity-dependent localized [Ca(2+)]cyt increases, implicating MS channels as intrinsic force measurement system. This mechanism may contribute to not only substrate rigidity sensing but also regulation of cell migration.

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

    SciTech Connect

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

    2013-07-15

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

  4. 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.

  5. RhoGAP68F controls transport of adhesion proteins in Rab4 endosomes to modulate epithelial morphogenesis of Drosophila leg discs

    PubMed Central

    de Madrid, Beatriz Hernandez; Greenberg, Lina; Hatini, Victor

    2015-01-01

    SUMMARY Elongation and invagination of epithelial tissues are fundamental developmental processes that contribute to the morphogenesis of embryonic and adult structures and are dependent on coordinated remodeling of cell-cell contacts. The morphogenesis of Drosophila leg imaginal discs depends on extensive remodeling of cell contacts and thus provides a useful system with which to investigate the underlying mechanisms. The small Rho GTPase regulator RhoGAP68F has been previously implicated in leg morphogenesis. It consists of an N-terminal Sec14 domain and a C-terminal GAP domain. Here we examined the molecular function and role of RhoGAP68F in epithelial remodeling. We find that depletion of RhoGAP68F impairs epithelial remodeling from a pseudostratified to simple, while overexpression of RhoGAP68F causes tears of lateral cell-cell contacts and thus impairs epithelial integrity. We show that the RhoGAP68F protein localizes to Rab4 recycling endosomes and forms a complex with the Rab4 protein. The Sec14 domain is sufficient for localizing to Rab4 endosomes, while the activity of the GAP domain is dispensable. RhoGAP68F, in turn, inhibits the scission and movement of Rab4 endosomes involved in transport the adhesion proteins Fasciclin3 and E-cadherin back to cell-cell contacts. Expression of RhoGAP68F is upregulated during prepupal development suggesting that RhoGAP68F decreases the transport of key adhesion proteins to the cell surface during this developmental stage to decrease the strength of adhesive cell-cell contacts and thereby facilitate epithelial remodeling and leg morphogenesis. PMID:25617722

  6. Abdominal Adhesions

    MedlinePlus

    ... Adhesions 1 Ward BC, Panitch A. Abdominal adhesions: current and novel therapies. Journal of Surgical Research. 2011;165(1):91–111. Seek Help for ... and how to participate, visit the NIH Clinical Research Trials and You website ... Foundation for Functional Gastrointestinal Disorders 700 West Virginia ...

  7. 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

  8. Proteomic dataset of the sea urchin Paracentrotus lividus adhesive organs and secreted adhesive

    PubMed Central

    Lebesgue, Nicolas; da Costa, Gonçalo; Ribeiro, Raquel Mesquita; Ribeiro-Silva, Cristina; Martins, Gabriel G.; Matranga, Valeria; Scholten, Arjen; Cordeiro, Carlos; Heck, Albert J.R.; Santos, Romana

    2016-01-01

    Sea urchins have specialized adhesive organs called tube feet, which mediate strong but reversible adhesion. Tube feet are composed by a disc, producing adhesive and de-adhesive secretions for substratum attachment, and a stem for movement. After detachment the secreted adhesive remains bound to the substratum as a footprint. Recently, a label-free quantitative proteomic approach coupled with the latest mass-spectrometry technology was used to analyze the differential proteome of Paracentrotus lividus adhesive organ, comparing protein expression levels in the tube feet adhesive part (the disc) versus the non-adhesive part (the stem), and also to profile the proteome of the secreted adhesive (glue). This data article contains complementary figures and results related to the research article “Deciphering the molecular mechanisms underlying sea urchin reversible adhesion: a quantitative proteomics approach” (Lebesgue et al., 2016) [1]. Here we provide a dataset of 1384 non-redundant proteins, their fragmented peptides and expression levels, resultant from the analysis of the tube feet differential proteome. Of these, 163 highly over-expressed tube feet disc proteins (>3-fold), likely representing the most relevant proteins for sea urchin reversible adhesion, were further annotated in order to determine the potential functions. In addition, we provide a dataset of 611 non-redundant proteins identified in the secreted adhesive proteome, as well as their functional annotation and grouping in 5 major protein groups related with adhesive exocytosis, and microbial protection. This list was further analyzed to identify the most abundant protein groups and pinpoint putative adhesive proteins, such as Nectin, the most abundant adhesive protein in sea urchin glue. The obtained data uncover the key proteins involved in sea urchins reversible adhesion, representing a step forward to the development of new wet-effective bio-inspired adhesives. PMID:27182547

  9. Proteomic dataset of the sea urchin Paracentrotus lividus adhesive organs and secreted adhesive.

    PubMed

    Lebesgue, Nicolas; da Costa, Gonçalo; Ribeiro, Raquel Mesquita; Ribeiro-Silva, Cristina; Martins, Gabriel G; Matranga, Valeria; Scholten, Arjen; Cordeiro, Carlos; Heck, Albert J R; Santos, Romana

    2016-06-01

    Sea urchins have specialized adhesive organs called tube feet, which mediate strong but reversible adhesion. Tube feet are composed by a disc, producing adhesive and de-adhesive secretions for substratum attachment, and a stem for movement. After detachment the secreted adhesive remains bound to the substratum as a footprint. Recently, a label-free quantitative proteomic approach coupled with the latest mass-spectrometry technology was used to analyze the differential proteome of Paracentrotus lividus adhesive organ, comparing protein expression levels in the tube feet adhesive part (the disc) versus the non-adhesive part (the stem), and also to profile the proteome of the secreted adhesive (glue). This data article contains complementary figures and results related to the research article "Deciphering the molecular mechanisms underlying sea urchin reversible adhesion: a quantitative proteomics approach" (Lebesgue et al., 2016) [1]. Here we provide a dataset of 1384 non-redundant proteins, their fragmented peptides and expression levels, resultant from the analysis of the tube feet differential proteome. Of these, 163 highly over-expressed tube feet disc proteins (>3-fold), likely representing the most relevant proteins for sea urchin reversible adhesion, were further annotated in order to determine the potential functions. In addition, we provide a dataset of 611 non-redundant proteins identified in the secreted adhesive proteome, as well as their functional annotation and grouping in 5 major protein groups related with adhesive exocytosis, and microbial protection. This list was further analyzed to identify the most abundant protein groups and pinpoint putative adhesive proteins, such as Nectin, the most abundant adhesive protein in sea urchin glue. The obtained data uncover the key proteins involved in sea urchins reversible adhesion, representing a step forward to the development of new wet-effective bio-inspired adhesives.

  10. 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.

  11. 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

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

    PubMed

    Xiao, Xiang; Mruk, Dolores D; Cheng, C Yan

    2013-01-15

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

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

    PubMed Central

    Xiao, Xiang; Mruk, Dolores D.

    2013-01-01

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

  14. The effect of stromelysin-1 (MMP-3) on non-collagenous extracellular matrix proteins of demineralized dentin and the adhesive properties of restorative resins.

    PubMed

    Boukpessi, T; Menashi, S; Camoin, L; Tencate, J M; Goldberg, M; Chaussain-Miller, C

    2008-11-01

    Dentin non-collagenous matrix components (NCPs) are structural proteins involved in the formation, the architecture and the mineralization of the extracellular matrix (ECM). We investigated here how recombinant metalloproteinase stromelysin-1, also termed MMP-3, initiates the release of ECM molecules from artificially demineralized human dentin. Analysis of the supernatants by Western blotting reveals that MMP-3 extracts PGs (decorin, biglycan), and also a series of phosphorylated proteins: dentin sialoprotein (DSP), osteopontin (OPN), bone sialoprotein (BSP) and MEPE, but neither dentin matrix protein-1 (DMP1), another member of the SIBLING family, nor osteocalcin (OC), a non-phosphorylated matrix molecule. After treatment of dentin surfaces by MMP-3, scanning electron microscope (SEM) examination of resin replica shows an increased penetration of the resin into the dentin tubules when compared to surfaces only treated by demineralizing solutions. This preclinical investigation suggests that MMP-3 may be used to improve the adhesive properties of restorative materials.

  15. Streptococcus suis Type 2 SSU0587 Protein is a Beta-Galactosidase That Contributes to Bacterial Adhesion but Not to Virulence in Mice

    PubMed Central

    TANG, Yulong; ZHANG, Xiaoyan; YIN, Yulong; HARDWIDGE, Philip R.; FANG, Weihuan

    2014-01-01

    ABSTRACT Bacterial surface proteins play key roles in virulence and often contribute to bacterial adhesion and invasion. We discovered that the Streptococcus suis type 2 (SS2) gene SSU0587 encodes a protein of 1,491 amino acids that possesses β-galactosidase activity. The surface association of the protein was dependent upon sortase activity. Deleting SSU0587 from clinical SS2 isolate JX081101 caused a loss of both β-galactosidase activity and adherence to microvascular endothelial cells. Deleting SSU0587 had no measurable impact on either invasion of microvascular endothelial cells or on virulence in a murine infection model, although the concentration of JX081101ΔSSU0587 was reduced in the brains of infected mice, as compared with the pathogen loads of the wild-type strain. PMID:24670993

  16. Streptococcus suis type 2 SSU0587 protein is a beta-galactosidase that contributes to bacterial adhesion but not to virulence in mice.

    PubMed

    Tang, Yulong; Zhang, Xiaoyan; Yin, Yulong; Hardwidge, Philip R; Fang, Weihuan

    2014-07-01

    Bacterial surface proteins play key roles in virulence and often contribute to bacterial adhesion and invasion. We discovered that the Streptococcus suis type 2 (SS2) gene SSU0587 encodes a protein of 1,491 amino acids that possesses β-galactosidase activity. The surface association of the protein was dependent upon sortase activity. Deleting SSU0587 from clinical SS2 isolate JX081101 caused a loss of both β-galactosidase activity and adherence to microvascular endothelial cells. Deleting SSU0587 had no measurable impact on either invasion of microvascular endothelial cells or on virulence in a murine infection model, although the concentration of JX081101ΔSSU0587 was reduced in the brains of infected mice, as compared with the pathogen loads of the wild-type strain.

  17. 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.

  18. Involvement of small G protein RhoB in the regulation of proliferation, adhesion and migration by dexamethasone in osteoblastic cells

    PubMed Central

    Wang, Yan; Li, Yidong; Xu, Weidong; Lu, Jian

    2017-01-01

    Long-term exposure to therapeutic doses of glucocorticoids (GCs) results in bone remodeling, which frequently causes osteoporosis and fracture healing retardation because of the abnormality of osteoblastic proliferation and differentiation. The mechanisms of GCs’ effect on osteoblasts are largely unknown. In this present study, we found that dexamethasone (Dex) could induce the expression of the small G protein, RhoB, in mRNA and protein levels in the osteoblast-derived osteosarcoma cell lines MG-63. The up-regulation of RhoB mRNA by Dex mainly occurs at posttranscriptional level by increasing its mRNA stability through PI-3K/Akt and p38 mitogen-activated protein kinase signaling pathways. Over-expression of RhoB in MG-63 cells magnified while down-regulation of RhoB level by RNA interference impaired Dex-induced growth inhibition but not differentiation. What’s more, over-expression of RhoB mimicked the effect of Dex on cell adhesion and migration. And interfering RhoB expression partially suppressed Dex-induced pro-adhesion and anti-migration in MG-63 cells. In conclusion, these results indicate that RhoB plays an important role in the pathological effect of Dex on osteoblastic growth and migration, which is a part of the mechanisms of GCs’ adverse effect on bone remodeling. PMID:28323887

  19. 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 (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 μM), a specific mitochondrial antioxidants, and cyclosporin A (1–5 μM), 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 μM), 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

  20. The Chlamydia outer membrane protein OmcB is required for adhesion and exhibits biovar-specific differences in glycosaminoglycan binding

    PubMed Central

    Moelleken, Katja; Hegemann, Johannes H

    2008-01-01

    Chlamydia pneumoniae, an obligate intracellular human pathogen, causes a number of respiratory diseases. We explored the role of the conserved OmcB protein in C. pneumoniae infections, using yeast display technology. (i) Yeast cells presenting OmcB were found to adhere to human epithelial cells. (ii) Pre-incubation of OmcB yeast cells with heparin, but not other glycosaminoglycans (GAGs), abrogated adhesion. (iii) Pre-treatment of the target cells with heparinase inhibited adherence, and GAG-deficient CHO cell lines failed to bind OmcB yeast. (iv) A heparin-binding motif present near the N-terminus of OmcB is required for host cell binding. (v) Pre-treatment of chlamydial elementary bodies (EBs) with anti-OmcB antibody or pre-incubation of target cells with recombinant OmcB protein reduced infectivity upon challenge with C. pneumoniae. (vi) Adhesion of fluorescently labelled EBs to epithelial or endothelial cells was abrogated by prior addition of heparin or OmcB protein. Thus, C. pneumoniae OmcB is an adhesin that binds heparan sulphate-like GAGs. OmcB from Chlamydia trachomatis serovar L1 also adheres to human cells in a heparin-dependent way, unlike its counterpart from serovar E. We show that a single position in the OmcB sequence determines heparin dependence/independence, and variations there may reflect differences between the two serovars in cell tropism and disease pattern. PMID:18086188

  1. Effects of the knockdown of death-associated protein 3 expression on cell adhesion, growth and migration in breast cancer cells.

    PubMed

    Wazir, Umar; Sanders, Andrew J; Wazir, Ahmad M A; Ye, Lin; Jiang, Wen G; Ster, Irina C; Sharma, Anup K; Mokbel, Kefah

    2015-05-01

    The death-associated protein 3 (DAP3) is a highly conserved phosphoprotein involved in the regulation of autophagy. A previous clinical study by our group suggested an association between low DAP3 expression and clinicopathological parameters of human breast cancer. In the present study, we intended to determine the role of DAP3 in cancer cell behaviour in the context of human breast cancer. We developed knockdown sub-lines of MCF7 and MDA-MB-231, and performed growth, adhesion, invasion assays and electric cell-substrate impedance sensing (ECIS) studies of post-wound migration of the cells. In addition, we studied the mRNA expression of caspase 8 and 9, death ligand signal enhancer (DELE), IFN-β promoter stimulator 1 (IPS1), cyclin D1 and p21 in the control and knockdown sub-lines. The knockdown sub-lines of MCF7 and MDA-MB-231 had significantly increased adhesion and decreased growth when compared to the controls. Furthermore, invasion and migration were significantly increased in the MDA-MB-231DAP3kd cells vs. the controls. The expression of caspase 9 and IPS1, known components of the apoptosis pathway, were significantly reduced in the MCF7DAP3kd cells (p=0.05 and p=0.003, respectively). We conclude that DAP3 silencing contributes to breast carcinogenesis by increasing cell adhesion, migration and invasion. It is possible that this may be due to the activity of focal adhesion kinase further downstream of the anoikis pathway. Further research in this direction would be beneficial in increasing our understanding of the mechanisms underlying human breast cancer.

  2. Berberine-induced anticancer activities in FaDu head and neck squamous cell carcinoma cells.

    PubMed

    Seo, Yo-Seob; Yim, Min-Ji; Kim, Bok-Hee; Kang, Kyung-Rok; Lee, Sook-Young; Oh, Ji-Su; You, Jae-Seek; Kim, Su-Gwan; Yu, Sang-Joun; Lee, Gyeong-Je; Kim, Do Kyung; Kim, Chun Sung; Kim, Jin-Soo; Kim, Jae-Sung

    2015-12-01

    In the present study, we investigated berberine‑induced apoptosis and the signaling pathways underlying its activity in FaDu head and neck squamous cell carcinoma cells. Berberine did not affect the viability of primary human normal oral keratinocytes. In contrast, the cytotoxicity of berberine was significantly increased in FaDu cells stimulated with berberine for 24 h. Furthermore, berberine increased nuclear condensation and apoptosis rates in FaDu cells than those in untreated control cells. Berberine also induced the upregulation of apoptotic ligands, such as FasL and TNF-related apoptosis-inducing ligand, and triggered the activation of caspase-8, -7 and -3, and poly(ADP ribose) polymerase, characteristic of death receptor-dependent extrinsic apoptosis. Moreover, berberine activated the mitochondria‑dependent apoptotic signaling pathway by upregulating pro-apoptotic factors, such as Bax, Bad, Apaf-1, and the active form of caspase-9, and downregulating anti-apoptotic factors, such as Bcl-2 and Bcl-xL. In addition, berberine increased the expression of the tumor suppressor p53 in FaDu cells. The pan-caspase inhibitor Z-VAD-fmk suppressed the activation of caspase-3 and prevented cytotoxicity in FaDu cells treated with berberine. Interestingly, berberine suppressed cell migration through downregulation of vascular endothelial growth factor (VEGF), matrix metalloproteinase (MMP)-2, and MMP-9. Moreover, the phosphorylation of extracellular signal-regulated kinase (ERK1/2) and p38, components of the mitogen-activated protein kinase pathway that are associated with the expression of MMP and VEGF, was suppressed in FaDu cells treated with berberine for 24 h. Therefore, these data suggested that berberine exerted anticancer effects in FaDu cells through induction of apoptosis and suppression of migration. Berberine may have potential applications as a chemotherapeutic agent for the management of head and neck squamous carcinoma.

  3. Mechanosensing through focal adhesion-anchored intermediate filaments.

    PubMed

    Gregor, Martin; Osmanagic-Myers, Selma; Burgstaller, Gerald; Wolfram, Michael; Fischer, Irmgard; Walko, Gernot; Resch, Guenter P; Jörgl, Almut; Herrmann, Harald; Wiche, Gerhard

    2014-02-01

    Integrin-based mechanotransduction involves a complex focal adhesion (FA)-associated machinery that is able to detect and respond to forces exerted either through components of the extracellular matrix or the intracellular contractile actomyosin network. Here, we show a hitherto unrecognized regulatory role of vimentin intermediate filaments (IFs) in this process. By studying fibroblasts in which vimentin IFs were decoupled from FAs, either because of vimentin deficiency (V0) or loss of vimentin network anchorage due to deficiency in the cytolinker protein plectin (P0), we demonstrate attenuated activation of the major mechanosensor molecule FAK and its downstream targets Src, ERK1/2, and p38, as well as an up-regulation of the compensatory feedback loop acting on RhoA and myosin light chain. In line with these findings, we show strongly reduced FA turnover rates in P0 fibroblasts combined with impaired directional migration, formation of protrusions, and up-regulation of "stretched" high-affinity integrin complexes. By exploiting tension-independent conditions, we were able to mechanistically link these defects to diminished cytoskeletal tension in both P0 and V0 cells. Our data provide important new insights into molecular mechanisms underlying cytoskeleton-regulated mechanosensing, a feature that is fundamental for controlled cell movement and tumor progression.

  4. Reduced PAK1 activity sensitizes FA/BRCA-proficient breast cancer cells to PARP inhibition.

    PubMed

    Villamar Cruz, Olga; Prudnikova, Tatiana Y; Araiza-Olivera, Daniela; Perez-Plasencia, Carlos; Johnson, Neil; Bernhardy, Andrea J; Slifker, Michael; Renner, Catherine; Chernoff, Jonathan; Arias-Romero, Luis E

    2016-11-22

    Cells that are deficient in homologous recombination, such as those that have mutations in any of the Fanconi Anemia (FA)/BRCA genes, are hypersensitive to inhibition of poly(ADP-ribose) polymerase (PARP). However, FA/BRCA-deficient tumors represent a small fraction of breast cancers, which might restrict the therapeutic utility of PARP inhibitor monotherapy. The gene encoding the serine-threonine protein kinase p21-activated kinase 1 (PAK1) is amplified and/or overexpressed in several human cancer types including 25-30% of breast tumors. This enzyme controls many cellular processes by phosphorylating both cytoplasmic and nuclear substrates. Here, we show that depletion or pharmacological inhibition of PAK1 down-regulated the expression of genes involved in the FA/BRCA pathway and compromised the ability of cells to repair DNA by Homologous Recombination (HR), promoting apoptosis and reducing colony formation. Combined inhibition of PAK1 and PARP in PAK1 overexpressing breast cancer cells had a synergistic effect, enhancing apoptosis, suppressing colony formation, and delaying tumor growth in a xenograft setting. Because reduced PAK1 activity impaired FA/BRCA function, inhibition of this kinase in PAK1 amplified and/or overexpressing breast cancer cells represents a plausible strategy for expanding the utility of PARP inhibitors to FA/BRCA-proficient cancers.

  5. Reduced PAK1 activity sensitizes FA/BRCA-proficient breast cancer cells to PARP inhibition

    PubMed Central

    Araiza-Olivera, Daniela; Perez-Plasencia, Carlos; Johnson, Neil; Bernhardy, Andrea J.; Slifker, Michael; Renner, Catherine; Chernoff, Jonathan; Arias, Luis E.

    2016-01-01

    Cells that are deficient in homologous recombination, such as those that have mutations in any of the Fanconi Anemia (FA)/BRCA genes, are hypersensitive to inhibition of poly(ADP-ribose) polymerase (PARP). However, FA/BRCA-deficient tumors represent a small fraction of breast cancers, which might restrict the therapeutic utility of PARP inhibitor monotherapy. The gene encoding the serine-threonine protein kinase p21-activated kinase 1 (PAK1) is amplified and/or overexpressed in several human cancer types including 25-30% of breast tumors. This enzyme controls many cellular processes by phosphorylating both cytoplasmic and nuclear substrates. Here, we show that depletion or pharmacological inhibition of PAK1 down-regulated the expression of genes involved in the FA/BRCA pathway and compromised the ability of cells to repair DNA by Homologous Recombination (HR), promoting apoptosis and reducing colony formation. Combined inhibition of PAK1 and PARP in PAK1 overexpressing breast cancer cells had a synergistic effect, enhancing apoptosis, suppressing colony formation, and delaying tumor growth in a xenograft setting. Because reduced PAK1 activity impaired FA/BRCA function, inhibition of this kinase in PAK1 amplified and/or overexpressing breast cancer cells represents a plausible strategy for expanding the utility of PARP inhibitors to FA/BRCA-proficient cancers. PMID:27740936

  6. The conserved dual phosphorylation sites of the Candida albicans Hog1 protein are crucial for white-opaque switching, mating, and pheromone-stimulated cell adhesion.

    PubMed

    Chang, Wen-Han; Liang, Shen-Huan; Deng, Fu-Sheng; Lin, Ching-Hsuan

    2016-08-01

    Candida albicans is an opportunistic human pathogen capable of causing life-threatening infections in immunocompromised patients. C. albicans has a unique morphological transition between white and opaque phases. These two cells differ in virulence, mating capability, biofilm formation, and host-cell interaction. Previous studies revealed that deletion of the SSK2, PBS2, or HOG1 gene resulted in 100% opaque cell formation and suppressed the mating response. Thr-174 and Tyr-176 of the Hog1 protein are important phosphoacceptors and can be activated in response to stimuli. In this study, we first demonstrated the importance of two conserved phosphorylation sites in white-opaque switching, mating, and pheromone-stimulated cell adhesion. Six Hog1 point-mutated strains were generated, including nonphosphorylated strains (Hog1(T174A), Hog1(Y176F), and Hog1(T174A,Y176F)) and negatively charged phosphorylated strains (Hog1(T174D), Hog1(Y176D), and Hog1(T174D,Y176D)). Point mutation on Thr-174, Tyr-176 or in combination with the Hog1 protein in C. albicans MTL homozygous strains stimulated opaque cell formation at a frequency of 100%. Furthermore, mating projections of point-mutated strains were significantly shorter and their mating efficiencies and pheromone-stimulated cell adhesive numbers were lower than those of the wild-type. By investigating the effects of Hog1 phosphorylation in ssk1Δ and sln1Δ, we also demonstrate that the phosphorylation intensity of Hog1p is directly involved in the white-opaque switching. Taken together, the results of our study demonstrate that dual phosphorylation sites of C. albicans are crucial for white-opaque transition, sexual mating, and pheromone-induced cell adhesion.

  7. Bio-active molecules modified surfaces enhanced mesenchymal stem cell adhesion and proliferation.

    PubMed

    Mobasseri, Rezvan; Tian, Lingling; Soleimani, Masoud; Ramakrishna, Seeram; Naderi-Manesh, Hossein

    2017-01-29

    Surface modification of the substrate as a component of in vitro cell culture and tissue engineering, using bio-active molecules including extracellular matrix (ECM) proteins or peptides derived ECM proteins can modulate the surface properties and thereby induce the desired signaling pathways in cells. The aim of this study was to evaluate the behavior of human bone marrow mesenchymal stem cells (hBM-MSCs) on glass substrates modified with fibronectin (Fn), collagen (Coll), RGD peptides (RGD) and designed peptide (R-pept) as bio-active molecules. The glass coverslips were coated with fibronectin, collagen, RGD peptide and R-peptide. Bone marrow mesenchymal stem cells were cultured on different substrates and the adhesion behavior in early incubation times was investigated using scanning electron microscopy (SEM) and confocal microscopy. The MTT assay was performed to evaluate the effect of different bio-active molecules on MSCs proliferation rate during 24 and 72 h. Formation of filopodia and focal adhesion (FA) complexes, two steps of cell adhesion process, were observed in MSCs cultured on bio-active molecules modified coverslips, specifically in Fn coated and R-pept coated groups. SEM image showed well adhesion pattern for MSCs cultured on Fn and R-pept after 2 h incubation, while the shape of cells cultured on Coll and RGD substrates indicated that they might experience stress condition in early hours of culture. Investigation of adhesion behavior, as well as proliferation pattern, suggests R-peptide as a promising bio-active molecule to be used for surface modification of substrate in supporting and inducing cell adhesion and proliferation.

  8. Immunolocalization of keratin-associated beta-proteins (beta-keratins) in pad lamellae of geckos suggest that glycine-cysteine-rich proteins contribute to their flexibility and adhesiveness.

    PubMed

    Alibardi, Lorenzo

    2013-03-01

    The epidermis of digital pads in geckos comprises superficial microornamentation from the oberhautchen layer that form long setae allowing these lizards to climb vertical surfaces. The beta-layer is reduced in pad lamellae but persists up to the apical free margin. Setae are made of different proteins including keratin-associated beta-proteins, formerly indicated as beta-keratins. In order to identify specific setal proteins the present ultrastructural study on geckos pad lamellae analyzes the immunolocalization of three beta-proteins previously found in the epidermis and adhesive setae of the green anolis. A protein rich in glycine but poor in cysteine (HgG5-like) is absent or masked in gecko pad lamellae. Another protein rich in glycine and cysteine (HgGC3-like) is weakly present in setae, oberhautchen and beta-layer. A glycine and cysteine medium rich beta-protein (HgGC10-like) is present in the lower part of the beta-layer but is absent in the oberhautchen, setae, and mesos layer. The latter two proteins may form intermolecular bonds that contribute to the flexibility of the corneous material sustaining the setae. The pliable alpha-layer present beneath the thin beta-layer and in the hinge region of the pad lamellae also contains HgGC10-like proteins. Based on the possibility that some HgGC3-like or other cys-rich beta-proteins are charged in the setae it is suggested that their charges influence the mechanism of adhesion increasing the induction of dipoles on the substrate and enhancing attractive van der Waals forces.

  9. The Src Homology 3 Domain Is Required for Junctional Adhesion Molecule Binding to the Third PDZ Domain of the Scaffolding Protein ZO-1

    SciTech Connect

    Nomme, Julian; Fanning, Alan S.; Caffrey, Michael; Lye, Ming F.; Anderson, James M.; Lavie, Arnon

    2012-01-20

    Tight junctions are cell-cell contacts that regulate the paracellular flux of solutes and prevent pathogen entry across cell layers. The assembly and permeability of this barrier are dependent on the zonula occludens (ZO) membrane-associated guanylate kinase (MAGUK) proteins ZO-1, -2, and -3. MAGUK proteins are characterized by a core motif of protein-binding domains that include a PDZ domain, a Src homology 3 (SH3) domain, and a region of homology to guanylate kinase (GUK); the structure of this core motif has never been determined for any MAGUK. To better understand how ZO proteins organize the assembly of protein complexes we have crystallized the entire PDZ3-SH3-GUK core motif of ZO-1. We have also crystallized this core motif in complex with the cytoplasmic tail of the ZO-1 PDZ3 ligand, junctional adhesion molecule A (JAM-A) to determine how the activity of different domains is coordinated. Our study shows a new feature for PDZ class II ligand binding that implicates the two highly conserved Phe{sup -2} and Ser{sup -3} residues of JAM. Our x-ray structures and NMR experiments also show for the first time a role for adjacent domains in the binding of ligands to PDZ domains in the MAGUK proteins family.

  10. Nectin/PRR: an immunoglobulin-like cell adhesion molecule recruited to cadherin-based adherens junctions through interaction with Afadin, a PDZ domain-containing protein.

    PubMed

    Takahashi, K; Nakanishi, H; Miyahara, M; Mandai, K; Satoh, K; Satoh, A; Nishioka, H; Aoki, J; Nomoto, A; Mizoguchi, A; Takai, Y

    1999-05-03

    We have isolated a novel actin filament-binding protein, named afadin, localized at cadherin-based cell-cell adherens junctions (AJs) in various tissues and cell lines. Afadin has one PDZ domain, three proline-rich regions, and one actin filament-binding domain. We found here that afadin directly interacted with a family of the immunoglobulin superfamily, which was isolated originally as the poliovirus receptor-related protein (PRR) family consisting of PRR1 and -2, and has been identified recently to be the alphaherpes virus receptor. PRR has a COOH-terminal consensus motif to which the PDZ domain of afadin binds. PRR and afadin were colocalized at cadherin-based cell-cell AJs in various tissues and cell lines. In E-cadherin-expressing EL cells, PRR was recruited to cadherin-based cell-cell AJs through interaction with afadin. PRR showed Ca2+-independent cell-cell adhesion activity. These results indicate that PRR is a cell-cell adhesion molecule of the immunoglobulin superfamily which is recruited to cadherin-based cell-cell AJs through interaction with afadin. We rename PRR as nectin (taken from the Latin word "necto" meaning "to connect").

  11. Protein Kinase C Phosphorylation of a γ-Protocadherin C-terminal Lipid Binding Domain Regulates Focal Adhesion Kinase Inhibition and Dendrite Arborization.

    PubMed

    Keeler, Austin B; Schreiner, Dietmar; Weiner, Joshua A

    2015-08-21

    The γ-protocadherins (γ-Pcdhs) are a family of 22 adhesion molecules with multiple critical developmental functions, including the proper formation of dendritic arbors by forebrain neurons. The γ-Pcdhs bind to and inhibit focal adhesion kinase (FAK) via a constant C-terminal cytoplasmic domain shared by all 22 proteins. In cortical neurons lacking the γ-Pcdhs, aberrantly high activity of FAK and of PKC disrupts dendrite arborization. Little is known, however, about how γ-Pcdh function is regulated by other factors. Here we show that PKC phosphorylates a serine residue situated within a phospholipid binding motif at the shared γ-Pcdh C terminus. Western blots using a novel phospho-specific antibody against this site suggest that a portion of γ-Pcdh proteins is phosphorylated in the cortex in vivo. We find that PKC phosphorylation disrupts both phospholipid binding and the γ-Pcdh inhibition of (but not binding to) FAK. Introduction of a non-phosphorylatable (S922A) γ-Pcdh construct into wild-type cortical neurons significantly increases dendrite arborization. This same S922A construct can also rescue dendrite arborization defects in γ-Pcdh null neurons cell autonomously. Consistent with these data, introduction of a phosphomimetic (S/D) γ-Pcdh construct or treatment with a PKC activator reduces dendrite arborization in wild-type cortical neurons. Together, these data identify a novel mechanism through which γ-Pcdh control of a signaling pathway important for dendrite arborization is regulated.

  12. Identification of the Ulex europaeus agglutinin-I-binding protein as a unique glycoform of the neural cell adhesion molecule in the olfactory sensory axons of adults rats.

    PubMed

    Pestean, A; Krizbai, I; Böttcher, H; Párducz, A; Joó, F; Wolff, J R

    1995-08-04

    Histochemical localization of two lectins, Ulex europaeus agglutinin-I (UEA-I) and Tetragonolobus purpureus (TPA), was studied in the olfactory bulb of adult rats. In contrast to TPA, UEA-I detected a fucosylated glycoprotein that is only present in the surface membranes of olfactory sensory cells including the whole course of their neurites up to the final arborization in glomeruli. Immunoblotting revealed that UEA-I binds specifically to a protein of 205 kDa, while TPA stains several other glycoproteins. Affinity chromatography with the use of a UEA-I column identified the 205 kDa protein as a glycoform of neural cell adhesion molecule (N-CAM), specific for the rat olfactory sensory nerves.

  13. Differential Protection of Cry1Fa Toxin against Spodoptera frugiperda Larval Gut Proteases by Cadherin Orthologs Correlates with Increased Synergism

    PubMed Central

    Rahman, Khalidur; Abdullah, Mohd Amir F.; Ambati, Suresh; Taylor, Milton D.

    2012-01-01

    The Cry proteins produced by Bacillus thuringiensis (Bt) are the most widely used biopesticides effective against a range of crop pests and disease vectors. Like chemical pesticides, development of resistance is the primary threat to the long-term efficacy of Bt toxins. Recently discovered cadherin-based Bt Cry synergists showed the potential to augment resistance management by improving efficacy of Cry toxins. However, the mode of action of Bt Cry synergists is thus far unclear. Here we elucidate the mechanism of cadherin-based Cry toxin synergism utilizing two cadherin peptides, Spodoptera frugiperda Cad (SfCad) and Manduca sexta Cad (MsCad), which differentially enhance Cry1Fa toxicity to Spodoptera frugiperda neonates. We show that differential SfCad- and MsCad-mediated protection of Cry1Fa toxin in the Spodoptera frugiperda midgut correlates with differential Cry1Fa toxicity enhancement. Both peptides exhibited high affinity for Cry1Fa toxin and an increased rate of Cry1Fa-induced pore formation in S. frugiperda. However, only SfCad bound the S. frugiperda brush border membrane vesicle and more effectively prolonged the stability of Cry1Fa toxin in the gut, explaining higher Cry1Fa enhancement by this peptide. This study shows that cadherin fragments may enhance B. thuringiensis toxicity by at least two different mechanisms or a combination thereof: (i) protection of Cry toxin from protease degradation in the insect midgut and (ii) enhancement of pore-forming ability of Cry toxin. PMID:22081566

  14. 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 20μg/ml) and 16% (for HS 10μg/ml); HBP Mf (35.2% for 10μg/ml and 25.4% for 20μg/ml) and HBP Ff (10.0% for 10μg/ml and 31.4% for 20μg/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

  15. Proteome profiling of the dimorphic fungus Penicillium marneffei extracellular proteins and identification of glyceraldehyde-3-phosphate dehydrogenase as an important adhesion factor for conidial attachment.

    PubMed

    Lau, Susanna K P; Tse, Herman; Chan, Joanna S Y; Zhou, Anna C; Curreem, Shirly O T; Lau, Candy C Y; Yuen, Kwok-Yung; Woo, Patrick C Y

    2013-12-01

    Despite being the most important thermal dimorphic fungus causing systemic mycosis in Southeast Asia, the pathogenic mechanisms of Penicillium marneffei remain largely unknown. By comparing the extracellular proteomes of P. marneffei in mycelial and yeast phases, we identified 12 differentially expressed proteins among which glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and heat shock protein 60 (HSP60) were found to be upregulated in mycelial and yeast phases respectively. Based on previous findings in other pathogens, we hypothesized that these two extracellular proteins may be involved in adherence during P. marneffei-host interaction. Using inhibition assays with recombinant GAPDH (rGAPDH) proteins and anti-rGAPDH sera, we demonstrated that adhesion of P. marneffei conidia to fibronectin and laminin was inhibited by rGAPDH or rabbit anti-rGAPDH serum in a dose-dependent manner. Similarly, a dose-dependent inhibition of conidial adherence to A549 pneumocytes by rGAPDH or rabbit anti-rGAPDH serum was observed, suggesting that P. marneffei GAPDH can mediate binding of conidia to human extracellular matrix proteins and pneumocytes. However, HSP60 did not exhibit similar inhibition on conidia adherence, and neither GAPDH norHSP60 exhibited inhibition on adherence to J774 or THP-1 macrophage cell lines. This report demonstrates GAPDH as an adherence factor in P. marneffei by mediating conidia adherence to host bronchoalveolar epithelium during the early establishment phase of infection.

  16. Ankyrin-binding proteins related to nervous system cell adhesion molecules: candidates to provide transmembrane and intercellular connections in adult brain

    PubMed Central

    1993-01-01

    A major class of ankyrin-binding glycoproteins have been identified in adult rat brain of 186, 155, and 140 kD that are alternatively spliced products of the same pre-mRNA. Characterization of cDNAs demonstrated that ankyrin-binding glycoproteins (ABGPs) share 72% amino acid sequence identity with chicken neurofascin, a membrane-spanning neural cell adhesion molecule in the Ig super-family expressed in embryonic brain. ABGP polypeptides have the following features consistent with a role as ankyrin-binding proteins in vitro and in vivo: (a) ABGPs and ankyrin associate as pure proteins in a 1:1 molar stoichiometry; (b) the ankyrin-binding site is located in the COOH-terminal 21 kD of ABGP186 which contains the predicted cytoplasmic domain; (c) ABGP186 is expressed at approximately the same levels as ankyrin (15 pmoles/milligram of membrane protein); and (d) ABGP polypeptides are co- expressed with the adult form of ankyrinB late in postnatal development and are colocalized with ankyrinB by immunofluorescence. Similarity in amino acid sequence and conservation of sites of alternative splicing indicate that genes encoding ABGPs and neurofascin share a common ancestor. However, the major differences in developmental expression reported for neurofascin in embryos versus the late postnatal expression of ABGPs suggest that ABGPs and neurofascin represent products of gene duplication events that have subsequently evolved in parallel with distinct roles. The predicted cytoplasmic domains of rat ABGPs and chicken neurofascin are nearly identical to each other and closely related to a group of nervous system cell adhesion molecules with variable extracellular domains, which includes L1, Nr-CAM, and Ng- CAM of vertebrates, and neuroglian of Drosophila. The ankyrin-binding site of rat ABGPs is localized to the C-terminal 200 residues which encompass the cytoplasmic domain, suggesting the hypothesis that ability to associate with ankyrin may be a shared feature of neurofascin and

  17. 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.

  18. The adaptor protein SAP directly associates with PECAM-1 and regulates PECAM-1-mediated-cell adhesion in T-like cell lines.

    PubMed

    Proust, Richard; Crouin, Catherine; Gandji, Leslie Yewakon; Bertoglio, Jacques; Gesbert, Franck

    2014-04-01

    SAP is a small cytosolic adaptor protein expressed in hematopoietic lineages whose main function is to regulate intracellular signaling pathways induced by the triggering of members of the SLAM receptor family. In this paper, we have identified the adhesion molecule PECAM-1 as a new partner for SAP in a conditional yeast two-hybrid screen. PECAM-1 is an immunoglobulin-like molecule expressed by endothelial cells and leukocytes, which possesses both pro- and anti-inflammatory properties. However, little is known about PECAM-1 functions in T cells. We show that SAP directly and specifically interacts with the cytosolic tyrosine 686 of PECAM-1. We generated different T-like cell lines in which SAP or PECAM-1 are expressed or down modulated and we demonstrate that a diminished SAP expression correlates with a diminished PECAM-1-mediated adhesion. Although SAP has mainly been shown to associate with SLAM receptors, we evidence here that SAP is a new actor downstream of PECAM-1.

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

    PubMed

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

    2013-01-01

    The Dictyostelium discoideum ampA gene encodes a multifunctional regulator protein that modulates cell-cell and cell-substrate 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 cell-cell 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.

  20. 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 μM) 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 μM Y-27632 and 10 μM Y-27632 retained their cell shape. At a concentration of 30 μM Y-27632, the cell shape became irregular. Cell adhesion ratios, in 1 μM Y-27632 (36.84%), 10 μM Y-27632 (84.21%) and 30 μM 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 μM or 30 μM 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 μM Y-27632 (P<0.01). Cell apoptosis was inhibited when 10 μM 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 μM 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

  1. Cryogenic/high temperature structural adhesives. [for space shuttles

    NASA Technical Reports Server (NTRS)

    Vaughan, R. W.; Sheppard, C. H.

    1974-01-01

    Results are described of the work performed to develop a structural adhesive system which possesses useful properties over a 20K (-423 F) to 589 K (600 F) temperature range. Adhesives systems based on polyimide, polyphenylquinoxaline polyquinoxaline, polybenzothiazole and polybenzimidazole polymers first were screened for suitability. Detailed evaluation of two polyimide adhesive sytems, Br34/FM34 and P4/A5F or P4A/A5FA, and one polyphenylquinoxaline adhesive system, PPQ II (IMW), then was performed. Property information was generated over the full temperature range for shear strength, stressed and unstressed thermal aging, thermal shock and coefficient of thermal expansion. Both polyimide adhesive systems were identified as being capable of providing structural adhesive joints for cryogenic/high temperature service.

  2. Hyaluronan-mediated cellular adhesion

    NASA Astrophysics Data System (ADS)

    Curtis, Jennifer

    2005-03-01

    Many cells surround themselves with a cushioning halo of polysaccharides that is further strengthened and organized by proteins. In fibroblasts and chrondrocytes, the primary component of this pericellular matrix is hyaluronan, a large linear polyanion. Hyaluronan production is linked to a variety of disease, developmental, and physiological processes. Cells manipulate the concentration of hyaluronan and hyaluronan receptors for numerous activities including modulation of cell adhesion, cell motility, and differentiation. Recent investigations by identify hyaluronan's role in mediating early-stage cell adhesion. An open question is how the cell removes the 0.5-10 micron thick pericellular matrix to allow for further mature adhesion events requiring nanometer scale separations. In this investigation, holographic optical tweezers are used to study the adhesion and viscoelastic properties of chondrocytes' pericellular matrix. Ultimately, we aim to shed further light on the spatial and temporal details of the dramatic transition from micron to nanometer gaps between the cell and its adhesive substrate.

  3. Wickerhamiella brachini f.a., sp. nov., Wickerhamiella pterostichi f.a., sp. nov. and Wickerhamiella qilinensis f.a., sp. nov., three yeast species isolated from insects.

    PubMed

    Liu, Xiao-Jing; Wang, Yun; Ren, Yong-Cheng; Hui, Feng-Li

    2016-10-01

    Eight strains representing three novel yeast species were isolated from insects distributed in three localities in Nanyang, Henan Province, Central China during 2014 and 2015. Sequence analysis of the D1/D2 domains of the large subunit (LSU) rRNA gene revealed that these species are members of the Wickerhamiella clade. These three novel species have a greater than 2.5 % difference from each other or their closest known species in the D1/D2 sequences. The three yeast species can also be separated from their closest known species in terms of physiological characteristics. Moreover, a sexual state could not be found in these three novel yeast species on various sporulation media. Therefore, the three novel species are described as Wickerhamiella brachini f.a., sp. nov. (type strain, NYNU 15885T=CICC 33092T=CBS 14176T), Wickerhamiellapterostichi f.a., sp. nov. (type strain, NYNU 15896T=CICC 33093T=CBS 14177T) and Wickerhamiellaqilinensis f.a., sp. nov. (type strain, NYNU 146103T=CICC 33062T=CBS 13929T). The MycoBank numbers of Wickerhamiella brachini f.a., sp. nov., Wickerhamiellapterostichi f.a., sp. nov. and Wickerhamiellaqilinensis f.a., sp. nov. are MB 816962, MB 816963 and MB 816964, respectively.

  4. Mutations in contactin-1, a neural adhesion and neuromuscular junction protein, cause a familial form of lethal congenital myopathy.

    PubMed

    Compton, Alison G; Albrecht, Douglas E; Seto, Jane T; Cooper, Sandra T; Ilkovski, Biljana; Jones, Kristi J; Challis, Daniel; Mowat, David; Ranscht, Barbara; Bahlo, Melanie; Froehner, Stanley C; North, Kathryn N

    2008-12-01

    We have previously reported a group of patients with congenital onset weakness associated with a deficiency of members of the syntrophin-alpha-dystrobrevin subcomplex and have demonstrated that loss of syntrophin and dystrobrevin from the sarcolemma of skeletal muscle can also be associated with denervation. Here, we have further studied four individuals from a consanguineous Egyptian family with a lethal congenital myopathy inherited in an autosomal-recessive fashion and characterized by a secondary loss of beta2-syntrophin and alpha-dystrobrevin from the muscle sarcolemma, central nervous system involvement, and fetal akinesia. We performed homozygosity mapping and candidate gene analysis and identified a mutation that segregates with disease within CNTN1, the gene encoding for the neural immunoglobulin family adhesion molecule, contactin-1. Contactin-1 transcripts were markedly decreased on gene-expression arrays of muscle from affected family members compared to controls. We demonstrate that contactin-1 is expressed at the neuromuscular junction (NMJ) in mice and man in addition to the previously documented expression in the central and peripheral nervous system. In patients with secondary dystroglycanopathies, we show that contactin-1 is abnormally localized to the sarcolemma instead of exclusively at the NMJ. The cntn1 null mouse presents with ataxia, progressive muscle weakness, and postnatal lethality, similar to the affected members in this family. We propose that loss of contactin-1 from the NMJ impairs communication or adhesion between nerve and muscle resulting in the severe myopathic phenotype. This disorder is part of the continuum in the clinical spectrum of congenital myopathies and congenital myasthenic syndromes.

  5. Clustering PPI data by combining FA and SHC method

    PubMed Central

    2015-01-01

    Clustering is one of main methods to identify functional modules from protein-protein interaction (PPI) data. Nevertheless traditional clustering methods may not be effective for clustering PPI data. In this paper, we proposed a novel method for clustering PPI data by combining firefly algorithm (FA) and synchronization-based hierarchical clustering (SHC) algorithm. Firstly, the PPI data are preprocessed via spectral clustering (SC) which transforms the high-dimensional similarity matrix into a low dimension matrix. Then the SHC algorithm is used to perform clustering. In SHC algorithm, hierarchical clustering is achieved by enlarging the neighborhood radius of synchronized objects continuously, while the hierarchical search is very difficult to find the optimal neighborhood radius of synchronization and the efficiency is not high. So we adopt the firefly algorithm to determine the optimal threshold of the neighborhood radius of synchronization automatically. The proposed algorithm is tested on the MIPS PPI dataset. The results show that our proposed algorithm is better than the traditional algorithms in precision, recall and f-measure value. PMID:25707632

  6. Clustering PPI data by combining FA and SHC method.

    PubMed

    Lei, Xiujuan; Ying, Chao; Wu, Fang-Xiang; Xu, Jin

    2015-01-01

    Clustering is one of main methods to identify functional modules from protein-protein interaction (PPI) data. Nevertheless traditional clustering methods may not be effective for clustering PPI data. In this paper, we proposed a novel method for clustering PPI data by combining firefly algorithm (FA) and synchronization-based hierarchical clustering (SHC) algorithm. Firstly, the PPI data are preprocessed via spectral clustering (SC) which transforms the high-dimensional similarity matrix into a low dimension matrix. Then the SHC algorithm is used to perform clustering. In SHC algorithm, hierarchical clustering is achieved by enlarging the neighborhood radius of synchronized objects continuously, while the hierarchical search is very difficult to find the optimal neighborhood radius of synchronization and the efficiency is not high. So we adopt the firefly algorithm to determine the optimal threshold of the neighborhood radius of synchronization automatically. The proposed algorithm is tested on the MIPS PPI dataset. The results show that our proposed algorithm is better than the traditional algorithms in precision, recall and f-measure value.

  7. Synthesis and SAR study of new thiazole derivatives as vascular adhesion protein-1 (VAP-1) inhibitors for the treatment of diabetic macular edema: part 2.

    PubMed

    Inoue, Takayuki; Morita, Masataka; Tojo, Takashi; Nagashima, Akira; Moritomo, Ayako; Imai, Keisuke; Miyake, Hiroshi

    2013-05-01

    Novel thiazole derivatives were synthesized and evaluated as vascular adhesion protein-1 (VAP-1) inhibitors. Although our previous compound 1 showed potent VAP-1 inhibitory activity, the activity differed between humans and rats. This issue was overcome by a hybrid design using human VAP-1 specific inhibitor 2, which was found by high-throughput screening (HTS), a docking study of a human VAP-1 homology model, and an analysis of sequence information for humans and rats. As a result, we identified compound 35c, which showed strong VAP-1 inhibitory activity (human IC(50) of 20 nM; rat IC(50) of 72 nM) and significant inhibitory effects in the ex vivo test.

  8. 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.

  9. 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 μM, rat IC50=0.0051 μM). 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.

  10. Integrated proteomics identified up-regulated focal adhesion-mediated proteins in human squamous cell carcinoma in an orthotopic murine model.

    PubMed

    Granato, Daniela C; Zanetti, Mariana R; Kawahara, Rebeca; Yokoo, Sami; Domingues, Romênia R; Aragão, Annelize Z; Agostini, Michelle; Carazzolle, Marcelo F; Vidal, Ramon O; Flores, Isadora L; Korvala, Johanna; Cervigne, Nilva K; Silva, Alan R S; Coletta, Ricardo D; Graner, Edgard; Sherman, Nicholas E; Paes Leme, Adriana F

    2014-01-01

    Understanding the molecular mechanisms of oral carcinogenesis will yield important advances in diagnostics, prognostics, effective treatment, and outcome of oral cancer. Hence, in this study we have investigated the proteomic and peptidomic profiles by combining an orthotopic murine model of oral squamous cell carcinoma (OSCC), mass spectrometry-based proteomics and biological network analysis. Our results indicated the up-regulation of proteins involved in actin cytoskeleton organization and cell-cell junction assembly events and their expression was validated in human OSCC tissues. In addition, the functional relevance of talin-1 in OSCC adhesion, migration and invasion was demonstrated. Taken together, this study identified specific processes deregulated in oral cancer and provided novel refined OSCC-targeting molecules.

  11. Integrated Proteomics Identified Up-Regulated Focal Adhesion-Mediated Proteins in Human Squamous Cell Carcinoma in an Orthotopic Murine Model

    PubMed Central

    Granato, Daniela C.; Zanetti, Mariana R.; Kawahara, Rebeca; Yokoo, Sami; Domingues, Romênia R.; Aragão, Annelize Z.; Agostini, Michelle; Carazzolle, Marcelo F.; Vidal, Ramon O.; Flores, Isadora L.; Korvala, Johanna; Cervigne, Nilva K.; Silva, Alan R. S.; Coletta, Ricardo D.; Graner, Edgard; Sherman, Nicholas E.; Leme, Adriana F. Paes

    2014-01-01

    Understanding the molecular mechanisms of oral carcinogenesis will yield important advances in diagnostics, prognostics, effective treatment, and outcome of oral cancer. Hence, in this study we have investigated the proteomic and peptidomic profiles by combining an orthotopic murine model of oral squamous cell carcinoma (OSCC), mass spectrometry-based proteomics and biological network analysis. Our results indicated the up-regulation of proteins involved in actin cytoskeleton organization and cell-cell junction assembly events and their expression was validated in human OSCC tissues. In addition, the functional relevance of talin-1 in OSCC adhesion, migration and invasion was demonstrated. Taken together, this study identified specific processes deregulated in oral cancer and provided novel refined OSCC-targeting molecules. PMID:24858105

  12. Amino Acid Sequences Mediating Vascular Cell Adhesion Molecule 1 Binding to Integrin Alpha 4: Homologous DSP Sequence Found for JC Polyoma VP1 Coat Protein

    PubMed Central

    Meyer, Michael Andrew

    2013-01-01

    The JC polyoma viral coat protein VP1 was analyzed for amino acid sequences homologies to the IDSP sequence which mediates binding of VLA-4 (integrin alpha 4) to vascular cell adhesion molecule 1. Although the full sequence was not found, a DSP sequence was located near the critical arginine residue linked to infectivity of the virus and binding to sialic acid containing molecules such as integrins (3). For the JC polyoma virus, a DSP sequence was found at residues 70, 71 and 72 with homology also noted for the mouse polyoma virus and SV40 virus. Three dimensional modeling of the VP1 molecule suggests that the DSP loop has an accessible site for interaction from the external side of the assembled viral capsid pentamer. PMID:24147211

  13. Polyporus squamosus Lectin 1a (PSL1a) Exhibits Cytotoxicity in Mammalian Cells by Disruption of Focal Adhesions, Inhibition of Protein Synthesis and Induction of Apoptosis

    PubMed Central

    Torgersen, Maria L.; Cordara, Gabriele; Künzler, Markus; Krengel, Ute; Sandvig, Kirsten

    2017-01-01

    PSL1a is a lectin from the mushroom Polyporus squamosus that binds to sialylated glycans and glycoconjugates with high specificity and selectivity. In addition to its N-terminal carbohydrate-binding domain, PSL1a possesses a Ca2+-dependent proteolytic activity in the C-terminal domain. In the present study, we demonstrate that PSL1a has cytotoxic effects on mammalian cancer cells, and we show that the cytotoxicity is dependent on the cysteine protease activity. PSL1a treatment leads to cell rounding and detachment from the substratum, concomitant with disruption of vinculin complexes in focal adhesions. We also demonstrate that PSL1a inhibits protein synthesis and induces apoptosis in HeLa cells, in a time- and concentration-dependent manner. PMID:28114329

  14. Novel hydrazine molecules as tools to understand the flexibility of vascular adhesion protein-1 ligand-binding site: toward more selective inhibitors.

    PubMed

    Nurminen, Elisa M; Pihlavisto, Marjo; Lázár, László; Pentikäinen, Ulla; Fülöp, Ferenc; Pentikäinen, Olli T

    2011-04-14

    Vascular adhesion protein-1 (VAP-1) belongs to a family of amine oxidases. It plays a role in leukocyte trafficking and in amine compound metabolism. VAP-1 is linked to various diseases, such as Alzheimer's disease, psoriasis, depression, diabetes, and obesity. Accordingly, selective inhibitors of VAP-1 could potentially be used to treat those diseases. In this study, eight novel VAP-1 hydrazine derivatives were synthesized and their VAP-1 and monoamine oxidase (MAO) inhibition ability was determined in vitro. MD simulations of VAP-1 with these new molecules reveal that the VAP-1 ligand-binding pocket is flexible and capable of fitting substantially larger ligands than was previously believed. The increase in the size of the VAP-1 ligands, together with the methylation of the secondary nitrogen atom of the hydrazine moiety, improves the VAP-1 selectivity over MAO.

  15. Amino Acid Sequences Mediating Vascular Cell Adhesion Molecule 1 Binding to Integrin Alpha 4: Homologous DSP Sequence Found for JC Polyoma VP1 Coat Protein.

    PubMed

    Meyer, Michael Andrew

    2013-01-01

    The JC polyoma viral coat protein VP1 was analyzed for amino acid sequences homologies to the IDSP sequence which mediates binding of VLA-4 (integrin alpha 4) to vascular cell adhesion molecule 1. Although the full sequence was not found, a DSP sequence was located near the critical arginine residue linked to infectivity of the virus and binding to sialic acid containing molecules such as integrins (3). For the JC polyoma virus, a DSP sequence was found at residues 70, 71 and 72 with homology also noted for the mouse polyoma virus and SV40 virus. Three dimensional modeling of the VP1 molecule suggests that the DSP loop has an accessible site for interaction from the external side of the assembled viral capsid pentamer.

  16. Natural Underwater Adhesives.

    PubMed

    Stewart, Russell J; Ransom, Todd C; Hlady, Vladimir

    2011-06-01

    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

  17. 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

  18. 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

  19. Two Distinct Actin Networks Mediate Traction Oscillations to Confer Focal Adhesion Mechanosensing.

    PubMed

    Wu, Zhanghan; Plotnikov, Sergey V; Moalim, Abdiwahab Y; Waterman, Clare M; Liu, Jian

    2017-02-28

    Focal adhesions (FAs) are integrin-based transmembrane assemblies that connect a cell to its extracellular matrix (ECM). They are mechanosensors through which cells exert actin cytoskeleton-mediated traction forces to sense the ECM stiffness. Interestingly, FAs themselves are dynamic structures that adapt their growth in response to mechanical force. It is unclear how the cell manages the plasticity of the FA structure and the associated traction force to accurately sense ECM stiffness. Strikingly, FA traction forces oscillate in time and space, and govern the cell mechanosensing of ECM stiffness. However, precisely how and why the FA traction oscillates is unknown. We developed a model of FA growth that integrates the contributions of the branched actin network and stress fibers (SFs). Using the model in combination with experimental tests, we show that the retrograde flux of the branched actin network promotes the proximal growth of the FA and contributes to a traction peak near the FA's distal tip. The resulting traction gradient within the growing FA favors SF formation near the FA's proximal end. The SF-mediated actomyosin contractility further stabilizes the FA and generates a second traction peak near the center of the FA. Formin-mediated SF elongation negatively feeds back with actomyosin contractility, resulting in central traction peak oscillation. This underpins the observed FA traction oscillation and, importantly, broadens the ECM stiffness range over which FAs can accurately adapt to traction force generation. Actin cytoskeleton-mediated FA growth and maturation thus culminate with FA traction oscillation to drive efficient FA mechanosensing.

  20. Mass spectrometry of the lithium adducts of diacylglycerols containing hydroxy FA in castor oil and two normal FA

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Castor oil can be used in industry. The molecular species of triacylglycerols containing hydroxy fatty acids (FA) in castor oil have been identified. We report here the identification of twelve diacylglycerols (DAG) containing hydroxy FA in castor oil using positive ion electrospray ionization mass ...

  1. 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

  2. Broadly distributed T cell reactivity, with no immunodominant loci, to the pre-erythrocytic antigen thrombospondin-related adhesive protein of Plasmodium falciparum in West Africans.

    PubMed

    Flanagan, K L; Plebanski, M; Akinwunmi, P; Lee, E A; Reece, W H; Robson, K J; Hill, A V; Pinder, M

    1999-06-01

    Protective immunity to malaria has been achieved in human volunteers utilizing the pre-erythrocytic Plasmodium falciparum antigen, the circumsporozoite protein (CS). However, T cell reactivity to CS is focused on several highly polymorphic T cell epitope regions, potentially limiting the efficacy of any vaccine to specific malaria strains. Another important pre-erythrocytic malaria antigen, the thrombospondin-related adhesive protein (TRAP), can induce protection in animal models of malaria, but knowledge of human T cell responses is limited to the identification of CD8 T cell epitopes, with no CD4 epitopes identified to date. This comprehensive study assessed reactivity to overlapping peptides spanning almost the whole of P. falciparum TRAP (PfTRAP), as well as peptides selected on the basis of HLA class II-binding motifs. A total of 50 naturally exposed Gambian adults were assessed to define 26 T cell epitopes in PfTRAP capable of inducing rapid IFN-gamma or IL-4 production, as assessed by enzyme-linked immunospot assays. In contrast to the CS protein, this reactivity was broadly distributed along the length of TRAP. Moreover, of the 26 epitopes identified, 10 were found to be conserved in West Africa.

  3. Cbf11 and Cbf12, the fission yeast CSL proteins, play opposing roles in cell adhesion and coordination of cell and nuclear division.

    PubMed

    Prevorovský, Martin; Grousl, Tomás; Stanurová, Jana; Rynes, Jan; Nellen, Wolfgang; Půta, Frantisek; Folk, Petr

    2009-05-01

    The CSL (CBF1/RBP-Jkappa/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+ and cbf12+, 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+ and cbf12+ 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 GTGA/GGAA in vitro. The deletion of cbf11+ 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.

  4. Marine Bioinspired Underwater Contact Adhesion.

    PubMed

    Clancy, Sean K; Sodano, Antonio; Cunningham, Dylan J; Huang, Sharon S; Zalicki, Piotr J; Shin, Seunghan; Ahn, B Kollbe

    2016-05-09

    Marine mussels and barnacles are sessile biofouling organisms that adhere to a number of surfaces in wet environments and maintain remarkably strong bonds. Previous synthetic approaches to mimic biological wet adhesive properties have focused mainly on the catechol moiety, present in mussel foot proteins (mfps), and especially rich in the interfacial mfps, for example, mfp-3 and -5, found at the interface between the mussel plaque and substrate. Barnacles, however, do not use Dopa for their wet adhesion, but are instead rich in noncatecholic aromatic residues. Due to this anomaly, we were intrigued to study the initial contact adhesion properties of copolymerized acrylate films containing the key functionalities of barnacle cement proteins and interfacial mfps, for example, aromatic (catecholic or noncatecholic), cationic, anionic, and nonpolar residues. The initial wet contact adhesion of the copolymers was measured using a probe tack testing apparatus with a flat-punch contact geometry. The wet contact adhesion of an optimized, bioinspired copolymer film was ∼15.0 N/cm(2) in deionized water and ∼9.0 N/cm(2) in artificial seawater, up to 150 times greater than commercial pressure-sensitive adhesive (PSA) tapes (∼0.1 N/cm(2)). Furthermore, maximum wet contact adhesion was obtained at ∼pH 7, suggesting viability for biomedical applications.

  5. 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.

  6. Biochemical features of the adhesion G protein-coupled receptor CD97 related to its auto-proteolysis and HeLa cell attachment activities.

    PubMed

    Yang, Li-Yun; Liu, Xiao-Fang; Yang, Yang; Yang, Lin-Lin; Liu, Kai-Wen; Tang, Yu-Bo; Zhang, Min; Tan, Min-Jia; Cheng, Shan-Mei; Xu, Ye-Chun; Yang, Huai-Yu; Liu, Zhi-Jie; Song, Gao-Jie; Huang, Wei

    2017-01-01

    CD97 belongs to the adhesion GPCR family characterized by a long ECD linked to the 7TM via a GPCR proteolytic site (GPS) and plays important roles in modulating cell migration and invasion. CD97 (EGF1-5) is a splicing variant of CD97 that recognizes a specific ligand chondroitin sulfate on cell membranes and the extracellular matrix. The aim of this study was to elucidate the extracellular molecular basis of the CD97 EGF1-5 isoform in protein expression, auto-proteolysis and cell adhesion, including epidermal growth factor (EGF)-like domain, GPCR autoproteolysis-inducing (GAIN) domain, as well as GPS mutagenesis and N-glycosylation. Both wild-type (WT) CD97-ECD and its truncated, GPS mutated, PNGase F-deglycosylated, and N-glycosylation site mutated forms were expressed and purified. The auto-proteolysis of the proteins was analyzed with Western blotting and SDS-PAGE. Small angle X-ray scattering (SAXS) and molecular modeling were used to determine a structural profile of the properly expressed receptor. Potential N-glycosylation sites were identified using MS and were modulated with PNGase F digestion and glyco-site mutations. A flow cytometry-based HeLa cell attachment assay was used for all aforementioned CD97 variants to elucidate the molecular basis of CD97-HeLa interactions. A unique concentration-dependent GPS auto-proteolysis was observed in CD97 EGF1-5 isoform with the highest concentration (4 mg/mL) per sample was self-cleaved much faster than the lower concentration (0.1 mg/mL), supporting an intermolecular mechanism of auto-proteolysis that is distinct to the reported intramolecular mechanism for other CD97 isoforms. N-glycosylation affected the auto-proteolysis of CD97 EGF1-5 isoform in a similar way as the other previously reported CD97 isoforms. SAXS data for WT and deglycosylated CD97ECD revealed a spatula-like shape with GAIN and EGF domains constituting the body and handle, respectively. Structural modeling indicated a potential interaction

  7. Vascular remodeling alters adhesion protein and cytoskeleton reactions to inflammatory stimuli resulting in enhanced permeability increases in rat venules.

    PubMed

    Yuan, Dong; He, Pingnian

    2012-10-01

    Vascular remodeling has been implicated in many inflammation-involved diseases. This study aims to investigate the microvascular remodeling-associated alterations in cell-cell adhesion and cytoskeleton reactions to inflammatory stimuli and their impact on microvessel permeability. Experiments were conducted in individually perfused rat mesenteric venules. Microvessel permeability was determined by measuring hydraulic conductivity (Lp), and endothelial intracellular calcium concentration, [Ca(2+)](i), was measured in fura-2-perfused vessels. Alterations in VE-cadherin and F-actin arrangement were examined by confocal imaging. Vascular wall cellular composition and structural changes were evaluated by electron microscopy. Vessels exposed to platelet activating factor (PAF) on day 1 were reevaluated 3 days later in rats that had undergone survival surgery. Initial PAF exposure and surgical disturbance increased microvascular wall thickness along with perivascular cell proliferation and altered F-actin arrangement. Although basal permeability was not changed, upon reexposure to PAF, peak endothelial [Ca(2+)](i) was augmented and the peak Lp was 9.3 ± 1.7 times higher than that of day 1. In contrast to patterns of PAF-induced stress fiber formation and VE-cadherin redistribution observed in day 1 vessels, the day 4 vessels at the potentiated Lp peak exhibited wide separations of VE-cadherin between endothelial cells and striking stress fibers throughout the vascular walls. Confocal images and ultrastructural micrographs also revealed that the largely separated VE-cadherin and endothelial gaps were completely covered by F-actin bundles in extended pericyte processes at the PAF-induced Lp peak. These results indicate that inflammation-induced vascular remodeling increased endothelial susceptibility to inflammatory stimuli with augmented Ca(2+) response resulting in upregulated contractility and potentiated permeability increase. Weakened adhesions between the endothelial

  8. Adhesive plasters

    DOEpatents

    Holcombe, Jr., Cressie E.; Swain, Ronald L.; Banker, John G.; Edwards, Charlene C.

    1978-01-01

    Adhesive plaster compositions are provided by treating particles of Y.sub.2 O.sub.3, Eu.sub.2 O.sub.3, Gd.sub.2 O.sub.3 or Nd.sub.2 O.sub.3 with dilute acid solutions. The resulting compositions have been found to spontaneously harden into rigid reticulated masses resembling plaster of Paris. Upon heating, the hardened material is decomposed into the oxide, yet retains the reticulated rigid structure.

  9. Monoubiquitination-dependent chromatin loading of FancD2 in silkworms, a species lacking the FA core complex.

    PubMed

    Sugahara, Ryohei; Mon, Hiroaki; Lee, Jae Man; Kusakabe, Takahiro

    2012-06-15

    The Fanconi anemia (FA) pathway is required for activation and operation of the DNA interstrand cross-link (ICL) repair pathway, although the precise mechanism of the FA pathway remains largely unknown. A critical step in the FA pathway is the monoubiquitination of FANCD2 catalyzed by a FA core complex. This modification appears to allow FANCD2 to coordinate ICL repair with other DNA repair proteins on chromatin. Silkworm, Bombyx mori, lacks apparent homologues of the FA core complex. However, BmFancD2 and BmFancI, the putative substrates of the complex, and BmFancL, the putative catalytic E3 ubiquitin ligase, are conserved. Here, we report that the silkworm FancD2 is monoubiquitinated depending on FancI and FancL, and stabilized on chromatin, following MMC treatment. A substitution of BmFancD2 at lysine 519 to arginine abolishes the monoubiquitination, but not the interaction between the FancD2 and FancI. In addition, we demonstrated that depletion of BmFancD2, BmFancI or BmFancL had effects on cell proliferation in the presence of MMC. These results suggest that the FA pathway in B. mori works in the same manner as that in vertebrates.

  10. Differential Role of β1C and β1A Integrin Cytoplasmic Variants in Modulating Focal Adhesion Kinase, Protein Kinase B/AKT, and Ras/Mitogen-activated Protein Kinase Pathways

    PubMed Central

    Fornaro, Mara; Steger, Craig A.; Bennett, Anton M.; Wu, J. Julie; Languino, Lucia R.

    2000-01-01

    The integrin cytoplasmic domain modulates cell proliferation, adhesion, migration, and intracellular signaling. The β1 integrin subunits, β1C and β1A, that contain variant cytoplasmic domains differentially affect cell proliferation; β1C inhibits proliferation, whereas β1A promotes it. We investigated the ability of β1C and β1A to modulate integrin-mediated signaling events that affect cell proliferation and survival in Chinese hamster ovary stable cell lines expressing either human β1C or human β1A. The different cytodomains of either β1C or β1A did not affect either association with the endogenous α2, αV, and α5 subunits or cell adhesion to fibronectin or TS2/16, a mAb to human β1. Upon engagement of endogenous and exogenous integrins by fibronectin, cells expressing β1C showed significantly inhibited extracellular signal–regulated kinase (ERK) 2 activation compared with β1A stable cell lines. In contrast, focal adhesion kinase phosphorylation and Protein Kinase B/AKT activity were not affected. Selective engagement of the exogenously expressed β1C by TS2/16 led to stimulation of Protein Kinase B/AKT phosphorylation but not of ERK2 activation; in contrast, β1A engagement induced activation of both proteins. We show that Ras activation was strongly reduced in β1C stable cell lines in response to fibronectin adhesion and that expression of constitutively active Ras, Ras 61 (L), rescued β1C-mediated down-regulation of ERK2 activation. Inhibition of cell proliferation in β1C stable cell lines was attributable to an inhibitory effect of β1C on the Ras/MAP kinase pathway because expression of activated MAPK kinase rescued β1C antiproliferative effect. These findings show that the β1C variant, by means of a unique signaling mechanism, selectively inhibits the MAP kinase pathway by preventing Ras activation without affecting either survival signals stimulated by integrins or cellular interactions with the extracellular matrix. These findings

  11. Exploring natural silk protein sericin for regenerative medicine: an injectable, photoluminescent, cell-adhesive 3D hydrogel

    NASA Astrophysics Data System (ADS)

    Wang, Zheng; Zhang, Yeshun; Zhang, Jinxiang; Huang, Lei; Liu, Jia; Li, Yongkui; Zhang, Guozheng; Kundu, Subhas C.; Wang, Lin

    2014-11-01

    Sericin, a major component of silk, has a long history of being discarded as a waste during silk processing. The value of sericin for tissue engineering is underestimated and its potential application in regenerative medicine has just begun to be explored. Here we report the successful fabrication and characterization of a covalently-crosslinked 3D pure sericin hydrogel for delivery of cells and drugs. This hydrogel is injectable, permitting its implantation through minimally invasive approaches. Notably, this hydrogel is found to exhibit photoluminescence, enabling bioimaging and in vivo tracking. Moreover, this hydrogel system possesses excellent cell-adhesive capability, effectively promoting cell attachment, proliferation and long-term survival of various types of cells. Further, the sericin hydrogel releases bioactive reagents in a sustained manner. Additionally, this hydrogel demonstrates good elasticity, high porosity, and pH-dependent degradation dynamics, which are advantageous for this sericin hydrogel to serve as a delivery vehicle for cells and therapeutic drugs. With all these unique features, it is expected that this sericin hydrogel will have wide utility in the areas of tissue engineering and regenerative medicine.

  12. Exploring natural silk protein sericin for regenerative medicine: an injectable, photoluminescent, cell-adhesive 3D hydrogel.

    PubMed

    Wang, Zheng; Zhang, Yeshun; Zhang, Jinxiang; Huang, Lei; Liu, Jia; Li, Yongkui; Zhang, Guozheng; Kundu, Subhas C; Wang, Lin

    2014-11-20

    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.

  13. 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 the LIM domains. Consistent with this notion, PLM synaptogenesis was independent of α-actinin and ENA-VASP, both of which bind to the N-terminal domain of zyxin. Our results demonstrate that the LIM domain moiety of zyxin functions autonomously to mediate responses to mechanical stress and provide in vivo evidence for a role of zyxin in neuronal development. PMID:25252943

  14. The conserved LIM domain-containing focal adhesion protein ZYX-1 regulates synapse maintenance in Caenorhabditis elegans.

    PubMed

    Luo, Shuo; Schaefer, Anneliese M; Dour, Scott; Nonet, Michael L

    2014-10-01

    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 the LIM domains. Consistent with this notion, PLM synaptogenesis was independent of α-actinin and ENA-VASP, both of which bind to the N-terminal domain of zyxin. Our results demonstrate that the LIM domain moiety of zyxin functions autonomously to mediate responses to mechanical stress and provide in vivo evidence for a role of zyxin in neuronal development.

  15. Liver oxidation and inflammation in Fa/Fa rats fed glucomannan/spirulina-surimi.

    PubMed

    Vázquez-Velasco, Miguel; González-Torres, Laura; López-Gasco, Patricia; Bastida, Sara; Benedí, Juana; Sánchez-Reus, María Isabel; González-Muñoz, María José; Sánchez-Muniz, Francisco J

    2014-09-15

    The effect of high-fat squid-surimi diets enriched in glucomannan or glucomannan-spirulina on lipemia, liver glutathione status, antioxidant enzymes and inflammation biomarkers was determined in Zucker Fa/Fa rats. Groups of eight rats each received for 7weeks the squid-surimi control (C), glucomannan-enriched squid-surimi (G) and glucomannan-spirulina enriched squid-surimi (GS). Liver weight, cytochrome P450 7A1 expression and cholesterolemia were decreased in G and GS vs. C, improving glutathione red-ox index (p<0.05). G also showed increased glutathione reductase (GR) levels vs. C, but reduced the endothelial (eNOS) and increased the inducible nitric oxide synthase (iNOS) and tumor necrosis factor-alpha (TNF-α) levels (p<0.05). The GS diet improved superoxide dismutase, catalase, glutathione peroxidase and GR activities and eNOS, iNOS and TNF-α levels (p<0.05). The glucomannan enriched surimi-diet induced hypocholesterolemic, antioxidant and proinflammatory effects, while the addition of 3g/kg spirulina kept those hypocholesterolemic and antioxidant effects but reduced the inflammation observed.

  16. Epigenetics and development of food allergy (FA) in early childhood.

    PubMed

    Hong, Xiumei; Wang, Xiaobin

    2014-09-01

    This review aims to highlight the latest advance on epigenetics in the development of food allergy (FA) and to offer future perspectives. FA, a condition caused by an immunoglobulin (Ig) E-mediated hypersensitivity reaction to food, has emerged as a major clinical and public health problem worldwide in light of its increasing prevalence, potential fatality, and significant medical and economic impact. Current evidence supports that epigenetic mechanisms are involved in immune regulation and that the epigenome may represent a key "missing piece" of the etiological puzzle for FA. There are a growing number of population-based epigenetic studies on allergy-related phenotypes, mostly focused on DNA methylation. Previous studies mostly applied candidate-gene approaches and have demonstrated that epigenetic marks are associated with multiple allergic diseases and/or with early-life exposures relevant to allergy development (such as early-life smoking exposure, air pollution, farming environment, and dietary fat). Rapid technological advancements have made unbiased genome-wide DNA methylation studies highly feasible, although there are substantial challenge in study design, data analyses, and interpretation of findings. In conclusion, epigenetics represents both an important knowledge gap and a promising research area for FA. Due to the early onset of FA, epigenetic studies of FA in prospective birth cohorts have the potential to better understand gene-environment interactions and underlying biological mechanisms in FA during critical developmental windows (preconception, in utero, and early childhood) and may lead to new paradigms in the diagnosis, prevention, and management of FA and provide novel targets for future drug discovery and therapies for FA.

  17. Decipher the dynamic coordination between enzymatic activity and structural modulation at focal adhesions in living cells

    NASA Astrophysics Data System (ADS)

    Lu, Shaoying; Seong, Jihye; Wang, Yi; Chang, Shiou-Chi; Eichorst, John Paul; Ouyang, Mingxing; Li, Julie Y.-S.; Chien, Shu; Wang, Yingxiao

    2014-07-01

    Focal adhesions (FAs) are dynamic subcellular structures crucial for cell adhesion, migration and differentiation. It remains an enigma how enzymatic activities in these local complexes regulate their structural remodeling in live cells. Utilizing biosensors based on fluorescence resonance energy transfer (FRET), we developed a correlative FRET imaging microscopy (CFIM) approach to quantitatively analyze the subcellular coordination between the enzymatic Src activation and the structural FA disassembly. CFIM reveals that the Src kinase activity only within the microdomain of lipid rafts at the plasma membrane is coupled with FA dynamics. FA disassembly at cell periphery was linearly dependent on this raft-localized Src activity, although cells displayed heterogeneous levels of response to stimulation. Within lipid rafts, the time delay between Src activation and FA disassembly was 1.2 min in cells seeded on low fibronectin concentration ([FN]) and 4.3 min in cells on high [FN]. CFIM further showed that the level of Src-FA coupling, as well as the time delay, was regulated by cell-matrix interactions, as a tight enzyme-structure coupling occurred in FA populations mediated by integrin αvβ3, but not in those by integrin α5β1. Therefore, different FA subpopulations have distinctive regulation mechanisms between their local kinase activity and structural FA dynamics.

  18. Study of the degradation activity and the strategies to promote the bioavailability of phenanthrene by Sphingomonas paucimobilis strain 20006FA.

    PubMed

    Coppotelli, Bibiana M; Ibarrolaza, Agustin; Dias, Romina L; Del Panno, Maria T; Berthe-Corti, Luise; Morelli, Irma S

    2010-02-01

    The present study describes the phenanthrene-degrading activity of Sphingomonas paucimobilis 20006FA and its ability to promote the bioavailability of phenanthrene. S. paucimobilis 20006FA was isolated from a phenanthrene-contaminated soil microcosm. The strain was able to grow in liquid mineral medium saturated with phenanthrene as the sole carbon source, showing high phenanthrene elimination (52.9% of the supplied phenanthrene within 20 days). The accumulation of 1-hydroxy-2-naphthoic acid and salicylic acid as major phenanthrene metabolites and the capacity of the strain to grow with sodium salicylate as the sole source of carbon and energy indicated that the S. paucimobilis 20006FA possesses a complete phenanthrene degradation pathway. However, under the studied conditions, the strain was able to mineralize only the 10% of the consumed phenanthrene. Investigations on the cell ability to promote bioavailability of phenanthrene showed that the S. paucimobilis strain 20006FA exhibited low cell hydrophobicity (0.13), a pronounced chemotaxis toward phenanthrene, and it was able to reduce the surface tension of mineral liquid medium supplemented with phenanthrene as sole carbon source. Scanning electron micrographs revealed that: (1) in suspension cultures, cells formed flocks and showed small vesicles on the cell surface and (2) cells were also able to adhere to phenanthrene crystals and to produce biofilms. Clearly, the strain seems to exhibit two different mechanisms to enhance phenanthrene bioavailability: biosurfactant production and adhesion to the phenanthrene crystals.

  19. Arginine-glycine-aspartic acid- and fibrinogen gamma-chain carboxyterminal peptides inhibit platelet adherence to arterial subendothelium at high wall shear rates. An effect dissociable from interference with adhesive protein binding.

    PubMed Central

    Lawrence, J B; Kramer, W S; McKeown, L P; Williams, S B; Gralnick, H R

    1990-01-01

    Arg-Gly-Asp (RGD)- and fibrinogen gamma-chain carboxyterminal (GQQHHLGGAKQAGDV) peptides inhibit fibrinogen, fibronectin (Fn), vitronectin, and von Willebrand factor (vWF) binding to the platelet glycoprotein IIb-IIIa complex (GP IIb-IIIa). GP IIb-IIIa, vWF, and Fn are essential for normal platelet adherence to subendothelium. We added peptides to normal citrated whole blood before perfusion over human umbilical artery subendothelium and evaluated platelet adherence morphometrically at high (2,600 s-1) and low (800 s-1) wall shear rates. We also examined the effects of the peptides on platelet adhesion to collagen in a static system. At the high wall shear rate, RGDS and GQQHHLGGAKQAGDV caused dose-dependent reduction in the surface coverage with spread and adherent platelets. Amino acid transposition and conservative substitutions of RGD peptides and the AGDV peptide significantly inhibited platelet adherence at 2,600 s-1. By contrast, the modified RGD peptides and AGDV do not affect adhesive protein binding to platelets. None of the native or modified RGD- or fibrinogen gamma-chain peptides significantly inhibited either platelet adherence to subendothelium at 800 s-1 or platelet adhesion to collagen. Our findings demonstrate that peptides that interfere with adhesive protein binding to GP IIb-IIIa inhibit platelet adherence to vascular subendothelium with flowing blood only at high wall shear rates. Platelet adherence to subendothelium at high wall shear rates appears to be mediated by different recognition specificities from those required for fluid-phase adhesive protein binding or static platelet adhesion. PMID:2243140

  20. Genetic evidence for the adhesion protein IgSF9/Dasm1 to regulate inhibitory synapse development independent of its intracellular domain.

    PubMed

    Mishra, Archana; Traut, Matthias H; Becker, Lore; Klopstock, Thomas; Stein, Valentin; Klein, Rüdiger

    2014-03-19

    Normal brain function requires balanced development of excitatory and inhibitory synapses. An imbalance in synaptic transmission underlies many brain disorders such as epilepsy, schizophrenia, and autism. Compared with excitatory synapses, relatively little is known about the molecular control of inhibitory synapse development. We used a genetic approach in mice to identify the Ig superfamily member IgSF9/Dasm1 as a candidate homophilic synaptic adhesion protein that regulates inhibitory synapse development. IgSF9 is expressed in pyramidal cells and subsets of interneurons in the CA1 region of hippocampus. Electrophysiological recordings of acute hippocampal slices revealed that genetic inactivation of the IgSF9 gene resulted in fewer functional inhibitory synapses; however, the strength of the remaining synapses was unaltered. These physiological abnormalities were correlated with decreased expression of inhibitory synapse markers in IgSF9(-/-) mice, providing anatomical evidence for a reduction in inhibitory synapse numbers, whereas excitatory synapse development was normal. Surprisingly, knock-in mice expressing a mutant isoform of IgSF9 lacking the entire cytoplasmic domain (IgSF9(ΔC/ΔC) mice) had no defects in inhibitory synapse development, providing genetic evidence that IgSF9 regulates synapse development via ectodomain interactions rather than acting itself as a signaling receptor. Further, we found that IgSF9 mediated homotypic binding and cell aggregation, but failed to induce synapse formation, suggesting that IgSF9 acts as a cell adhesion molecule (CAM) to maintain synapses. Juvenile IgSF9(-/-) mice exhibited increased seizure susceptibility indicative of an imbalance in synaptic excitation and inhibition. These results provide genetic evidence for a specific role of IgSF9 in inhibitory synapse development/maintenance, presumably by its CAM-like activity.

  1. Focal adhesion kinase and mitogen-activated protein kinases are involved in chondrocyte activation by the 29-kDa amino-terminal fibronectin fragment.

    PubMed

    Gemba, Takefumi; Valbracht, Jean; Alsalameh, Saifeddin; Lotz, Martin

    2002-01-11

    The 29-kDa amino-terminal fibronectin fragment (FN-f) has a potent chondrolytic effect and is thought to be involved in cartilage degradation in arthritis. However, little is known about signal transduction pathways that are activated by FN-f. Here we demonstrated that FN-f induced nitric oxide (NO) production from human articular chondrocytes. Expression of inducible nitric-oxide synthase (iNOS) mRNA and NO production were observed at 6 and 48 h after FN-f treatment, respectively. Interleukin-1beta (IL-1beta) mRNA up-regulation was stimulated by FN-f in human chondrocytes. To address the possibility that FN-f-induced NO release is mediated by IL-1beta production, the effect of IL-1 receptor antagonist (IL-1ra) was determined. IL-1ra partially inhibited FN-f-induced NO release although it almost completely inhibited IL-1beta-induced NO release. Tyrosine phosphorylation of focal adhesion kinase was induced transiently by FN-f treatment. Blocking antibodies to alpha(5) or beta(1) integrin and Arg-Gly-Asp-containing peptides did not inhibit FN-f-induced NO production. PP2, a Src family kinase inhibitor, or cytochalasin D, which selectively disrupts the network of actin filaments, inhibited both FAK phosphorylation and NO production induced by FN-f, but the phosphatidylinositol 3-kinase inhibitor wortmannin had no effect. Analysis of mitogen-activated protein kinases (MAPK) showed activation of extracellular signal-regulated kinase (ERK), c-Jun NH(2)-terminal kinase, and p38 MAPK. High concentrations of SB203580, which inhibit both JNK and p38 MAPK, and PD98059 a selective inhibitor of MEK1/2 that blocks ERK activation, inhibited FN-f induced NO production. These data suggest that focal adhesion kinase and MAPK mediate FN-f induced activation of human articular chondrocytes.

  2. Field-Evolved Mode 1 Resistance of the Fall Armyworm to Transgenic Cry1Fa-Expressing Corn Associated with Reduced Cry1Fa Toxin Binding and Midgut Alkaline Phosphatase Expression

    PubMed Central

    Jakka, Siva R. K.; Gong, Liang; Hasler, James; Banerjee, Rahul; Sheets, Joel J.; Narva, Kenneth; Blanco, Carlos A.

    2015-01-01

    Insecticidal protein genes from the bacterium Bacillus thuringiensis (Bt) are expressed by transgenic Bt crops (Bt crops) for effective and environmentally safe pest control. The development of resistance to these insecticidal proteins is considered the most serious threat to the sustainability of Bt crops. Resistance in fall armyworm (Spodoptera frugiperda) populations from Puerto Rico to transgenic corn producing the Cry1Fa insecticidal protein resulted, for the first time in the United States, in practical resistance, and Bt corn was withdrawn from the local market. In this study, we used a field-collected Cry1Fa corn-resistant strain (456) of S. frugiperda to identify the mechanism responsible for field-evolved resistance. Binding assays detected reduced Cry1Fa, Cry1Ab, and Cry1Ac but not Cry1Ca toxin binding to midgut brush border membrane vesicles (BBMV) from the larvae of strain 456 compared to that from the larvae of a susceptible (Ben) strain. This binding phenotype is descriptive of the mode 1 type of resistance to Bt toxins. A comparison of the transcript levels for putative Cry1 toxin receptor genes identified a significant downregulation (>90%) of a membrane-bound alkaline phosphatase (ALP), which translated to reduced ALP protein levels and a 75% reduction in ALP activity in BBMV from 456 compared to that of Ben larvae. We cloned and heterologously expressed this ALP from susceptible S. frugiperda larvae and demonstrated that it specifically binds with Cry1Fa toxin. This study provides a thorough mechanistic description of field-evolved resistance to a transgenic Bt crop and supports an association between resistance and reduced Cry1Fa toxin binding and levels of a putative Cry1Fa toxin receptor, ALP, in the midguts of S. frugiperda larvae. PMID:26637593

  3. Osteopontin inhibits osteoblast responsiveness through the downregulation of focal adhesion kinase mediated by the induction of low molecular weight-protein tyrosine phosphatase.

    PubMed

    Kusuyama, Joji; Bandow, Kenjiro; Ohnishi, Tomokazu; Hisadome, Mitsuhiro; Shima, Kaori; Semba, Ichiro; Matsuguchi, Tetsuya

    2017-03-22

    Osteopontin (OPN) is an osteogenic marker protein. Osteoblast functions are affected by inflammatory cytokines and pathological conditions. OPN is highly expressed in bone legions such as rheumatoid arthritis. However, local regulatory effects of OPN on osteoblasts remain ambiguous. Here, we examined how OPN influences osteoblast responses to mechanical stress and growth factors. Expression of NO synthase 1 (Nos1) and Nos2 was increased by low intensity pulsed ultrasound (LIPUS) in MC3T3-E1 cells and primary osteoblasts. The increase of Nos1/2 expression was abrogated by both exogenous OPN overexpression and recombinant OPN treatment, whereas it was promoted by OPN-specific siRNA and OPN antibody. Moreover, LIPUS-induced phosphorylation of focal adhesion kinase (FAK), a crucial regulator of mechano-responses, was downregulated by OPN treatments. OPN also attenuated hepatocyte growth factor (HGF)-induced vitamin D receptor (Vdr) expression and platelet-derived growth factor (PDGF)-induced cell mobility through the repression of FAK activity. Notably, the expression of low molecular-weight protein tyrosine phosphatase (LMW-PTP), a FAK phosphatase, was increased in both OPN-treated and differentiated osteoblasts. CD44 was a specific OPN receptor for LWW-PTP induction. Consistently, the suppressive influence of OPN on osteoblast responsiveness was abrogated by LMW-PTP knockdown. Taken together, these results have revealed novel functions of OPN on osteoblast physiology.

  4. Heterologous expression of newly identified galectin-8 from sea urchin embryos produces recombinant protein with lactose binding specificity and anti-adhesive activity

    PubMed Central

    Karakostis, Kostantinos; Costa, Caterina; Zito, Francesca; Matranga, Valeria

    2015-01-01

    Galectin family members specifically bind beta-galactoside derivatives and are involved in different cellular events, including cell communication, signalling, apoptosis, and immune responses. Here, we report a tandem-repeat type galectin from the Paracentrotus lividus sea urchin embryo, referred to as Pl-GAL-8. The 933nt sequence encodes a protein of 34.73 kDa, containing the conserved HFNPRF and WGxExR motifs in the two highly similar carbohydrate-recognition domains (CRD). The three-dimensional protein structure model of the N-CRD confirms the high evolutionary conservation of carbohydrate binding sites. The temporal gene expression is regulated during development and transcripts localize at the tip of the archenteron at gastrula stage, in a subset of the secondary mesenchyme cells that differentiate into blastocoelar (immune) cells. Functional studies using a recombinant Pl-GAL-8 expressed in bacteria demonstrate its hemo-agglutinating activity on human red blood cells through the binding to lactose, as well as its ability in inhibiting the adhesion of human Hep-G2 cells to the substrate. The recent implications in autoimmune diseases and inflammatory disorders make Gal-8 an attractive candidate for therapeutic purposes. Our results offer a solid basis for addressing the use of the new Pl-GAL-8 in functional and applicative studies, respectively in the developmental and biomedical fields. PMID:26640155

  5. The rhizobial adhesion protein RapA1 is involved in adsorption of rhizobia to plant roots but not in nodulation.

    PubMed

    Mongiardini, Elías J; Ausmees, Nora; Pérez-Giménez, Julieta; Julia Althabegoiti, María; Ignacio Quelas, Juan; López-García, Silvina L; Lodeiro, Aníbal R

    2008-08-01

    The effect of the rhizobium adhesion protein RapA1 on Rhizobium leguminosarum bv. trifolii adsorption to Trifolium pratense (red clover) roots was investigated. We altered RapA1 production by cloning its encoding gene under the plac promoter into the stable vector pHC60. After introducing this plasmid in R. leguminosarum bv. trifolii, three to four times more RapA1 was produced, and two to five times higher adsorption to red clover roots was obtained, as compared with results for the empty vector. Enhanced adsorption was also observed on soybean and alfalfa roots, not related to R. leguminosarum cross inoculation groups. Although the presence of 1 mM Ca2+ during rhizobial growth enhanced adsorption, it was unrelated to RapA1 level. Similar effects were obtained when the same plasmid was introduced in Rhizobium etli for its adsorption to bean roots. Although root colonization by the RapA1-overproducing strain was also higher, nodulation was not enhanced. In addition, in vitro biofilm formation was similar to the wild-type both on polar and on hydrophobic surfaces. These results suggest that RapA1 receptors are present in root but not on inert surfaces, and that the function of this protein is related to rhizosphere colonization.

  6. The intermediate filament protein vimentin binds specifically to a recombinant integrin {alpha}2/{beta}1 cytoplasmic tail complex and co-localizes with native {alpha}2/{beta}1 in endothelial cell focal adhesions

    SciTech Connect

    Kreis, Stephanie; Schoenfeld, Hans-Joachim; Melchior, Chantal; Steiner, Beat; Kieffer, Nelly . E-mail: kieffer@cu.lu

    2005-04-15

    Integrin receptors are crucial players in cell adhesion and migration. Identification and characterization of cellular proteins that interact with their short {alpha} and {beta} cytoplasmic tails will help to elucidate the molecular mechanisms by which integrins mediate bi-directional signaling across the plasma membrane. Integrin {alpha}2{beta}1 is a major collagen receptor but to date, only few proteins have been shown to interact with the {alpha}2 cytoplasmic tail or with the {alpha}2{beta}1 complex. In order to identify novel binding partners of a {alpha}2{beta}1cytoplasmic domain complex, we have generated recombinant GST-fusion proteins, incorporating the leucine zipper heterodimerization cassettes of Jun and Fos. To ascertain proper functionality of the recombinant proteins, interaction with natural binding partners was tested. GST-{alpha}2 and GST-Jun {alpha}2 bound His-tagged calreticulin while GST-{beta}1 and GST-Fos {beta}1 proteins bound talin. In screening assays for novel binding partners, the immobilized GST-Jun {alpha}2/GST-Fos {beta}1 heterodimeric complex, but not the single subunits, interacted specifically with endothelial cell-derived vimentin. Vimentin, an abundant intermediate filament protein, has previously been shown to co-localize with {alpha}v{beta}3-positive focal contacts. Here, we provide evidence that this interaction also occurs with {alpha}2{beta}1-enriched focal adhesions and we further show that this association is lost after prolonged adhesion of endothelial cells to collagen.

  7. Reduced Hepatic Carcinoembryonic Antigen-Related Cell Adhesion Molecule 1 Level in Obesity

    PubMed Central

    Heinrich, Garrett; Muturi, Harrison T.; Rezaei, Khadijeh; Al-Share, Qusai Y.; DeAngelis, Anthony M.; Bowman, Thomas A.; Ghadieh, Hilda E.; Ghanem, Simona S.; Zhang, Deqiang; Garofalo, Robert S.; Yin, Lei; Najjar, Sonia M.

    2017-01-01

    Impairment of insulin clearance is being increasingly recognized as a critical step in the development of insulin resistance and metabolic disease. The carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM1) promotes insulin clearance. Null deletion or liver-specific inactivation of Ceacam1 in mice causes a defect in insulin clearance, insulin resistance, steatohepatitis, and visceral obesity. Immunohistological analysis revealed reduction of hepatic CEACAM1 in obese subjects with fatty liver disease. Thus, we aimed to determine whether this occurs at the hepatocyte level in response to systemic extrahepatic factors and whether this holds across species. Northern and Western blot analyses demonstrate that CEACAM1 mRNA and protein levels are reduced in liver tissues of obese individuals compared to their lean age-matched counterparts. Furthermore, Western analysis reveals a comparable reduction of CEACAM1 protein in primary hepatocytes derived from the same obese subjects. Similar to humans, Ceacam1 mRNA level, assessed by quantitative RT-PCR analysis, is significantly reduced in the livers of obese Zucker (fa/fa, ZDF) and Koletsky (f/f) rats relative to their age-matched lean counterparts. These studies demonstrate that the reduction of hepatic CEACAM1 in obesity occurs at the level of hepatocytes and identify the reduction of hepatic CEACAM1 as a common denominator of obesity across multiple species.

  8. F/A-18 FAST Offers Advanced System Test Capability

    NASA Video Gallery

    NASA's Dryden Flight Research Center has modified an F/A-18A Hornet aircraft with additional research flight control computer systems for use as a Full-scale Advanced Systems Test Bed. Previously f...

  9. F/A-18 Automated Aerial Refueling (AAR) Phase 1

    NASA Video Gallery

    Engineers at NASA's Dryden Flight Research Center are evaluating the capability of an F/A-18A aircraft as an in-flight refueling tanker to develop analytical models for an automated aerial refuelin...

  10. NASA Researches the 'FaINT' Side of Sonic Booms

    NASA Video Gallery

    As the latest in a continuing progression of NASA supersonics research projects aimed at reducing or mitigating the effect of sonic booms, the Farfield Investigation of No Boom Threshold, or FaINT,...

  11. Total Risk Integrated Methodology (TRIM) - TRIM.FaTE

    EPA Pesticide Factsheets

    TRIM.FaTE is a spatially explicit, compartmental mass balance model that describes the movement and transformation of pollutants over time, through a user-defined, bounded system that includes both biotic and abiotic compartments.

  12. TRIM.FaTE Public Reference Library Documentation

    EPA Pesticide Factsheets

    TRIM.FaTE is a spatially explicit, compartmental mass balance model that describes the movement and transformation of pollutants over time, through a user-defined, bounded system that includes both biotic and abiotic compartments.

  13. Knockdown of μ-calpain in Fanconi Anemia, FA-A, cells by siRNA Restores αII Spectrin levels and Corrects Chromosomal Instability and Defective DNA Interstrand Cross-link Repair†

    PubMed Central

    Zhang, Pan; Sridharan, Deepa; Lambert, Muriel W.

    2010-01-01

    We have previously shown that there is a deficiency in the structural protein, nonerythroid α spectrin (αIISp), in cells from patients with Fanconi anemia (FA). These studies indicate that this deficiency is due to reduced stability of αIISp and correlates with decreased repair of DNA interstrand cross-links and chromosomal instability in FA cells. An important factor in the stability of αIISp is its susceptibility to cleavage by the protease, μ-calpain. We hypothesized that increased μ-calpain cleavage of αIISp in FA cells leads to increased breakdown of αIISp and that knocking-down expression of μ-calpain in FA cells should restore levels of αIISp and correct a number of the phenotypic defects observed. The results showed that there is increased μ-calpain activity in FA-A, FA-C, FA-D2, FA-F and FA-G cells which could account for the deficiency in αIISp in these FA cells. Protein interaction studies indicated that FANCA and FANCG bind directly to μ-calpain. We hypothesize that this binding may lead to inhibition of μ-calpain activity in normal cells. Knocking-down μ-calpain by siRNA in FA-A cells restored levels of αIISp to normal and reversed a number of the cellular deficiencies in these cells. It corrected the DNA repair defect and the chromosomal instability observed after exposure to a DNA interstrand cross-linking agent. These studies indicated that FA proteins may play an important role in maintaining stability of αIISp in the cell by regulating its cleavage by μ-calpain. Thus by reducing breakdown of αIISp in FA cells, it may be possible to reverse a number of the cellular deficiencies observed in this disorder. PMID:20518497

  14. Fulvic Acid Attenuates Resistin-Induced Adhesion of HCT-116 Colorectal Cancer Cells to Endothelial Cells

    PubMed Central

    Huang, Wen-Shih; Yang, Jen-Tsung; Lu, Chien-Chang; Chang, Shun-Fu; Chen, Cheng-Nan; Su, Yu-Ping; Lee, Ko-Chao

    2015-01-01

    A high level of serum resistin has recently been found in patients with a number of cancers, including colorectal cancer (CRC). Hence, resistin may play a role in CRC development. Fulvic acid (FA), a class of humic substances, possesses pharmacological properties. However, the effect of FA on cancer pathophysiology remains unclear. The aim of this study was to investigate the effect of resistin on the endothelial adhesion of CRC and to determine whether FA elicits an antagonistic mechanism to neutralize this resistin effect. Human HCT-116 (p53-negative) and SW-48 (p53-positive) CRC cells and human umbilical vein endothelial cells (HUVECs) were used in the experiments. Treatment of both HCT-116 and SW-48 cells with resistin increases the adhesion of both cells to HUVECs. This result indicated that p53 may not regulate this resistin effect. A mechanistic study in HCT-116 cells further showed that this resistin effect occurs via the activation of NF-κB and the expression of intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1). Co-treating cells with both FA and resistin revealed that FA significantly attenuated the resistin-increased NF-κB activation and ICAM-1/VCAM-1 expression and the consequent adhesion of HCT-116 cells to HUVECs. These results demonstrate the role of resistin in promoting HCT-116 cell adhesion to HUVECs and indicate that FA might be a potential candidate for the inhibition of the endothelial adhesion of CRC in response to resistin. PMID:26690142

  15. Functional characterization of FaNIP1;1 gene, a ripening-related and receptacle-specific aquaporin in strawberry fruit.

    PubMed

    Molina-Hidalgo, Francisco J; Medina-Puche, Laura; Gelis, Samuel; Ramos, José; Sabir, Farzana; Soveral, Graça; Prista, Catarina; Iglesias-Fernández, Raquel; Caballero, José L; Muñoz-Blanco, Juan; Blanco-Portales, Rosario

    2015-09-01

    Strawberry fruit (Fragaria × ananassa) is a soft fruit with high water content at ripe stage (more than 90% of its fresh weight). Aquaporins play an important role in plant water homeostasis, through the facilitation of water transport and solutes. We report the role played by FaNIP1;1 in the receptacle ripening process. The analysis by qRT-PCR of FaNIP1;1 showed that this gene is mainly expressed in fruit receptacle and has a ripening-related expression pattern that was accompanied by an increase in both the abscisic acid and water content of the receptacle throughout fruit ripening. Moreover, FaNIP1;1 was induced in situations of water deficit. Additionally, we show that FaNIP1;1 expression was positively regulated by abscisic acid and negatively regulated by auxins. The water transport capacity of FaNIP1;1 was determined by a stopped-flow spectroscopy in yeast over-expressing FaNIP1;1. Glycerol, H2O2 and boron transport were also demonstrated in yeast. On the other hand, GFP-FaNIP1;1 fusion protein was located in plasma membrane. In conclusion, FaNIP1;1 seems to play an important role increasing the plasma membrane permeability, that allows the water accumulation in the strawberry fruit receptacle throughout the ripening process.

  16. Adhesive Performance of Biomimetic Adhesive-Coated Biologic Scaffolds

    PubMed Central

    Murphy, John L.; Vollenweider, Laura; Xu, Fangmin; Lee, Bruce P.

    2010-01-01

    Surgical repair of a discontinuity in traumatized or degenerated soft tissues is traditionally accomplished using sutures. A current trend is to reinforce this primary repair with surgical grafts, meshes, or patches secured with perforating mechanical devices (i.e., sutures, staples, or tacks). These fixation methods frequently lead to chronic pain and mesh detachment. We developed a series of biodegradable adhesive polymers that are synthetic mimics of mussel adhesive proteins (MAPs), composed of 3,4-dihydroxyphenylalanine (DOPA)-derivatives, polyethylene glycol (PEG), and polycaprolactone (PCL). These polymers can be cast into films, and their mechanical properties, extent of swelling, and degradation rate can be tailored through the composition of the polymers as well as blending with additives. When coated onto a biologic mesh used for hernia repair, these adhesive constructs demonstrated adhesive strengths significantly higher than fibrin glue. With further development, a pre-coated bioadhesive mesh may represent a new surgical option for soft tissue repair. PMID:20919699

  17. High-Frequency Mechanostimulation of Cell Adhesion.

    PubMed

    Kadem, Laith F; Suana, K Grace; Holz, Michelle; Wang, Wei; Westerhaus, Hannes; Herges, Rainer; Selhuber-Unkel, Christine

    2017-01-02

    Cell adhesion is regulated by molecularly defined protein interactions and by mechanical forces, which can activate a dynamic restructuring of adhesion sites. Previous attempts to explore the response of cell adhesion to forces have been limited to applying mechanical stimuli that involve the cytoskeleton. In contrast, we here apply a new, oscillatory type of stimulus through push-pull azobenzenes. Push-pull azobenzenes perform a high-frequency, molecular oscillation upon irradiation with visible light that has frequently been applied in polymer surface relief grating. We here use these oscillations to address single adhesion receptors. The effect of molecular oscillatory forces on cell adhesion has been analyzed using single-cell force spectroscopy and gene expression studies. Our experiments demonstrate a reinforcement of cell adhesion as well as upregulated expression levels of adhesion-associated genes as a result of the nanoscale "tickling" of integrins. This novel type of mechanical stimulus provides a previously unprecedented molecular control of cellular mechanosensing.

  18. Adhesive mechanisms in cephalopods: a review.

    PubMed

    von Byern, Janek; Klepal, Waltraud

    2006-01-01

    Several genera of cephalopods (Nautilus, Sepia, Euprymna and Idiosepius) produce adhesive secretions, which are used for attachment to the substratum, for mating and to capture prey. These adhesive structures are located in different parts of the body, viz. in the digital tentacles (Nautilus), in the ventral surface of the mantle and fourth arm pair (Sepia), in the dorsal epidermis (Euprymna), or in the dorsal mantle side and partly on the fins (Idiosepius). Adhesion in Sepia is induced by suction of dermal structures on the mantle, while for Nautilus, Euprymna and Idiosepius adhesion is probably achieved by chemical substances. Histochemical studies indicate that in Nautilus and Idiosepius secretory cells that appear to be involved in adhesion stain for carbohydrates and protein, whilst in Euprymna only carbohydrates are detectable. De-adhesion is either achieved by muscle contraction of the tentacles and mantle (Nautilus and Sepia) or by secretion of substances (Euprymna). The de-adhesive mechanism used by Idiosepius remains unknown.

  19. Adhesion and Cohesion

    PubMed Central

    von Fraunhofer, J. Anthony

    2012-01-01

    The phenomena of adhesion and cohesion are reviewed and discussed with particular reference to dentistry. This review considers the forces involved in cohesion and adhesion together with the mechanisms of adhesion and the underlying molecular processes involved in bonding of dissimilar materials. The forces involved in surface tension, surface wetting, chemical adhesion, dispersive adhesion, diffusive adhesion, and mechanical adhesion are reviewed in detail and examples relevant to adhesive dentistry and bonding are given. Substrate surface chemistry and its influence on adhesion, together with the properties of adhesive materials, are evaluated. The underlying mechanisms involved in adhesion failure are covered. The relevance of the adhesion zone and its importance with regard to adhesive dentistry and bonding to enamel and dentin is discussed. PMID:22505913

  20. 3,4-Methylenedioxy-β-nitrostyrene inhibits adhesion and migration of human triple-negative breast cancer cells by suppressing β1 integrin function and surface protein disulfide isomerase.

    PubMed

    Chen, I-Hua; Chang, Fang-Rong; Wu, Yang-Chang; Kung, Po-Hsiung; Wu, Chin-Chung

    2015-03-01

    Triple negative breast cancer (TNBC) exhibits an aggressive clinical course by high metastatic potential. It is known that integrin-mediated cell adhesion and migration are important for cancer metastasis. In the present study, a synthetic compound, 3, 4-methyenedioxy-β-nitrostyrene (MNS), significantly inhibited adhesion of TNBC cell lines to different extracellular matrix (ECM) components. The antimetastatic capacity of MNS was also observed through reducing TNBC cells migration and invasion without affecting cell viability. Confocal microscopy revealed that MNS disrupted the formation of focal adhesion complex and actin stress fiber networks. Consistent with this finding, MNS inhibited phosphorylation of focal adhesion kinase (FAK) and paxillin as detected by Western blot analysis. In exploring the underlying mechanism, we found that MNS inhibited phosphorylation of FAK as a result of reducing β1 integrin activation and clustering. A cell-impermeable dithiol reagent, 2, 3-dimercaptopropane-1-sulfonic acid abrogated all of MNS's actions, indicating that MNS may react with thiol groups of cell surface proteins that are involved in regulation of β1 integrin function as well as cell adhesion and migration. Cell surface protein disulfide isomerase (PDI) has been reported to be essential for the affinity modulation of β integrins. We also demonstrated that MNS inhibited PDI activity both in a pure enzyme system and in intact cancer cells. Taken together, our results suggest that MNS inhibits in vitro metastatic properties of TNBC cells through suppression of β1 integrin activation and focal adhesion signaling. Moreover, inhibition of surface PDI may contribute, at least in part, to the actions of MNS. These results suggest that MNS has a potential to be developed as an anticancer agent for treatment of TNBC.

  1. Interfacial adhesion in rayon/nylon sheath/core composite fibers

    SciTech Connect

    Tao Weiying.

    1991-01-01

    The fibers with enhanced adhesion were produced using a wire coating type process. One objective was to determine an effective coupling agent and the most-appropriate application conditions for maximum interfacial adhesion in the rayon/nylon bicomponent fibers. The second objective was to characterize the interfacial adhesion between the core fiber and the rayon skin. After removal of the spin finish by water washing, the nylon core fibers were pretreated with fumaric acid (FA) as an adhesion promoter and then were coated with viscose rayon. The results indicated that the interfacial adhesion in the rayon/nylon composite fibers was significantly improved under the application conditions of 1.0% with 36 second pretreatment time, 1.5% with 18 second pretreatment, and 2% with 9 second pretreatment time. A fiber pull adhesion test method was developed to test the interfacial adhesion. This method effectively determined the adhesion between the core and the skin.

  2. Members of a Novel Protein Family Containing Microneme Adhesive Repeat Domains Act as Sialic Acid-binding Lectins during Host Cell Invasion by Apicomplexan Parasites*

    PubMed Central

    Friedrich, Nikolas; Santos, Joana M.; Liu, Yan; Palma, Angelina S.; Leon, Ester; Saouros, Savvas; Kiso, Makoto; Blackman, Michael J.; Matthews, Stephen; Feizi, Ten; Soldati-Favre, Dominique

    2010-01-01

    Numerous intracellular pathogens exploit cell surface glycoconjugates for host cell recognition and entry. Unlike bacteria and viruses, Toxoplasma gondii and other parasites of the phylum Apicomplexa actively invade host cells, and this process critically depends on adhesins (microneme proteins) released onto the parasite surfac