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Sample records for fibronectin drives focal

  1. Micrometer scale spacings between fibronectin nanodots regulate cell morphology and focal adhesions

    NASA Astrophysics Data System (ADS)

    Horzum, Utku; Ozdil, Berrin; Pesen-Okvur, Devrim

    2014-04-01

    Cell adhesion to extracellular matrix is an important process for both health and disease states. Surface protein patterns that are topographically flat, and do not introduce other chemical, topographical or rigidity related functionality and, more importantly, that mimic the organization of the in vivo extracellular matrix are desired. Previous work showed that vinculin and cytoskeletal organization are modulated by size and shape of surface nanopatterns. However, quantitative analysis on cell morphology and focal adhesions as a function of micrometer scale spacings of FN nanopatterns was absent. Here, electron beam lithography was used to pattern fibronectin nanodots with micrometer scale spacings on a K-casein background on indium tin oxide coated glass which, unlike silicon, is transparent and thus suitable for many light microscopy techniques. Exposure times were significantly reduced using the line exposure mode with micrometer scale step sizes. Micrometer scale spacings of 2, 4 and 8 μm between fibronectin nanodots proved to modulate cell adhesion through modification of cell area, focal adhesion number, size and circularity. Overall, cell behavior was shown to shift at the apparent threshold of 4 μm spacing. The findings presented here offer exciting new opportunities for cell biology research.

  2. Mechanotransduction through fibronectin-integrin focal adhesion in microvascular smooth muscle cells: is calcium essential?

    PubMed Central

    Li, Zhaohui; Meininger, Gerald A.

    2012-01-01

    It is believed that increased transmural pressure exerts force on vascular smooth muscle cells (VSMCs) and triggers Ca2+ signaling as an initiating event responsible for the arteriolar myogenic response. However, the mechanisms linking the pressure increase to Ca2+ signaling are unclear. We have shown previously using atomic force microscopy (AFM) that mechanical force induces a VSMC contractile response when applied to single fibronectin (FN; Sun Z, Martinez-Lemus LA, Hill MA, Meininger GA. Am J Physiol Cell Physiol 295; C268–C278, 2008) focal adhesion sites. This current study seeks to determine whether application of force to single focal adhesions can cause a change in VSMC Ca2+. Experiments were performed in low passage (p3∼10) as well as in freshly isolated skeletal muscle arteriole VSMCs. AFM-attached microbeads (5 μm) were coated with FN or collagen type I (CN-I) or type IV (CN-IV) and placed on a VSMC for 20 min, resulting in formation of a focal adhesion between the cell and the microbead. In low passage VSMCs, mechanically pulling on the FN-coated beads (800∼3000 pN) did not induce a Ca2+ increase but did cause a contractile response. In freshly isolated VSMCs, application of an FN or CN-I-coated bead onto the cell surface induced global Ca2+ increases. However, these Ca2+ increases were not correlated with the application of AFM pulling force to the bead or with the VSMC contractile responses to FN-coupled pulling. Chelating cytosolic Ca2+ using BAPTA loading had no negative effect on the focal adhesion-related contractile response in both freshly isolated and low passage VSMCs, while the Rho-kinase inhibitor Y27632 abolished the micromyogenic response in both cases. These observations suggest that, in freshly isolated and cultured VSMCs, application of mechanical force to a focal adhesion does not invoke an acute global Ca2+ increase. On the other hand, our data support a role for Rho-linked signaling mechanism involved in mechanotransduction

  3. Full-Length Fibronectin Drives Fibroblast Accumulation at the Surface of Collagen Microtissues during Cell-Induced Tissue Morphogenesis.

    PubMed

    Foolen, Jasper; Shiu, Jau-Ye; Mitsi, Maria; Zhang, Yang; Chen, Christopher S; Vogel, Viola

    2016-01-01

    Generating and maintaining gradients of cell density and extracellular matrix (ECM) components is a prerequisite for the development of functionality of healthy tissue. Therefore, gaining insights into the drivers of spatial organization of cells and the role of ECM during tissue morphogenesis is vital. In a 3D model system of tissue morphogenesis, a fibronectin-FRET sensor recently revealed the existence of two separate fibronectin populations with different conformations in microtissues, i.e. 'compact and adsorbed to collagen' versus 'extended and fibrillar' fibronectin that does not colocalize with the collagen scaffold. Here we asked how the presence of fibronectin might drive this cell-induced tissue morphogenesis, more specifically the formation of gradients in cell density and ECM composition. Microtissues were engineered in a high-throughput model system containing rectangular microarrays of 12 posts, which constrained fibroblast-populated collagen gels, remodeled by the contractile cells into trampoline-shaped microtissues. Fibronectin's contribution during the tissue maturation process was assessed using fibronectin-knockout mouse embryonic fibroblasts (Fn-/- MEFs) and floxed equivalents (Fnf/f MEFs), in fibronectin-depleted growth medium with and without exogenously added plasma fibronectin (full-length, or various fragments). In the absence of full-length fibronectin, Fn-/- MEFs remained homogenously distributed throughout the cell-contracted collagen gels. In contrast, in the presence of full-length fibronectin, both cell types produced shell-like tissues with a predominantly cell-free compacted collagen core and a peripheral surface layer rich in cells. Single cell assays then revealed that Fn-/- MEFs applied lower total strain energy on nanopillar arrays coated with either fibronectin or vitronectin when compared to Fnf/f MEFs, but that the presence of exogenously added plasma fibronectin rescued their contractility. While collagen decoration of

  4. Full-Length Fibronectin Drives Fibroblast Accumulation at the Surface of Collagen Microtissues during Cell-Induced Tissue Morphogenesis

    PubMed Central

    Foolen, Jasper; Shiu, Jau-Ye; Mitsi, Maria; Zhang, Yang; Chen, Christopher S.; Vogel, Viola

    2016-01-01

    Generating and maintaining gradients of cell density and extracellular matrix (ECM) components is a prerequisite for the development of functionality of healthy tissue. Therefore, gaining insights into the drivers of spatial organization of cells and the role of ECM during tissue morphogenesis is vital. In a 3D model system of tissue morphogenesis, a fibronectin-FRET sensor recently revealed the existence of two separate fibronectin populations with different conformations in microtissues, i.e. ‘compact and adsorbed to collagen’ versus ‘extended and fibrillar’ fibronectin that does not colocalize with the collagen scaffold. Here we asked how the presence of fibronectin might drive this cell-induced tissue morphogenesis, more specifically the formation of gradients in cell density and ECM composition. Microtissues were engineered in a high-throughput model system containing rectangular microarrays of 12 posts, which constrained fibroblast-populated collagen gels, remodeled by the contractile cells into trampoline-shaped microtissues. Fibronectin’s contribution during the tissue maturation process was assessed using fibronectin-knockout mouse embryonic fibroblasts (Fn-/- MEFs) and floxed equivalents (Fnf/f MEFs), in fibronectin-depleted growth medium with and without exogenously added plasma fibronectin (full-length, or various fragments). In the absence of full-length fibronectin, Fn-/- MEFs remained homogenously distributed throughout the cell-contracted collagen gels. In contrast, in the presence of full-length fibronectin, both cell types produced shell-like tissues with a predominantly cell-free compacted collagen core and a peripheral surface layer rich in cells. Single cell assays then revealed that Fn-/- MEFs applied lower total strain energy on nanopillar arrays coated with either fibronectin or vitronectin when compared to Fnf/f MEFs, but that the presence of exogenously added plasma fibronectin rescued their contractility. While collagen

  5. Introduction of four different drive systems used in LAMOST focal plane

    NASA Astrophysics Data System (ADS)

    Wang, Guomin; Jiang, Xiang; Wang, Yuefei; Li, Guoping; Gu, Bozhong

    2006-06-01

    This paper describes four different drive systems adopted in LAMOST focal plane mechanism to achieve four movements: field derotation, focal plane attitude adjustment, focusing and move aside out of light path for optical checking. Different type drive systems, such as worm gear drive, spur gear drive, friction drive and direct drive, which were devised and used in telescopes in the past years, have their own inherent characteristics and their working conditions. According to feasibility, reliability, suitability and cost effective, friction drive, worm gear drive, ball screw drive and chain drive are selected to as the drive systems for the above four movements. The on-shop test results show that all the drive systems have met the design goals with the accuracy of image field derotation 0.45 arcsec, attitude adjustment 0.24 arcsec, focusing 2 microns and move aside 0.02mm.

  6. SYSTEMATIC ANALYSIS OF REAL-WORLD DRIVING BEHAVIOR FOLLOWING FOCAL BRAIN LESIONS

    PubMed Central

    Thompson, Kelsey; Read, Katherine; Anderson, Steven; Rizzo, Matthew

    2012-01-01

    Summary Many patients with circumscribed brain injuries, such as those caused by stroke or focal trauma, return to driving after a period of acute recovery. These persons often have chronic residual cognitive deficits that may impact on driving safety, but little is known about their driving behavior in the real world. Extant studies tend to rely on driving simulators or controlled on-road drives. These methods of observation are not able to capture the complexities of the typical driving environment, and may not accurately represent a driver’s usual behavior on the road. The current study used a video event-activated data recorder (VEADR) system to observe drivers with focal brain lesions in their normal daily driving environment over a three-month period. In the context of primarily safe driving behavior, we were able to document a number of relatively infrequent and hitherto unobserved high risk behaviors and traffic violations. These findings demonstrate the feasibility and value of sampling real-world driving in neurologic patient populations such as those with focal brain lesions, and highlight the critical importance of evaluating unsafe driving behaviors which may occur with insufficient frequency to be captured by relatively brief simulator or controlled on-road evaluations. PMID:25309966

  7. Fast focal zooming scheme for direct drive fusion implemented by inserting KD2PO4 crystal

    NASA Astrophysics Data System (ADS)

    Zhong, Zheqiang; Hu, Xiaochuan; Zhang, Bin

    2016-06-01

    The highly required uniformity of target in direct-drive fusion is difficult to achieve and maintain during the entire laser fusion implosion. To mitigate the increasing nonuniformity, the fast focal zooming scheme implemented by inserting an electro-optic (EO) crystal in the front end of beamline has been proposed. Functioning as a phase plate, the specifically designed EO crystal may add the induced spherical wavefront to the laser beam and alter its focusing characteristics. However, in order to zoom out the focal spot by half, the required voltage for KD2PO4 (DKDP) with single pair of electrodes is relatively high. In order to decrease the voltage while maintaining the zooming performance, the DKDP cylinder with multi pairs of electrodes has been presented. The continuous phase plate (CPP) is designed according to both the injected beam and the output field. However, the conventional CPP is designed based on the assumption of an injected beam without wavefront distortion, which would zoom in the focal spot variation in the focal zooming scheme. In order to zoom out the focal spot, a redesigned CPP has been proposed by adding a spherical wavefront to the phase variation of the conventional CPP and further optimizing. On the basis, the focusing characteristics of laser beam during the fast focal zooming process have been analyzed. Results indicate that the focal spot size decreases with the increasing voltage on DKDP crystal, meanwhile the uniformity maintains high during the focal zooming process.

  8. Whole-genome plasma sequencing reveals focal amplifications as a driving force in metastatic prostate cancer.

    PubMed

    Ulz, Peter; Belic, Jelena; Graf, Ricarda; Auer, Martina; Lafer, Ingrid; Fischereder, Katja; Webersinke, Gerald; Pummer, Karl; Augustin, Herbert; Pichler, Martin; Hoefler, Gerald; Bauernhofer, Thomas; Geigl, Jochen B; Heitzer, Ellen; Speicher, Michael R

    2016-01-01

    Genomic alterations in metastatic prostate cancer remain incompletely characterized. Here we analyse 493 prostate cancer cases from the TCGA database and perform whole-genome plasma sequencing on 95 plasma samples derived from 43 patients with metastatic prostate cancer. From these samples, we identify established driver aberrations in a cancer-related gene in nearly all cases (97.7%), including driver gene fusions (TMPRSS2:ERG), driver focal deletions (PTEN, RYBP and SHQ1) and driver amplifications (AR and MYC). In serial plasma analyses, we observe changes in focal amplifications in 40% of cases. The mean time interval between new amplifications was 26.4 weeks (range: 5-52 weeks), suggesting that they represent rapid adaptations to selection pressure. An increase in neuron-specific enolase is accompanied by clonal pattern changes in the tumour genome, most consistent with subclonal diversification of the tumour. Our findings suggest a high plasticity of prostate cancer genomes with newly occurring focal amplifications as a driving force in progression. PMID:27328849

  9. Whole-genome plasma sequencing reveals focal amplifications as a driving force in metastatic prostate cancer

    PubMed Central

    Ulz, Peter; Belic, Jelena; Graf, Ricarda; Auer, Martina; Lafer, Ingrid; Fischereder, Katja; Webersinke, Gerald; Pummer, Karl; Augustin, Herbert; Pichler, Martin; Hoefler, Gerald; Bauernhofer, Thomas; Geigl, Jochen B.; Heitzer, Ellen; Speicher, Michael R.

    2016-01-01

    Genomic alterations in metastatic prostate cancer remain incompletely characterized. Here we analyse 493 prostate cancer cases from the TCGA database and perform whole-genome plasma sequencing on 95 plasma samples derived from 43 patients with metastatic prostate cancer. From these samples, we identify established driver aberrations in a cancer-related gene in nearly all cases (97.7%), including driver gene fusions (TMPRSS2:ERG), driver focal deletions (PTEN, RYBP and SHQ1) and driver amplifications (AR and MYC). In serial plasma analyses, we observe changes in focal amplifications in 40% of cases. The mean time interval between new amplifications was 26.4 weeks (range: 5–52 weeks), suggesting that they represent rapid adaptations to selection pressure. An increase in neuron-specific enolase is accompanied by clonal pattern changes in the tumour genome, most consistent with subclonal diversification of the tumour. Our findings suggest a high plasticity of prostate cancer genomes with newly occurring focal amplifications as a driving force in progression. PMID:27328849

  10. Three-dimensional imaging system by using a low-voltage-driving LC lens with a tunable focal length

    NASA Astrophysics Data System (ADS)

    Kawamura, Marenori; Ishikuro, Shunsuke

    2015-09-01

    We develop a three-dimensional imaging system by using a low-voltage-driving liquid crystal (LC) lens for determining depth mapping properties of three-dimensional objects. The sequential photo images without the magnification and reduction are taken by electrically controlling a focal plane along a depth direction with no mechanical movements. The depth mapping properties can be obtained by processing an image digital filter from the different focal images.

  11. Inverted formin 2 in focal adhesions promotes dorsal stress fiber and fibrillar adhesion formation to drive extracellular matrix assembly

    PubMed Central

    Skau, Colleen T.; Plotnikov, Sergey V.; Doyle, Andrew D.; Waterman, Clare M.

    2015-01-01

    Actin filaments and integrin-based focal adhesions (FAs) form integrated systems that mediate dynamic cell interactions with their environment or other cells during migration, the immune response, and tissue morphogenesis. How adhesion-associated actin structures obtain their functional specificity is unclear. Here we show that the formin-family actin nucleator, inverted formin 2 (INF2), localizes specifically to FAs and dorsal stress fibers (SFs) in fibroblasts. High-resolution fluorescence microscopy and manipulation of INF2 levels in cells indicate that INF2 plays a critical role at the SF–FA junction by promoting actin polymerization via free barbed end generation and centripetal elongation of an FA-associated actin bundle to form dorsal SF. INF2 assembles into FAs during maturation rather than during their initial generation, and once there, acts to promote rapid FA elongation and maturation into tensin-containing fibrillar FAs in the cell center. We show that INF2 is required for fibroblasts to organize fibronectin into matrix fibers and ultimately 3D matrices. Collectively our results indicate an important role for the formin INF2 in specifying the function of fibrillar FAs through its ability to generate dorsal SFs. Thus, dorsal SFs and fibrillar FAs form a specific class of integrated adhesion-associated actin structure in fibroblasts that mediates generation and remodeling of ECM. PMID:25918420

  12. Fibronectin and craniofacial surgery.

    PubMed

    Al-Qattan, Mohammad M; AlShomer, Feras; Alqahtani, Abdullah; Alhadlg, Ahmad

    2014-12-01

    Fibronectin is an essential component of the extracellular matrix. The role of fibronectin in craniofacial surgery has not been previously reviewed. Fibronectin mediates bone differentiation and development of the skull. Studies have shown that normal development of the skull requires a specific pattern of expression around the epithelial-mesenchymal interface of the neurocranium. Fibronectin is also essential in mediating the migration of neural crest cells to form the facial skeleton. The calvaria of patients with Apert and Crouzon syndromes have an abnormally elevated collagen level. However, fibronectin levels are elevated in the former syndrome and decreased in the latter syndrome. The significance of this requires further research. Fibronectin gene expression is increased in port wine-derived fibroblasts in patients with Sturge-Weber syndrome. Normal palatogenesis also requires a specific pattern of expression of fibronectin around the maxillary process as well as the roof of the stomodeum, and several studies have linked the development of cleft lip/palate to an imbalance of fibronectin content of the extracellular matrix. Fibronectin mediates cell-to-cell attachment during repair of calvarial defects; hence, fibronectin has been used as a carrier for bone morphogenetic proteins to treat calvarial defects. Finally, fibronectin is now an essential component in stem cell technology related to craniofacial surgery. PMID:24322634

  13. Haematopoietic focal adhesion kinase deficiency alters haematopoietic homeostasis to drive tumour metastasis.

    PubMed

    Batista, Silvia; Maniati, Eleni; Reynolds, Louise E; Tavora, Bernardo; Lees, Delphine M; Fernandez, Isabelle; Elia, George; Casanovas, Oriol; Lo Celso, Cristina; Hagemann, Thorsten; Hodivala-Dilke, Kairbaan

    2014-01-01

    Metastasis is the main cause of cancer-related death and thus understanding the molecular and cellular mechanisms underlying this process is critical. Here, our data demonstrate, contrary to established dogma, that loss of haematopoietic-derived focal adhesion kinase (FAK) is sufficient to enhance tumour metastasis. Using both experimental and spontaneous metastasis models, we show that genetic ablation of haematopoietic FAK does not affect primary tumour growth but enhances the incidence of metastasis significantly. At a molecular level, haematopoietic FAK deletion results in an increase in PU-1 levels and decrease in GATA-1 levels causing a shift of hematopoietic homeostasis towards a myeloid commitment. The subsequent increase in circulating granulocyte number, with an increase in serum CXCL12 and granulocyte CXCR4 levels, was required for augmented metastasis in mice lacking haematopoietic FAK. Overall our findings provide a mechanism by which haematopoietic FAK controls cancer metastasis. PMID:25270220

  14. Quantitative changes in focal adhesion kinase and its inhibitor, FRNK, drive load-dependent expression of costamere components.

    PubMed

    Klossner, Stephan; Li, Ruowei; Ruoss, Severin; Durieux, Anne-Cécile; Flück, Martin

    2013-09-15

    Costameres are mechanosensory sites of focal adhesion in the sarcolemma that reinforce the muscle-fiber composite and provide an anchor for myofibrillogenesis. We hypothesized that elevated content of the integrin-associated regulator of costamere turnover in culture, focal adhesion kinase (FAK), drives changes in costamere component content in antigravity muscle in a load-dependent way in correspondence with altered muscle weight. The content of FAK in soleus muscle being phosphorylated at autoregulatory tyrosine 397 (FAK-pY397) was increased after 20 s of stretch. FAK-pY397 content remained elevated after 24 h of stretch-overload due to upregulated FAK content. Overexpression of FAK in soleus muscle fibers by means of gene electrotransfer increased the β1-integrin (+56%) and meta-vinculin (+88%) content. α7-Integrin (P = 0.46) and γ-vinculin (P = 0.18) content was not altered after FAK overexpression. Co-overexpression of the FAK inhibitor FAK-related nonkinase (FRNK) reduced FAK-pY397 content by 33% and increased the percentage of fast-type fibers that arose in connection with hybrid fibers with gene transfer. Transplantation experiments confirmed the association of FRNK expression with slow-to-fast fiber transformation. Seven days of unloading blunted the elevation of FAK-pY397, β1-integrin, and meta-vinculin content with FAK overexpression, and this was reversed by 1 day of reloading. The results highlight that the expression of components for costameric attachment sites of myofibrils is under load- and fiber type-related control via FAK and its inhibitor FRNK. PMID:23904105

  15. Fibronectin adsorption, cell adhesion, and proliferation on nanostructured tantalum surfaces.

    PubMed

    Dolatshahi-Pirouz, A; Jensen, T; Kraft, David Christian; Foss, Morten; Kingshott, Peter; Hansen, John Lundsgaard; Larsen, Arne Nylandsted; Chevallier, Jacques; Besenbacher, Flemming

    2010-05-25

    The interaction between dental pulp derived mesenchymal stem cells (DP-MSCs) and three different tantalum nanotopographies with and without a fibronectin coating is examined: sputter-coated tantalum surfaces with low surface roughness <0.2 nm, hut-nanostructured surfaces with a height of 2.9 +/- 0.6 nm and a width of 35 +/- 8 nm, and dome structures with a height of 13 +/- 2 nm and a width of 52 +/- 14 nm. Using ellipsometry, the adsorption and the availability of fibronectin cell-binding domains on the tantalum surfaces were examined, as well as cellular attachment, proliferation, and vinculin focal adhesion spot assembly on the respective surfaces. The results showed the highest fibronectin mass uptake on the hut structures, with a slightly higher availability of cell-binding domains and the most pronounced formation of vinculin focal adhesion spots as compared to the other surfaces. The proliferation of DP-MSCs was found to be significantly higher on dome and hut surfaces coated with fibronectin compared to the uncoated flat tantalum surfaces. Consequently, the results presented in this study indicate that fibronectin-coated nanotopographies with a vertical dimension of less than 5 nm influence cell adhesion. This rather interesting behavior is argued to originate from the more available fibronectin cell-binding domains observed on the hut structures. PMID:20443575

  16. Fibronectin regulates calvarial osteoblast differentiation

    NASA Technical Reports Server (NTRS)

    Moursi, A. M.; Damsky, C. H.; Lull, J.; Zimmerman, D.; Doty, S. B.; Aota, S.; Globus, R. K.

    1996-01-01

    The secretion of fibronectin by differentiating osteoblasts and its accumulation at sites of osteogenesis suggest that fibronectin participates in bone formation. To test this directly, we determined whether fibronectin-cell interactions regulate progressive differentiation of cultured fetal rat calvarial osteoblasts. Spatial distributions of alpha 5 integrin subunit, fibronectin, osteopontin (bone sialoprotein I) and osteocalcin (bone Gla-protein) were similar in fetal rat calvaria and mineralized, bone-like nodules formed by cultured osteoblasts. Addition of anti-fibronectin antibodies to cultures at confluence reduced subsequent formation of nodules to less than 10% of control values, showing that fibronectin is required for normal nodule morphogenesis. Anti-fibronectin antibodies selectively inhibited steady-state expression of mRNA for genes associated with osteoblast differentiation; mRNA levels for alkaline phosphatase and osteocalcin were suppressed, whereas fibronectin, type I collagen and osteopontin were unaffected. To identify functionally relevant domains of fibronectin, we treated cells with soluble fibronectin fragments and peptides. Cell-binding fibronectin fragments (type III repeats 6-10) containing the Arg-Gly-Asp (RGD) sequence blocked both nodule initiation and maturation, whether or not they contained a functional synergy site. In contrast, addition of the RGD-containing peptide GRGDSPK alone did not inhibit nodule initiation, although it did block nodule maturation. Thus, in addition to the RGD sequence, other features of the large cell-binding fragments contribute to the full osteogenic effects of fibronectin. Nodule formation and osteoblast differentiation resumed after anti-fibronectin antibodies or GRGDSPK peptides were omitted from the media, showing that the inhibition was reversible and the treatments were not cytotoxic. Outside the central cell-binding domain, peptides from the IIICS region and antibodies to the N terminus did not

  17. THE α4β1 INTEGRIN AND THE EDA DOMAIN OF FIBRONECTIN REGULATE A PROFIBROTIC PHENOTYPE IN DERMAL FIBROBLASTS*

    PubMed Central

    Shinde, Arti V.; Kelsh, Rhiannon; Peters, John H.; Sekiguchi, Kiyotoshi; Van De Water, Livingston; McKeown-Longo, Paula J.

    2015-01-01

    Prompt deposition of fibronectin-rich extracellular matrix is a critical feature of normal development and the host-response to injury. Fibronectin isoforms that include the EDA and EDB domains are prominent in these fibronectin matrices. We now report using human dermal fibroblast cultures that the EDA domain of fibronectin or EDA-derived peptides modeled after the C-C’ loop promote stress fiber formation and myosin-light chain phosphorylation. These changes are accompanied by an increase in fibronectin synthesis and fibrillogenesis. These effects are blocked by pretreating cells with either siRNA or blocking antibody to the α4 integrin. Our data indicate that the interaction between the α4β1 integrin and the EDA domain of fibronectin helps to drive tissue fibrosis by promoting a contractile phenotype and an increase in fibronectin synthesis and deposition. PMID:25433338

  18. The α4β1 integrin and the EDA domain of fibronectin regulate a profibrotic phenotype in dermal fibroblasts.

    PubMed

    Shinde, Arti V; Kelsh, Rhiannon; Peters, John H; Sekiguchi, Kiyotoshi; Van De Water, Livingston; McKeown-Longo, Paula J

    2015-01-01

    Prompt deposition of fibronectin-rich extracellular matrix is a critical feature of normal development and the host-response to injury. Fibronectin isoforms that include the EDA and EDB domains are prominent in these fibronectin matrices. We now report using human dermal fibroblast cultures that the EDA domain of fibronectin or EDA-derived peptides modeled after the C-C' loop promote stress fiber formation and myosin-light chain phosphorylation. These changes are accompanied by an increase in fibronectin synthesis and fibrillogenesis. These effects are blocked by pretreating cells with either siRNA or blocking antibody to the α4 integrin. Our data indicate that the interaction between the α4β1 integrin and the EDA domain of fibronectin helps to drive tissue fibrosis by promoting a contractile phenotype and an increase in fibronectin synthesis and deposition. PMID:25433338

  19. Dynamic Structure of Plasma Fibronectin

    PubMed Central

    Maurer, Lisa M.; Ma, Wenjiang; Mosher, Deane F.

    2016-01-01

    Fibronectin is a large vertebrate glycoprotein that is found in soluble and insoluble forms and involved in diverse processes. Protomeric fibronectin is a dimer of subunits, each of which comprises 29 to 31 modules—12 type I, two type II, and 15-17 type III. Plasma fibronectin is secreted by hepatocytes and circulates in a compact conformation before it binds to cell surfaces, converts to an extended conformation, and is assembled into fibronectin fibrils. Here we review biophysical and structural studies that have shed light on how plasma fibronectin transitions from the compact to the extended conformation. The three types of modules each have a well-organized secondary and tertiary structure as defined by NMR and crystallography and have been likened to “beads on a string”. There are flexible sequences in the N-terminal tail, between the fifth and sixth type I modules, between the first two and last two of the type III modules, and at the C-terminus. Several specific module-module interactions have been identified that likely maintain the compact quaternary structure of circulating fibronectin. The quaternary structure is perturbed in response to binding events, including binding of fibronectin to the surface of vertebrate cells for fibril assembly and to bacterial adhesins. PMID:27185500

  20. Collagenofibrotic glomerulonephropathy with fibronectin deposition in a dog.

    PubMed

    Kamiie, J; Yasuno, K; Ogihara, K; Nakamura, A; Tamahara, S; Fujino, Y; Ono, K; Shirota, K

    2009-07-01

    We report herein a case of collagenofibrotic glomerulonephropathy in a 3-year-old Shiba Inu with severe proteinuria. Histologically, renal glomeruli were enlarged with massive deposition of a homogeneous eosinophilic substance within the mesangium and capillary walls. The deposits reacted weakly with periodic acid-Schiff, stained deep blue with Masson's trichrome, and were positive by immunofluorescence for type III collagen and fibronectin. Ultrastructurally, the deposits consisted of fibrils and amorphous material in the mesangial matrix and beneath the glomerular capillary endothelium. The fibrils had transverse bands analogous to those of collagen fibrils. Electron microscopy also revealed focal detachment of podocytes and foot process effacement in glomerular tufts, which suggested that podocyte injury had contributed to the development of proteinuria in this dog. The current case resembles collagenofibrotic glomerulonephropathy (CFGN) in humans in histopathologic, immunofluorescence, and electron microscopic findings. This is the first report of CFGN in a nonhuman species with glomerular deposition of fibronectin and type III collagen. PMID:19276051

  1. Genetics Home Reference: fibronectin glomerulopathy

    MedlinePlus

    ... any age. It eventually leads to irreversible kidney failure (end-stage renal disease). Individuals with fibronectin glomerulopathy usually have blood ... Health Topic: High Blood Pressure Health Topic: Kidney Failure ... Renal Disease (ESRD) KidsHealth from Nemours: Renal Tubular Acidosis ...

  2. Engineering of Self-Assembled Fibronectin Matrix Protein and Its Effects on Mesenchymal Stem Cells

    PubMed Central

    Yun, Ye-Rang; Pham, Le B. Hang; Yoo, Yie-Ri; Lee, Sujin; Kim, Hae-Won; Jang, Jun-Hyeog

    2015-01-01

    Fibronectin (FN) contributes to cell adhesion, proliferation, and differentiation in various cell types. To enhance the activity of fibronectin at the sites of focal adhesion, we engineered a novel recombinant fibronectin (FNIII10) fragment connected to the peptide amphiphile sequence (PA), LLLLLLCCCGGDS. In this study, the effects of FNIII10-PA on rat mesenchymal stem cells (rMSCs) were compared with those of FNIII10. FNIII10-PA showed the prominent protein adhesion activity. In addition, FNIII10-PA showed a significantly higher effect on adhesion, proliferation, and differentiation of rMSCs than FNIII10. Taken together, the FNIII10-containing self-assembled sequence enhanced rMSCs adhesion, proliferation, and differentiation. PMID:26295389

  3. Matrix Gla Protein Binds to Fibronectin and Enhances Cell Attachment and Spreading on Fibronectin

    PubMed Central

    Nishimoto, Satoru Ken; Nishimoto, Miyako

    2014-01-01

    Background. Matrix Gla protein (MGP) is a vitamin K-dependent, extracellular matrix protein. MGP is a calcification inhibitor of arteries and cartilage. However MGP is synthesized in many tissues and is especially enriched in embryonic tissues and in cancer cells. The presence of MGP in those instances does not correlate well with the calcification inhibitory role. This study explores a potential mechanism for MGP to bind to matrix proteins and alter cell matrix interactions. Methods. To determine whether MGP influences cell behavior through interaction with fibronectin, we studied MGP binding to fibronectin, the effect of MGP on fibronectin mediated cell attachment and spreading and immunolocalized MGP and fibronectin. Results. First, MGP binds to fibronectin. The binding site for MGP is in a specific fibronectin fragment, called III1-C or anastellin. The binding site for fibronectin is in a MGP C-terminal peptide comprising amino acids 61–77. Second, MGP enhances cell attachment and cell spreading on fibronectin. MGP alone does not promote cell adhesion. Third, MGP is present in fibronectin-rich regions of tissue sections. Conclusions. MGP binds to fibronectin. The presence of MGP increased cell-fibronectin interactions. PMID:25210519

  4. The evolution of tenascins and fibronectin.

    PubMed

    Adams, Josephine C; Chiquet-Ehrismann, Ruth; Tucker, Richard P

    2015-01-01

    Tenascins are extracellular matrix glycoproteins that act both as integrin ligands and as modifiers of fibronectin-integrin interactions to regulate cell adhesion, migration, proliferation and differentiation. In tetrapods, both tenascins and fibronectin bind to integrins via RGD and LDV-type tripeptide motifs found in exposed loops in their fibronectin-type III domains. We previously showed that tenascins appeared early in the chordate lineage and are represented by single genes in extant cephalochordates and tunicates. Here we have examined the genomes of the coelacanth Latimeria chalumnae, the elephant shark Callorhinchus milii as well as the lampreys Petromyzon marinus and Lethenteron japonicum to learn more about the evolution of the tenascin gene family as well as the timing of the appearance of fibronectin during chordate evolution. The coelacanth has 4 tenascins that are more similar to tetrapod tenascins than are tenascins from ray-finned fishes. In contrast, only 2 tenascins were identified in the elephant shark and the Japanese lamprey L. japonicum. An RGD motif exposed to integrin binding is observed in tenascins from many, but not all, classes of chordates. Tetrapods that lack this RGD motif in tenascin-C have a similar motif in the paralog tenascin-W, suggesting the potential for some overlapping function. A predicted fibronectin with the same domain organization as the fibronectin from tetrapods is found in the sea lamprey P. marinus but not in tunicates, leading us to infer that fibronectin first appeared in vertebrates. The motifs that recognize LDV-type integrin receptors are conserved in fibronectins from a broad spectrum of vertebrates, but the RGD integrin-binding motif may have evolved in gnathostomes. PMID:25482621

  5. The evolution of tenascins and fibronectin

    PubMed Central

    Adams, Josephine C; Chiquet-Ehrismann, Ruth; Tucker, Richard P

    2015-01-01

    Tenascins are extracellular matrix glycoproteins that act both as integrin ligands and as modifiers of fibronectin-integrin interactions to regulate cell adhesion, migration, proliferation and differentiation. In tetrapods, both tenascins and fibronectin bind to integrins via RGD and LDV-type tripeptide motifs found in exposed loops in their fibronectin-type III domains. We previously showed that tenascins appeared early in the chordate lineage and are represented by single genes in extant cephalochordates and tunicates. Here we have examined the genomes of the coelacanth Latimeria chalumnae, the elephant shark Callorhinchus milii as well as the lampreys Petromyzon marinus and Lethenteron japonicum to learn more about the evolution of the tenascin gene family as well as the timing of the appearance of fibronectin during chordate evolution. The coelacanth has 4 tenascins that are more similar to tetrapod tenascins than are tenascins from ray-finned fishes. In contrast, only 2 tenascins were identified in the elephant shark and the Japanese lamprey L. japonicum. An RGD motif exposed to integrin binding is observed in tenascins from many, but not all, classes of chordates. Tetrapods that lack this RGD motif in tenascin-C have a similar motif in the paralog tenascin-W, suggesting the potential for some overlapping function. A predicted fibronectin with the same domain organization as the fibronectin from tetrapods is found in the sea lamprey P. marinus but not in tunicates, leading us to infer that fibronectin first appeared in vertebrates. The motifs that recognize LDV-type integrin receptors are conserved in fibronectins from a broad spectrum of vertebrates, but the RGD integrin-binding motif may have evolved in gnathostomes. PMID:25482621

  6. Fibronectin phosphorylation by ecto-protein kinase

    SciTech Connect

    Imada, Sumi; Sugiyama, Yayoi; Imada, Masaru )

    1988-12-01

    The presence of membrane-associated, extracellular protein kinase (ecto-protein kinase) and its substrate proteins was examined with serum-free cultures of Swiss 3T3 fibroblast. When cells were incubated with ({gamma}-{sup 32})ATP for 10 min at 37{degree}C, four proteins with apparent molecular weights between 150 and 220 kDa were prominently phosphorylated. These proteins were also radiolabeled by lactoperoxidase catalyzed iodination and were sensitive to mild tryptic digestion, suggesting that they localized on the cell surface or in the extracellular matrix. Phosphorylation of extracellular proteins with ({gamma}-{sup 32}P)ATP in intact cell culture is consistent with the existence of ecto-protein kinase. Anti-fibronectin antibody immunoprecipitated one of the phosphoproteins which comigrated with a monomer and a dimer form of fibronectin under reducing and nonreducing conditions of electrophoresis, respectively. The protein had affinity for gelatin as demonstrated by retention with gelatin-conjugated agarose. This protein substrate of ecto-protein kinase was thus concluded to be fibronectin. The sites of phosphorylation by ecto-protein kinase were compared with those of intracellularly phosphorylated fibronectin by the analysis of radiolabeled amino acids and peptides. Ecto-protein kinase phosphorylated fibronectin at serine and threonine residues which were distinct from the sites of intracellular fibronectin phosphorylation.

  7. Dynamic Modeling of Cell Migration and Spreading Behaviors on Fibronectin Coated Planar Substrates and Micropatterned Geometries

    PubMed Central

    Kim, Min-Cheol; Neal, Devin M.; Kamm, Roger D.; Asada, H. Harry

    2013-01-01

    An integrative cell migration model incorporating focal adhesion (FA) dynamics, cytoskeleton and nucleus remodeling, actin motor activity, and lamellipodia protrusion is developed for predicting cell spreading and migration behaviors. This work is motivated by two experimental works: (1) cell migration on 2-D substrates under various fibronectin concentrations and (2) cell spreading on 2-D micropatterned geometries. These works suggest (1) cell migration speed takes a maximum at a particular ligand density (∼1140 molecules/µm2) and (2) that strong traction forces at the corners of the patterns may exist due to combined effects exerted by actin stress fibers (SFs). The integrative model of this paper successfully reproduced these experimental results and indicates the mechanism of cell migration and spreading. In this paper, the mechanical structure of the cell is modeled as having two elastic membranes: an outer cell membrane and an inner nuclear membrane. The two elastic membranes are connected by SFs, which are extended from focal adhesions on the cortical surface to the nuclear membrane. In addition, the model also includes ventral SFs bridging two focal adhesions on the cell surface. The cell deforms and gains traction as transmembrane integrins distributed over the outer cell membrane bond to ligands on the ECM surface, activate SFs, and form focal adhesions. The relationship between the cell migration speed and fibronectin concentration agrees with existing experimental data for Chinese hamster ovary (CHO) cell migrations on fibronectin coated surfaces. In addition, the integrated model is validated by showing persistent high stress concentrations at sharp geometrically patterned edges. This model will be used as a predictive model to assist in design and data processing of upcoming microfluidic cell migration assays. PMID:23468612

  8. High-affinity binding of fibronectin to cultured Kupffer cells

    SciTech Connect

    Cardarelli, P.M.; Blumenstock, F.A.; McKeown-Longo, P.J.; Saba, T.M.; Mazurkiewicz, J.E.; Dias, J.A. )

    1990-11-01

    Hepatic Kupffer cells are a major component of the reticuloendothelial or macrophage system. They were the first phagocytic cell type whose phagocytosis was shown to be influenced by plasma fibronectin, a dimeric opsonic glycoprotein. In the current study, the binding of soluble radioiodinated fibronectin purified from rat serum to isolated rat hepatic Kupffer cells was investigated using a cultured Kupffer cell monolayer technique. Binding was specific, since unlabeled purified fibronectin competed in a dose-dependent manner with the 125I-fibronectin for binding to the Kupffer cells. Addition of gelatin enhanced the binding of 125I-fibronectin to Kupffer cells. The phagocytosis of gelatinized-coated red cells by Kupffer cells was increased either by preopsonizing the target particles with purified fibronectin or by the addition of purified fibronectin to the culture medium. In contrast, exposure of the Kupffer cells to medium containing purified fibronectin followed by wash-removal of the fibronectin did not increase the uptake of gelatin-coated red blood cells, even though fibronectin was detected on the surface of the Kupffer cells by immunofluorescence. Trypsinized monolayers expressed decreased capacity to bind 125I-fibronectin as well as fibronectin-coated sheep erythrocytes. The binding of 125I-fibronectin-gelatin complexes was inhibited by excess unlabeled fibronectin. We calculated that specific high-affinity (Kd = 7.46 x 10(-9) M) binding sites for fibronectin exist on Kupffer cells. There are approximately 2,800-3,500 binding sites or putative fibronectin receptors per Kupffer cell. These sites appear to mediate the enhanced phagocytosis of gelatin-coated particles opsonized by fibronectin.

  9. Fibronectin is a survival factor for differentiated osteoblasts

    NASA Technical Reports Server (NTRS)

    Globus, R. K.; Doty, S. B.; Lull, J. C.; Holmuhamedov, E.; Humphries, M. J.; Damsky, C. H.

    1998-01-01

    The skeletal extracellular matrix produced by osteoblasts contains the glycoprotein fibronectin, which regulates the adhesion, differentiation and function of various adherent cells. Interactions with fibronectin are required for osteoblast differentiation in vitro, since fibronectin antagonists added to cultures of immature fetal calvarial osteoblasts inhibit their progressive differentiation. To determine if fibronectin plays a unique role in fully differentiated osteoblasts, cultures that had already formed mineralized nodules in vitro were treated with fibronectin antagonists. Fibronectin antibodies caused >95% of the cells in the mature cultures to display characteristic features of apoptosis (nuclear condensation, apoptotic body formation, DNA laddering) within 24 hours. Cells appeared to acquire sensitivity to fibronectin antibody-induced apoptosis as a consequence of differentiation, since antibodies failed to kill immature cells and the first cells killed were those associated with mature nodules. Intact plasma fibronectin, as well as fragments corresponding to the amino-terminal, cell-binding, and carboxy-terminal domains of fibronectin, independently induced apoptosis of mature (day-13), but not immature (day-4), osteoblasts. Finally, transforming growth factor-beta1 partially protected cells from the apoptotic effects of fibronectin antagonists. Thus, in the course of maturation cultured osteoblasts switch from depending on fibronectin for differentiation to depending on fibronectin for survival. These data suggest that fibronectin, together with transforming growth factor-beta1, may affect bone formation, in part by regulating the survival of osteoblasts.

  10. Fibronectin mediates mesendodermal cell fate decisions

    PubMed Central

    Cheng, Paul; Andersen, Peter; Hassel, David; Kaynak, Bogac L.; Limphong, Pattraranee; Juergensen, Lonny; Kwon, Chulan; Srivastava, Deepak

    2013-01-01

    Non-cell-autonomous signals often play crucial roles in cell fate decisions during animal development. Reciprocal signaling between endoderm and mesoderm is vital for embryonic development, yet the key signals and mechanisms remain unclear. Here, we show that endodermal cells efficiently promote the emergence of mesodermal cells in the neighboring population through signals containing an essential short-range component. The endoderm-mesoderm interaction promoted precardiac mesoderm formation in mouse embryonic stem cells and involved endodermal production of fibronectin. In vivo, fibronectin deficiency resulted in a dramatic reduction of mesoderm accompanied by endodermal expansion in zebrafish embryos. This event was mediated by regulation of Wnt signaling in mesodermal cells through activation of integrin-β1. Our findings highlight the importance of the extracellular matrix in mediating short-range signals and reveal a novel function of endoderm, involving fibronectin and its downstream signaling cascades, in promoting the emergence of mesoderm. PMID:23715551

  11. Formation of focal adhesion-stress fibre complexes coordinated by adhesive and non-adhesive surface domains.

    PubMed

    Zimerman, B; Arnold, M; Ulmer, J; Blümmel, J; Besser, A; Spatz, J P; Geiger, B

    2004-04-01

    Cell motility consists of repeating cycles of protrusion of a leading edge in the direction of migration, attachment of the advancing membrane to the matrix, and pulling of the trailing edge forward. In this dynamic process there is a major role for the cytoskeleton, which drives the protrusive events via polymerisation of actin in the lamellipodium, followed by actomyosin contractility. To study the transition of the actin cytoskeleton from a 'protrusive' to 'retractive' form, we have monitored the formation of focal adhesions and stress fibres during cell migration on a micro-patterned surface. This surface consisted of parallel arrays of 2 microm-wide, fibronectin-coated gold stripes, separated by non-adhesive (poly(ethylene glycol)-coated) glass areas with variable width, ranging from 4-12 microm. Monitoring the spreading of motile cells indicated that cell spreading was equally effective along and across the adhesive stripes, as long as the non-adhesive spaces between them did not exceed 6 microm. When the width of the PEG region was 8 microm or more, cells became highly polarised upon spreading, and failed to reach the neighboring adhesive stripes. It was also noted that as soon as the protruding lamella successfully crossed the PEG-coated area and reached an adhesive region, the organisation of actin in that area was transformed from a diffuse meshwork into a bundle, oriented perpendicularly to the stripes and anchored at its ends in focal adhesions. This transition depends on actomyosin-based contractility and is apparently triggered by the adhesion to the rigid fibronectin surface. PMID:16475844

  12. Ethanol Enhances the Interaction of Breast Cancer Cells Over-Expressing ErbB2 With Fibronectin

    PubMed Central

    Xu, Mei; Bower, Kimberly A.; Chen, Gang; Shi, Xianglin; Dong, Zheng; Ke, Zunji; Luo, Jia

    2016-01-01

    Background Ethanol is a tumor promoter and may enhance the metastasis of breast cancer. However, the underlying cellular / molecular mechanisms remain unknown. Amplification of ErbB2 or HER2, a receptor tyrosine kinase of the ErbB family, is found in 20 to 30% of patients with breast cancer. We have previously demonstrated that the effect of ethanol on the migration / invasion of breast cancer cells positively correlated with the expression levels of ErbB2. Adhesion to the extracellular matrix (ECM) is an important initial step for cancer cell invasion and metastasis. In this study, we investigated the effects of ethanol on the adhesion of MCF7 breast cancer cells over-expressing ErbB2 (MCF7ErbB2) to human plasma fibronectin. Methods To test the hypothesis that ethanol may enhance the attachment of human breast cancer cells to fibronectin, an important component of the ECM, we evaluated the effect of ethanol on the expression of focal adhesions, cell attachment, and ErbB2 signaling in cultured MCF7ErbB2 cells. Results Exposure to ethanol drastically enhanced the adhesion of MCFErbB2 cells to fibronectin and increased the expression of focal adhesions. Ethanol induced phosphorylation of ErbB2 at Tyr1248, FAK at Tyr861, and cSrc at Try216. Ethanol promoted the interaction among ErbB2, FAK, and cSrc, and the formation of a focal complex. AG825, a selective ErbB2 inhibitor, attenuated the ethanol-induced phosphorylation of ErbB2 and its association with FAK. Furthermore, AG825 blocked ethanol-promoted cell / fibronectin adhesion as well as the expression of focal adhesions. Conclusions Our results suggest that ethanol enhances the adhesion of breast cancer cells to fibronectin in an ErbB2-dependent manner, and the FAK pathway plays an important role in ethanol-induced formation of a focal complex. PMID:20201928

  13. Recipient-derived EDA fibronectin promotes cardiac allograft fibrosis.

    PubMed

    Booth, Adam J; Wood, Sherri C; Cornett, Ashley M; Dreffs, Alyssa A; Lu, Guanyi; Muro, Andrés F; White, Eric S; Bishop, D Keith

    2012-03-01

    Advances in donor matching and immunosuppressive therapies have decreased the prevalence of acute rejection of cardiac grafts; however, chronic rejection remains a significant obstacle for long-term allograft survival. While initiating elements of anti-allograft immune responses have been identified, the linkage between these factors and the ultimate development of cardiac fibrosis is not well understood. Tissue fibrosis resembles an exaggerated wound healing response, in which extracellular matrix (ECM) molecules are central. One such ECM molecule is an alternatively spliced isoform of the ubiquitous glycoprotein fibronectin (FN), termed extra domain A-containing cellular fibronectin (EDA cFN). EDA cFN is instrumental in fibrogenesis; thus, we hypothesized that it might also regulate fibrotic remodelling associated with chronic rejection. We compared the development of acute and chronic cardiac allograft rejection in EDA cFN-deficient (EDA(-/-)) and wild-type (WT) mice. While EDA(-/-) mice developed acute cardiac rejection in a manner indistinguishable from WT controls, cardiac allografts in EDA(-/-) mice were protected from fibrosis associated with chronic rejection. Decreased fibrosis was not associated with differences in cardiomyocyte hypertrophy or intra-graft expression of pro-fibrotic mediators. Further, we examined expression of EDA cFN and total FN by whole splenocytes under conditions promoting various T-helper lineages. Conditions supporting regulatory T-cell (Treg) development were characterized by greatest production of total FN and EDA cFN, though EDA cFN to total FN ratios were highest in Th1 cultures. These findings indicate that recipient-derived EDA cFN is dispensable for acute allograft rejection responses but that it promotes the development of fibrosis associated with chronic rejection. Further, conditions favouring the development of regulatory T cells, widely considered graft-protective, may drive production of ECM molecules which enhance

  14. Material-driven fibronectin assembly for high-efficiency presentation of growth factors

    PubMed Central

    Llopis-Hernández, Virginia; Cantini, Marco; González-García, Cristina; Cheng, Zhe A.; Yang, Jingli; Tsimbouri, Penelope M; García, Andrés J.; Dalby, Matthew J.; Salmerón-Sánchez, Manuel

    2016-01-01

    Growth factors (GFs) are powerful signaling molecules with the potential to drive regenerative strategies, including bone repair and vascularization. However, GFs are typically delivered in soluble format at supraphysiological doses because of rapid clearance and limited therapeutic impact. These high doses have serious side effects and are expensive. Although it is well established that GF interactions with extracellular matrix proteins such as fibronectin control GF presentation and activity, a translation-ready approach to unlocking GF potential has not been realized. We demonstrate a simple, robust, and controlled material-based approach to enhance the activity of GFs during tissue healing. The underlying mechanism is based on spontaneous fibrillar organization of fibronectin driven by adsorption onto the polymer poly(ethyl acrylate). Fibrillar fibronectin on this polymer, but not a globular conformation obtained on control polymers, promotes synergistic presentation of integrin-binding sites and bound bone morphogenetic protein 2 (BMP-2), which enhances mesenchymal stem cell osteogenesis in vitro and drives full regeneration of a nonhealing bone defect in vivo at low GF concentrations. This simple and translatable technology could unlock the full regenerative potential of GF therapies while improving safety and cost-effectiveness. PMID:27574702

  15. Material-driven fibronectin assembly for high-efficiency presentation of growth factors.

    PubMed

    Llopis-Hernández, Virginia; Cantini, Marco; González-García, Cristina; Cheng, Zhe A; Yang, Jingli; Tsimbouri, Penelope M; García, Andrés J; Dalby, Matthew J; Salmerón-Sánchez, Manuel

    2016-08-01

    Growth factors (GFs) are powerful signaling molecules with the potential to drive regenerative strategies, including bone repair and vascularization. However, GFs are typically delivered in soluble format at supraphysiological doses because of rapid clearance and limited therapeutic impact. These high doses have serious side effects and are expensive. Although it is well established that GF interactions with extracellular matrix proteins such as fibronectin control GF presentation and activity, a translation-ready approach to unlocking GF potential has not been realized. We demonstrate a simple, robust, and controlled material-based approach to enhance the activity of GFs during tissue healing. The underlying mechanism is based on spontaneous fibrillar organization of fibronectin driven by adsorption onto the polymer poly(ethyl acrylate). Fibrillar fibronectin on this polymer, but not a globular conformation obtained on control polymers, promotes synergistic presentation of integrin-binding sites and bound bone morphogenetic protein 2 (BMP-2), which enhances mesenchymal stem cell osteogenesis in vitro and drives full regeneration of a nonhealing bone defect in vivo at low GF concentrations. This simple and translatable technology could unlock the full regenerative potential of GF therapies while improving safety and cost-effectiveness. PMID:27574702

  16. Small GTPase Rab21 Mediates Fibronectin Induced Actin Reorganization in Entamoeba histolytica: Implications in Pathogen Invasion

    PubMed Central

    Emmanuel, Merlyn; Nakano, Yumiko Saito; Nozaki, Tomoyoshi; Datta, Sunando

    2015-01-01

    The protozoan parasite Entamoeba histolytica causes a wide spectrum of intestinal infections. In severe cases, the trophozoites can breach the mucosal barrier, invade the intestinal epithelium and travel via the portal circulation to the liver, where they cause hepatic abscesses, which can prove fatal if left untreated. The host Extra Cellular Matrix (ECM) plays a crucial role in amoebic invasion by triggering an array of cellular responses in the parasite, including induction of actin rich adhesion structures. Similar actin rich protrusive structures, known as ‘invadosomes’, promote chemotactic migration of the metastatic cancer cells and non-transformed cells by remodeling the ECM. Recent studies showed a central role for Rab GTPases, the master regulators of vesicular trafficking, in biogenesis of invadosomes. Here, we showed that fibronectin, a major host ECM component induced actin remodeling in the parasite in a Rab21 dependent manner. The focalized actin structures formed were reminiscent of the mammalian invadosomes. By using various approaches, such as immunofluorescence confocal microscopy and scanning electron microscopy, along with in vitro invasion assay and matrix degradation assay, we show that the fibronectin induced formation of amoebic actin dots depend on the nucleotide status of the GTPase. The ECM components, fibronectin and collagen type I, displayed differential control over the formation of actin dots, with fibronectin positively and collagen type I negatively modulating it. The cell surface adhesion molecule Gal/GalNAc complex was also found to impose additional regulation on this process, which might have implication in collagen type I mediated suppression of actin dots. PMID:25730114

  17. Bonds between fibronectin and fibronectin-binding proteins on Staphylococcus aureus and Lactococcus lactis.

    PubMed

    Buck, Andrew W; Fowler, Vance G; Yongsunthon, Ruchirej; Liu, Jie; DiBartola, Alex C; Que, Yok-Ai; Moreillon, Philippe; Lower, Steven K

    2010-07-01

    Bacterial cell-wall-associated fibronectin binding proteins A and B (FnBPA and FnBPB) form bonds with host fibronectin. This binding reaction is often the initial step in prosthetic device infections. Atomic force microscopy was used to evaluate binding interactions between a fibronectin-coated probe and laboratory-derived Staphylococcus aureus that are (i) defective in both FnBPA and FnBPB (fnbA fnbB double mutant, DU5883), (ii) capable of expressing only FnBPA (fnbA fnbB double mutant complemented with pFNBA4), or (iii) capable of expressing only FnBPB (fnbA fnbB double mutant complemented with pFNBB4). These experiments were repeated using Lactococcus lactis constructs expressing fnbA and fnbB genes from S. aureus. A distinct force signature was observed for those bacteria that expressed FnBPA or FnBPB. Analysis of this force signature with the biomechanical wormlike chain model suggests that parallel bonds form between fibronectin and FnBPs on a bacterium. The strength and covalence of bonds were evaluated via nonlinear regression of force profiles. Binding events were more frequent (p < 0.01) for S. aureus expressing FnBPA or FnBPB than for the S. aureus double mutant. The binding force, frequency, and profile were similar between the FnBPA and FnBPB expressing strains of S. aureus. The absence of both FnBPs from the surface of S. aureus removed its ability to form a detectable bond with fibronectin. By contrast, ectopic expression of FnBPA or FnBPB on the surface of L. lactis conferred fibronectin binding characteristics similar to those of S. aureus. These measurements demonstrate that fibronectin-binding adhesins FnBPA and FnBPB are necessary and sufficient for the binding of S. aureus to prosthetic devices that are coated with host fibronectin. PMID:20218549

  18. Fibronectin Deposition Participates in Extracellular Matrix Assembly and Vascular Morphogenesis

    PubMed Central

    Hielscher, Abigail; Ellis, Kim; Qiu, Connie; Porterfield, Josh; Gerecht, Sharon

    2016-01-01

    The extracellular matrix (ECM) has been demonstrated to facilitate angiogenesis. In particular, fibronectin has been documented to activate endothelial cells, resulting in their transition from a quiescent state to an active state in which the cells exhibit enhanced migration and proliferation. The goal of this study is to examine the role of polymerized fibronectin during vascular tubulogenesis using a 3 dimensional (3D) cell-derived de-cellularized matrix. A fibronectin-rich 3D de-cellularized ECM was used as a scaffold to study vascular morphogenesis of endothelial cells (ECs). Confocal analyses of several matrix proteins reveal high intra- and extra-cellular deposition of fibronectin in formed vascular structures. Using a small peptide inhibitor of fibronectin polymerization, we demonstrate that inhibition of fibronectin fibrillogenesis in ECs cultured atop de-cellularized ECM resulted in decreased vascular morphogenesis. Further, immunofluorescence and ultrastructural analyses reveal decreased expression of stromal matrix proteins in the absence of polymerized fibronectin with high co-localization of matrix proteins found in association with polymerized fibronectin. Evaluating vascular kinetics, live cell imaging showed that migration, migration velocity, and mean square displacement, are disrupted in structures grown in the absence of polymerized fibronectin. Additionally, vascular organization failed to occur in the absence of a polymerized fibronectin matrix. Consistent with these observations, we tested vascular morphogenesis following the disruption of EC adhesion to polymerized fibronectin, demonstrating that block of integrins α5β1 and αvβ3, abrogated vascular morphogenesis. Overall, fibronectin deposition in a 3D cell-derived de-cellularized ECM appears to be imperative for matrix assembly and vascular morphogenesis. PMID:26811931

  19. Periostin promotes secretion of fibronectin from the endoplasmic reticulum.

    PubMed

    Kii, Isao; Nishiyama, Takashi; Kudo, Akira

    2016-02-19

    Extracellular matrix (ECM) proteins are synthesized in the endoplasmic reticulum (ER), transported to the extracellular milieu through the secretory pathway, and assembled into an extracellular architecture. A previous study of ours showed that periostin, a secretory protein, interacts with fibronectin and is involved in ECM remodeling. Here we show that periostin played a role in fibronectin secretion from the ER. Co-immunoprecipitation and in situ proximity ligation assays revealed an interaction between periostin and fibronectin in the ER. Although accumulation of fibronectin was detected in the ER of fibroblastic C3H10T1/2 cells, forced expression of periostin in those cells decreased the accumulation of fibronectin in the ER, suggesting that periostin promoted the secretion of fibronectin. A substitution mutant of tryptophan at the position 65 to alanine in the EMI domain of periostin, which caused periostin to lose its ability to interact with fibronectin, did not decrease the accumulation. Furthermore, targeted disruption of periostin in mice caused the non-fibrillar and ectopic deposition of fibronectin in the periodontal ligament. Thus, these results demonstrate a subcellular role of periostin in promotion of fibronectin secretion from the ER. PMID:26820539

  20. Sorption of fibronectin to human root surfaces in vitro

    SciTech Connect

    Mendieta, C.; Caravana, C.; Fine, D.H. )

    1990-05-01

    The purpose of this study was to determine the conditions that favor the sorption and retention of human plasma fibronectin to cementum. Rectangular root segments prepared from teeth extracted for orthodontic reasons were mounted on a capillary pipette and immersed in solutions of {sup 125}I fibronectin for assay of cementum sorption under various conditions. Kinetic studies showed sorption to be rapid, with 77% of the maximum fibronectin sorption occurring within 1 minute. Fibronectin sorption was reduced when added in conjunction with serum and was inhibited by monovalent ions (such as sodium), but enhanced in the presence of divalent cations (such as calcium). Exposure of cementum to serum partially blocked subsequent sorption of fibronectin, while cementum bound fibronectin was eluted by subsequent exposure to serum. Treatment of cementum with citric acid pH 1.1 (4 minutes) followed by 5% sodium hypochlorite (5 minutes) caused a significant increase in fibronectin sorption with maximum retention upon subsequent exposure to serum (P less than 0.05). Fibronectin sorption to cementum was: rapid, electrostatic in nature, competitive, reversible, Ca+(+)-facilitated, and maximized by prior treatment of the root with citric acid and sodium hypochlorite. It is concluded that sorption of fibronectin to cementum can be achieved for clinical gain; however, conditions of application can significantly influence both accumulation and subsequent release of root sorbed material.

  1. Plasma fibronectin deficiency during chemotherapy of acute myeloid leukaemia.

    PubMed

    Brodin, B; Liedén, G; Malm, C; Vikrot, O

    1983-03-01

    Plasma fibronectin was determined using a laser nephelometric method in 10 patients with acute myeloid leukaemia undergoing chemotherapy. There was a continuous fall during the first 3 weeks to about 50% of the normal level. The decrease of fibronectin may contribute to the lowered resistance against infection characteristic of these patients. PMID:6574587

  2. Molecular architecture of native fibronectin fibrils.

    PubMed

    Früh, Susanna Maria; Schoen, Ingmar; Ries, Jonas; Vogel, Viola

    2015-01-01

    Fibronectin fibrils within the extracellular matrix play central roles in physiological and pathological processes, yet many structural details about their hierarchical and molecular assembly remain unknown. Here we combine site-specific protein labelling with single-molecule localization by stepwise photobleaching or direct stochastic optical reconstruction microscopy (dSTORM), and determine the relative positions of various labelled sites within native matrix fibrils. Single end-labelled fibronectin molecules in fibrils display an average end-to-end distance of ∼133 nm. Sampling of site-specific antibody epitopes along the thinnest fibrils (protofibrils) shows periodic punctate label patterns with ∼95 nm repeats and alternating N- and C-terminal regions. These measurements suggest an antiparallel 30-40 nm overlap between N-termini, suggesting that the first five type I modules bind type III modules of the adjacent molecule. Thicker fibres show random bundling of protofibrils without a well-defined line-up. This super-resolution microscopy approach can be applied to other fibrillar protein assemblies of unknown structure. PMID:26041410

  3. Molecular architecture of native fibronectin fibrils

    PubMed Central

    Früh, Susanna Maria; Schoen, Ingmar; Ries, Jonas; Vogel, Viola

    2015-01-01

    Fibronectin fibrils within the extracellular matrix play central roles in physiological and pathological processes, yet many structural details about their hierarchical and molecular assembly remain unknown. Here we combine site-specific protein labelling with single-molecule localization by stepwise photobleaching or direct stochastic optical reconstruction microscopy (dSTORM), and determine the relative positions of various labelled sites within native matrix fibrils. Single end-labelled fibronectin molecules in fibrils display an average end-to-end distance of ∼133 nm. Sampling of site-specific antibody epitopes along the thinnest fibrils (protofibrils) shows periodic punctate label patterns with ∼95 nm repeats and alternating N- and C-terminal regions. These measurements suggest an antiparallel 30–40 nm overlap between N-termini, suggesting that the first five type I modules bind type III modules of the adjacent molecule. Thicker fibres show random bundling of protofibrils without a well-defined line-up. This super-resolution microscopy approach can be applied to other fibrillar protein assemblies of unknown structure. PMID:26041410

  4. Molecular architecture of native fibronectin fibrils

    NASA Astrophysics Data System (ADS)

    Früh, Susanna Maria; Schoen, Ingmar; Ries, Jonas; Vogel, Viola

    2015-06-01

    Fibronectin fibrils within the extracellular matrix play central roles in physiological and pathological processes, yet many structural details about their hierarchical and molecular assembly remain unknown. Here we combine site-specific protein labelling with single-molecule localization by stepwise photobleaching or direct stochastic optical reconstruction microscopy (dSTORM), and determine the relative positions of various labelled sites within native matrix fibrils. Single end-labelled fibronectin molecules in fibrils display an average end-to-end distance of ~133 nm. Sampling of site-specific antibody epitopes along the thinnest fibrils (protofibrils) shows periodic punctate label patterns with ~95 nm repeats and alternating N- and C-terminal regions. These measurements suggest an antiparallel 30-40 nm overlap between N-termini, suggesting that the first five type I modules bind type III modules of the adjacent molecule. Thicker fibres show random bundling of protofibrils without a well-defined line-up. This super-resolution microscopy approach can be applied to other fibrillar protein assemblies of unknown structure.

  5. Fibronectin as a Prognostic Indicator in Portal Hypertension

    PubMed Central

    Maharaj, D. P.; Garden, O. J.; Carter, D. C.

    1992-01-01

    Plasma fibronectin levels were measured in 33 patients with portal hypertension and c6mpared with modified Child’s grading and a previously described prognostic index. Outcome at one year from blood sampling was recorded. Mean plasma fibronectin level was 304.1 mg/ml (sem 24.3) and significantly lower levels were found in patients who had had a variceal bleed within the previous seven days. Plasma fibronectin levels tended to be lower in patients with poor liver function as assessed by modified Child’s grading but this did not achieve statistical significance. Plasma fibronectin alone was not an accurate predictor of one year survival in these patients but only one of seven patients who had a plasma fibronectin level below 300mg/l in association with a poor prognostic index survived for one year. PMID:1390363

  6. Fibroblast migration on fibronectin requires three distinct functional domains.

    PubMed

    Clark, Richard A F; An, Jian-Qiang; Greiling, Doris; Khan, Azim; Schwarzbauer, Jean E

    2003-10-01

    Mesenchymal cell movement is normally constrained; however, fibronectin can provide a pathway for stromal cell migration during embryogenesis, morphogenesis, and wound healing. Cells can adhere to fibronectin via integrin and nonintegrin receptors, which bind multiple unique peptide sequences. Synthetic peptides and recombinant proteins were used to delineate the functional domains needed for human fibroblast migration over fibronectin. The 9th and 10th fibronectin type III repeats, which contain RGD and PHSRN synergy cell attachment sequences, support almost maximal fibroblast attachment, but not migration of primary dermal fibroblasts. Specific sequences within the heparin domain and the IIICS region are also required for migration. These findings predict and additional data confirm the necessity for the cooperation of multiple integrin and nonintegrin receptors for fibroblast migration on fibronectin. Such stringency of migration most likely imposes an immense constraint on normal mesenchymal cell mobility in unperturbed tissue. Loss of such restraint may be critical for the migration cancer cells through the extracellular matrix. PMID:14632184

  7. Characterization of the fibronectin-attachment protein of Mycobacterium avium reveals a fibronectin-binding motif conserved among mycobacteria.

    PubMed

    Schorey, J S; Holsti, M A; Ratliff, T L; Allen, P M; Brown, E J

    1996-07-01

    Mycobacterium avium is an intracellular pathogen and a major opportunistic infectious agent observed in patients with acquired immune deficiency syndrome (AIDS). Evidence suggests that the initial portal of infection by M. avium is often the gastrointestinal tract. However, the mechanism by which the M. avium crosses the epithelial barrier is unclear. A possible mechanism is suggested by the ability of M. avium to bind fibronectin, an extracellular matrix protein that is a virulence factor for several extracellular pathogenic bacteria which bind to mucosal surfaces. To further characterize fibronectin binding by M. avium, we have cloned the M. avium fibronectin-attachment protein (FAP). The M. avium FAP (FAP-A) has an unusually large number of Pro and Ala residues (40% overall) and is 50% identical to FAP of both Mycobacterium leprae and Mycobacterium tuberculosis. Using recombinant FAP-A and FAP-A peptides, we show that two non-continuous regions in FAP-A bind fibronectin. Peptides from these regions and homologous sequences from M. leprae FAP inhibit fibronectin binding by both M. avium and Mycobacterium bovis Bacillus Calmette-Guerin (BCG). These regions have no homology to eukaryotic fibronectin-binding proteins and are only distantly related to fibronectin-binding peptides of Gram-positive bacteria. Nevertheless, these fibronectin-binding regions are highly conserved among the mycobacterial FAPs, suggesting an essential function for this interaction in mycobacteria infection of their metazoan hosts. PMID:8858587

  8. Utilizing Fibronectin Integrin-Binding Specificity to Control Cellular Responses

    PubMed Central

    Bachman, Haylee; Nicosia, John; Dysart, Marilyn; Barker, Thomas H.

    2015-01-01

    Significance: Cells communicate with the extracellular matrix (ECM) protein fibronectin (Fn) through integrin receptors on the cell surface. Controlling integrin–Fn interactions offers a promising approach to directing cell behavior, such as adhesion, migration, and differentiation, as well as coordinated tissue behaviors such as morphogenesis and wound healing. Recent Advances: Several different groups have developed recombinant fragments of Fn that can control epithelial to mesenchymal transition, sequester growth factors, and promote bone and wound healing. It is thought that these physiological responses are, in part, due to specific integrin engagement. Furthermore, it has been postulated that the integrin-binding domain of Fn is a mechanically sensitive switch that drives binding of one integrin heterodimer over another. Critical Issues: Although computational simulations have predicted the mechano-switch hypothesis and recent evidence supports the existence of varying strain states of Fn in vivo, experimental evidence of the Fn integrin switch is still lacking. Future Directions: Evidence of the integrin mechano-switch will enable the development of new Fn-based peptides in tissue engineering and wound healing, as well as deepen our understanding of ECM pathologies, such as fibrosis. PMID:26244106

  9. Recipient–derived EDA fibronectin promotes cardiac allograft fibrosis

    PubMed Central

    Booth, Adam J; Wood, Sherri C; Cornett, Ashley M; Dreffs, Alyssa A; Lu, Guanyi; Muro, Andrés F; White, Eric S; Bishop, D Keith

    2014-01-01

    Advances in donor matching and immunosuppressive therapies have decreased the prevalence of acute rejection of cardiac grafts; however, chronic rejection remains a significant obstacle for long-term allograft survival. While initiating elements of anti-allograft immune responses have been identified, the linkage between these factors and the ultimate development of cardiac fibrosis is not well understood. Tissue fibrosis resembles an exaggerated wound healing response, in which extracellular matrix (ECM) molecules are central. One such ECM molecule is an alternatively spliced isoform of the ubiquitous glycoprotein fibronectin (FN), termed extra domain A-containing cellular fibronectin (EDA cFN). EDA cFN is instrumental in fibrogenesis; thus, we hypothesized that it might also regulate fibrotic remodelling associated with chronic rejection. We compared the development of acute and chronic cardiac allograft rejection in EDA cFN-deficient (EDA−/−) and wild-type (WT) mice. While EDA−/− mice developed acute cardiac rejection in a manner indistinguishable from WT controls, cardiac allografts in EDA−/− mice were protected from fibrosis associated with chronic rejection. Decreased fibrosis was not associated with differences in cardiomyocyte hypertrophy or intra-graft expression of pro-fibrotic mediators. Further, we examined expression of EDA cFN and total FN by whole splenocytes under conditions promoting various T-helper lineages. Conditions supporting regulatory T-cell (Treg) development were characterized by greatest production of total FN and EDA cFN, though EDA cFN to total FN ratios were highest in Th1 cultures. These findings indicate that recipient-derived EDA cFN is dispensable for acute allograft rejection responses but that it promotes the development of fibrosis associated with chronic rejection. Further, conditions favouring the development of regulatory T cells, widely considered graft-protective, may drive production of ECM molecules which

  10. Differential deposition of fibronectin by asthmatic bronchial epithelial cells.

    PubMed

    Ge, Qi; Zeng, Qingxiang; Tjin, Gavin; Lau, Edmund; Black, Judith L; Oliver, Brian G G; Burgess, Janette K

    2015-11-15

    Altered ECM protein deposition is a feature in asthmatic airways. Fibronectin (Fn), an ECM protein produced by human bronchial epithelial cells (HBECs), is increased in asthmatic airways. This study investigated the regulation of Fn production in asthmatic or nonasthmatic HBECs and whether Fn modulated HBEC proliferation and inflammatory mediator secretion. The signaling pathways underlying transforming growth factor (TGF)-β1-regulated Fn production were examined using specific inhibitors for ERK, JNK, p38 MAPK, phosphatidylinositol 3 kinase, and activin-like kinase 5 (ALK5). Asthmatic HBECs deposited higher levels of Fn in the ECM than nonasthmatic cells under basal conditions, whereas cells from the two groups had similar levels of Fn mRNA and soluble Fn. TGF-β1 increased mRNA levels and ECM and soluble forms of Fn but decreased cell proliferation in both cells. The rate of increase in Fn mRNA was higher in nonasthmatic cells. However, the excessive amounts of ECM Fn deposited by asthmatic cells after TGF-β1 stimulation persisted compared with nonasthmatic cells. Inhibition of ALK5 completely prevented TGF-β1-induced Fn deposition. Importantly, ECM Fn increased HBEC proliferation and IL-6 release, decreased PGE2 secretion, but had no effect on VEGF release. Soluble Fn had no effect on cell proliferation and inflammatory mediator release. Asthmatic HBECs are intrinsically primed to produce more ECM Fn, which when deposited into the ECM, is capable of driving remodeling and inflammation. The increased airway Fn may be one of the key driving factors in the persistence of asthma and represents a novel, therapeutic target. PMID:26342086

  11. Cadherin-11 localizes to focal adhesions and promotes cell–substrate adhesion

    PubMed Central

    Langhe, Rahul P.; Gudzenko, Tetyana; Bachmann, Michael; Becker, Sarah F.; Gonnermann, Carina; Winter, Claudia; Abbruzzese, Genevieve; Alfandari, Dominique; Kratzer, Marie-Claire; Franz, Clemens M.; Kashef, Jubin

    2016-01-01

    Cadherin receptors have a well-established role in cell–cell adhesion, cell polarization and differentiation. However, some cadherins also promote cell and tissue movement during embryonic development and tumour progression. In particular, cadherin-11 is upregulated during tumour and inflammatory cell invasion, but the mechanisms underlying cadherin-11 stimulated cell migration are still incompletely understood. Here, we show that cadherin-11 localizes to focal adhesions and promotes adhesion to fibronectin in Xenopus neural crest, a highly migratory embryonic cell population. Transfected cadherin-11 also localizes to focal adhesions in different mammalian cell lines, while endogenous cadherin-11 shows focal adhesion localization in primary human fibroblasts. In focal adhesions, cadherin-11 co-localizes with β1-integrin and paxillin and physically interacts with the fibronectin-binding proteoglycan syndecan-4. Adhesion to fibronectin mediated by cadherin-11/syndecan-4 complexes requires both the extracellular domain of syndecan-4, and the transmembrane and cytoplasmic domains of cadherin-11. These results reveal an unexpected role of a classical cadherin in cell–matrix adhesion during cell migration. PMID:26952325

  12. Propolis modulates fibronectin expression in the matrix of thermal injury.

    PubMed

    Olczyk, Pawel; Komosinska-Vassev, Katarzyna; Wisowski, Grzegorz; Mencner, Lukasz; Stojko, Jerzy; Kozma, Ewa M

    2014-01-01

    The aim of the study was to assess the propolis effect on fibronectin metabolism in the course of burn wounds healing process. A model of burn wound healing of pig skin was applied. The amount of the released glycoprotein was assessed by a surface plasmon resonance. The profile of extracted fibronectin components was also assessed by an electrophoresis in polyacrylamide gel, with a subsequent immunodetection by Western Blotting. Propolis burn treatment decreased the release of fibronectin components from healing wounds in relation to damages treated with silver sulfadiazine. The main reason of decreased extraction of fibronectin components from wounds treated with propolis was a substantial decrease of degradation product release of the mentioned glycoprotein, which was observed particularly from the 3rd to 5th day of the repair. Wounds treatment with propolis demonstrated, especially in relation to damages treated with silver sulfadiazine, the decreased release of synthesized fibronectin molecules. The obtained results suggest that propolis modifies fibronectin metabolism in the course of wound healing process. The influence of propolis is reflected in prevention of fibronectin biosynthesis as well as its degradation in the wound area. The above-mentioned metabolic changes may decrease the risk of complications in the repair wounds process. PMID:24738072

  13. Guidance of myogenic cell migration by oriented deposits of fibronectin.

    PubMed

    Turner, D C; Lawton, J; Dollenmeier, P; Ehrismann, R; Chiquet, M

    1983-02-01

    Fibronectin mediates myoblast-substratum attachment; one region of the molecule binds directly to the cell surface, while others bind to collagen, glycosaminoglycans, and other fibronectin molecules. There is evidence to suggest that fibronectin-containing extracellular matrices guide cell migration in vivo. We describe a method for producing regular deposits of fibronectin in vitro that can serve as a model system for studying cell-substrate interactions, cell orientation, and contact guidance. The novel culture substrate is prepared by allowing an aqueous solution of fibronectin and urea to dry in a culture dish and then washing away the urea crystals. Myogenic cells in vitro adhere to, align with, and migrate along, parallel streaks of fibronectin. This leads to the formation of myotubes that are long and thin, with little branching. Myogenic clones are highly elongated in the direction of the deposits, in contrast with the roughly circular clones seen in conventional cultures. Fibroblasts and limb bud mesenchymal cells align with fibronectin deposits, assuming a bipolar shape. PMID:6825944

  14. Propolis Modulates Fibronectin Expression in the Matrix of Thermal Injury

    PubMed Central

    Komosinska-Vassev, Katarzyna; Wisowski, Grzegorz; Mencner, Lukasz; Stojko, Jerzy; Kozma, Ewa M.

    2014-01-01

    The aim of the study was to assess the propolis effect on fibronectin metabolism in the course of burn wounds healing process. A model of burn wound healing of pig skin was applied. The amount of the released glycoprotein was assessed by a surface plasmon resonance. The profile of extracted fibronectin components was also assessed by an electrophoresis in polyacrylamide gel, with a subsequent immunodetection by Western Blotting. Propolis burn treatment decreased the release of fibronectin components from healing wounds in relation to damages treated with silver sulfadiazine. The main reason of decreased extraction of fibronectin components from wounds treated with propolis was a substantial decrease of degradation product release of the mentioned glycoprotein, which was observed particularly from the 3rd to 5th day of the repair. Wounds treatment with propolis demonstrated, especially in relation to damages treated with silver sulfadiazine, the decreased release of synthesized fibronectin molecules. The obtained results suggest that propolis modifies fibronectin metabolism in the course of wound healing process. The influence of propolis is reflected in prevention of fibronectin biosynthesis as well as its degradation in the wound area. The above-mentioned metabolic changes may decrease the risk of complications in the repair wounds process. PMID:24738072

  15. Fibronectin maintains the balance between hemostasis and thrombosis.

    PubMed

    Wang, Yiming; Ni, Heyu

    2016-09-01

    Fibronectin is a dimeric protein widely distributed in solid tissues and blood. This major extracellular matrix protein is indispensable for embryogenesis and plays crucial roles in many physiological and pathological processes. Fibronectin pre-mRNA undergoes alternative splicing to generate over 20 splicing variants, which are categorized as either plasma fibronectin (pFn) or cellular fibronectin (cFn). All fibronectin variants contain integrin binding motifs, as well as N-terminus collagen and fibrin binding motifs. With motifs that can be recognized by platelet integrins and coagulation factors, fibronectin, especially pFn, has long been suspected to be involved in hemostasis and thrombosis, but the exact function of fibronectin in these processes is controversial. The advances made using intravital microscopy models and fibronectin deficient and mutant mice have greatly facilitated the direct investigation of fibronectin function in vivo. Recent studies revealed that pFn is a vital hemostatic factor that is especially crucial for hemostasis in both genetic and anticoagulant-induced deficiencies of fibrin formation. pFn may also be an important self-limiting regulator to prevent hemorrhage as well as excessive thrombus formation and vessel occlusion. In addition to pFn, cFn is found to be prothrombotic and may contribute to thrombotic complications in various diseases. Further investigations of the role of pFn and cFn in thrombotic and hemorrhagic diseases may provide insights into development of novel therapeutic strategies (e.g., pFn transfusion) for the maintenance of the fine balance between hemostasis and thrombosis. PMID:27098513

  16. Adhesion of fibroblasts to fibronectin stimulates both serine and tyrosine phosphorylation of paxillin.

    PubMed Central

    Bellis, S L; Perrotta, J A; Curtis, M S; Turner, C E

    1997-01-01

    Tyrosine phosphorylation of paxillin by the focal adhesion kinase (FAK) has been implicated as a signal transduction mechanism associated with cell adhesion and cytoskeletal reorganization. The potential role of serine phosphorylation of paxillin in these events has not been well characterized. In this study we have examined the phosphorylation profile of paxillin both in vitro and in vivo. By using glutathione S-transferase-paxillin fusion proteins in precipitation-kinase assays in vitro we observed that a fusion protein spanning amino acid residues 54-313 of paxillin, and containing a FAK-binding site, precipitated substantial serine kinase activity as well as FAK activity from a smooth-muscle lysate. Together these kinases phosphorylated paxillin on tyrosine residue 118, a site that has been identified previously as a target for FAK phosphorylation, and on serine residues 188 and/or 190. The binding site for the serine kinase, the identity of which is currently unknown, was further mapped to residues 168-191 of paxillin. To assess the physiological relevance of these sites phosphorylated in vitro, the profile of paxillin phosphorylation in vivo stimulated by seeding fibroblasts on fibronectin was characterized. As expected, plating cells on fibronectin enhanced the tyrosine phosphorylation of paxillin. However, 96% of the phosphorylation of paxillin occurred on serine residues. Comparison by two-dimensional phosphopeptide analyses indicated that the major sites of tyrosine and serine phosphorylation detected in the assays in vitro co-migrate with phosphopeptides derived from paxillin phosphorylated in vivo in response to plating cells on fibronectin. These findings support a role for both tyrosine and serine kinases in the signal transduction pathway linking integrin activation to paxillin phosphorylation. PMID:9230116

  17. Plasma fibronectin concentrations in dogs with disseminated intravascular coagulation.

    PubMed

    Feldman, B F; Thomson, D B; O'Neill, S

    1985-05-01

    Plasma fibronectin concentrations were significantly (P less than 0.001) below the reference range in dogs with disseminated intravascular coagulation (DIC) secondary to nonlymphomatous neoplasia, acute necrotizing pancreatitis, sepsis, chronic active hepatitis, and heat stroke. There was no statistical evidence of a group effect. Decrease in fibronectin concentration was associated with severe DIC, although no attempt was made to correlate fibronectin concentration with prognosis. These findings parallel those reported for severely ill human beings with diseases associated with DIC. They exemplify the potential of spontaneous diseases in animals as models for the study of human disease. PMID:4003893

  18. A fibronectin mimetic motif improves integrin mediated cell biding to recombinant spider silk matrices.

    PubMed

    Widhe, Mona; Shalaly, Nancy Dekki; Hedhammar, My

    2016-01-01

    The cell binding motif RGD is the most widely used peptide to improve cell binding properties of various biomaterials, including recombinant spider silk. In this paper we use genetic engineering to further enhance the cell supportive capacity of spider silk by presenting the RGD motif as a turn loop, similar to the one found in fibronectin (FN), but in the silk stabilized by cysteines, and therefore denoted FNCC. Human primary cells cultured on FNCC-silk showed increased attachment, spreading, stress fiber formation and focal adhesions, not only compared to RGD-silk, but also to silk fused with linear controls of the RGD containing motif from fibronectin. Cell binding to FNCC-silk was shown to involve the α5β1 integrin, and to support proliferation and migration of keratinocytes. The FNCC-silk protein allowed efficient assembly, and could even be transformed into free standing films, on which keratinocytes could readily form a monolayer culture. The results hold promise for future applications within tissue engineering. PMID:26461118

  19. Small Molecules Antagonise the MIA-Fibronectin Interaction in Malignant Melanoma.

    PubMed

    Yip, King Tuo; Zhong, Xue Yin; Seibel, Nadia; Pütz, Stefanie; Autzen, Jasmin; Gasper, Raphael; Hofmann, Eckhard; Scherkenbeck, Jürgen; Stoll, Raphael

    2016-01-01

    Melanoma inhibitory activity (MIA), an extracellular protein highly expressed by malignant melanoma cells, plays an important functional role in melanoma development, progression, and metastasis. After its secretion, MIA directly interacts with extracellular matrix proteins, such as fibronectin (FN). By this mechanism, MIA actively facilitates focal cell detachment from surrounding structures and strongly promotes tumour cell invasion and migration. Hence, the molecular understanding of MIA's function provides a promising target for the development of new strategies in malignant melanoma therapy. Here, we describe for the first time the discovery of small molecules that are able to disrupt the MIA-FN complex by selectively binding to a new druggable pocket, which we could identify on MIA by structural analysis and fragment-based screening. Our findings may inspire novel drug discovery efforts aiming at a therapeutically effective treatment of melanoma by targeting MIA. PMID:27151361

  20. Small Molecules Antagonise the MIA-Fibronectin Interaction in Malignant Melanoma

    PubMed Central

    Yip, King Tuo; Zhong, Xue Yin; Seibel, Nadia; Pütz, Stefanie; Autzen, Jasmin; Gasper, Raphael; Hofmann, Eckhard; Scherkenbeck, Jürgen; Stoll, Raphael

    2016-01-01

    Melanoma inhibitory activity (MIA), an extracellular protein highly expressed by malignant melanoma cells, plays an important functional role in melanoma development, progression, and metastasis. After its secretion, MIA directly interacts with extracellular matrix proteins, such as fibronectin (FN). By this mechanism, MIA actively facilitates focal cell detachment from surrounding structures and strongly promotes tumour cell invasion and migration. Hence, the molecular understanding of MIA’s function provides a promising target for the development of new strategies in malignant melanoma therapy. Here, we describe for the first time the discovery of small molecules that are able to disrupt the MIA-FN complex by selectively binding to a new druggable pocket, which we could identify on MIA by structural analysis and fragment-based screening. Our findings may inspire novel drug discovery efforts aiming at a therapeutically effective treatment of melanoma by targeting MIA. PMID:27151361

  1. Porins of Pseudomonas fluorescens MFO as fibronectin-binding proteins.

    PubMed

    Rebière-Huët, J; Guérillon, J; Pimenta, A L; Di Martino, P; Orange, N; Hulen, C

    2002-09-24

    Bacterial adherence is a complex phenomenon involving specific interactions between receptors, including matricial fibronectin, and bacterial ligands. We show here that fibronectin and outer membrane proteins of Pseudomonas fluorescens were able to inhibit adherence of P. fluorescens to fibronectin-coated wells. We identified at least six fibronectin-binding proteins with molecular masses of 70, 55, 44, 37, 32 and 28 kDa. The presence of native (32 kDa) and heat-modified forms (37 kDa) of OprF was revealed by immuno-analysis and the 44-kDa band was composed of three proteins, their N-terminal sequences showing homologies with Pseudomonas aeruginosa porins (OprD, OprE1 and OprE3). PMID:12393211

  2. Beamlet focal plane diagnostic

    SciTech Connect

    Caird, J.A.; Nielsen, N.D.; Patton, H.G.; Seppala, L.G.; Thompson, C.E.; Wegner, P.J.

    1996-12-01

    This paper describes the major optical and mechanical design features of the Beamlet Focal Plane Diagnostic system as well as measurements of the system performance, and typical data obtained to date. We also discuss the NIF requirements on the focal spot that we are interested in measuring, and some of our plans for future work using this system.

  3. In Vitro Analysis of Fibronectin-Modified Titanium Surfaces

    PubMed Central

    Chang, Yu-Chi; Lee, Wei-Fang; Feng, Sheng-Wei; Huang, Haw-Ming; Lin, Che-Tong; Teng, Nai-Chia; Chang, Wei Jen

    2016-01-01

    Background Glow discharge plasma (GDP) procedure is an effective method for grafting various proteins, including albumin, type I collagen, and fibronectin, onto a titanium surface. However, the behavior and impact of titanium (Ti) surface modification is yet to be unraveled. Purpose The purpose of this study is to evaluate and analyze the biological properties of fibronectin-grafted Ti surfaces treated by GDP. Materials and Methods Grade II Ti discs were initially cleaned and autoclaved to obtain original specimens. Subsequently, the specimens were GDP treated and grafted with fibronectin to form Ar-GDP (Argon GDP treatment only) and GDP-fib (fibronectin coating following GDP treatment) groups. Blood coagulation test and MG-63 cell culture were performed to evaluate the biological effects on the specimen. Results There was no significant difference between Ar-GDP and GDP-fib groups in blood compatibility analysis. While in the MTT test, cellular proliferation was benefited from the presence of fibronectin coating. The numbers of cells on Ar-GDP and GDP-fib specimens were greater than those in the original specimens after 24 h of culturing. Conclusions GDP treatment combined with fibronectin grafting favored MG-63 cell adhesion, migration, and proliferation on titanium surfaces, which could be attributed to the improved surface properties. PMID:26731536

  4. Fibronectin receptors of mononuclear phagocytes: Binding characteristics and biochemical isolation

    SciTech Connect

    Garcia-Pardo, A.; Ferreira, O.C.; Valinsky, J.; Bianco, C. )

    1989-04-01

    Fibronectin receptors on mononuclear phagocytes are involved in the localization of monocytes at inflammatory sites and in the subsequent expression of macrophage-like phenotypes. In this study, the authors have investigated the hypothesis that preteolytically derived fragments of fibronectin may interfere with binding of fibronectin to monocytes in the extracellular matrix. They report on the reactivity of U937 cells with an 80-kDa tryptic fragment of fibronectin which contains the cell-binding domain but lacks the gelatin/collagen-binding domain. U937 cells attached to surfaces coated with the 80-kDa fragment as well as with intact fibronectin. Preincubation of the cells with the 80-kDa fragment inhibited attachment to both surfaces while intact fibronectin had little or no inhibitory effect. This complex resolved into a single diffuse band of 144 kDa upon reduction. Binding of the protein complex to the affinity column required divalent cations. The complex bound to wheat germ agglutinin and could be specifically eluted by N-acetylglucosamine. Similar cell-surface proteins were isolated from peripheral blood monocytes.

  5. Fibronectin matrix assembly is essential for cell condensation during chondrogenesis

    PubMed Central

    Singh, Purva; Schwarzbauer, Jean E.

    2014-01-01

    ABSTRACT Mesenchymal cell condensation is the initiating event in endochondral bone formation. Cell condensation is followed by differentiation into chondrocytes, which is accompanied by induction of chondrogenic gene expression. Gene mutations involved in chondrogenesis cause chondrodysplasias and other skeletal defects. Using mesenchymal stem cells (MSCs) in an in vitro chondrogenesis assay, we found that knockdown of the diastrophic dysplasia (DTD) sulfate transporter (DTDST, also known as SLC26A2), which is required for normal cartilage development, blocked cell condensation and caused a significant reduction in fibronectin matrix. Knockdown of fibronectin with small interfering RNAs (siRNAs) also blocked condensation. Fibrillar fibronectin matrix was detected prior to cell condensation, and its levels increased during and after condensation. Inhibition of fibronectin matrix assembly by use of the functional upstream domain (FUD) of adhesin F1 from Streptococcus pyogenes prevented cell condensation by MSCs and also by the chondrogenic cell line ATDC5. Our data show that cell condensation and induction of chondrogenesis depend on fibronectin matrix assembly and DTDST, and indicate that this transporter is required earlier in chondrogenesis than previously appreciated. They also raise the possibility that certain of the skeletal defects in DTD patients might derive from the link between DTDST, fibronectin matrix and condensation. PMID:25146392

  6. Tendon synovial cells secrete fibronectin in vivo and in vitro

    SciTech Connect

    Banes, A.J.; Link, G.W.; Bevin, A.G.; Peterson, H.D.; Gillespie, Y.; Bynum, D.; Watts, S.; Dahners, L.

    1988-01-01

    The chemistry and cell biology of the tendon have been largely overlooked due to the emphasis on collagen, the principle structural component of the tendon. The tendon must not only transmit the force of muscle contraction to bone to effect movement, but it must also glide simultaneously over extratendonous tissues. Fibronectin is classified as a cell attachment molecule that induces cell spreading and adhesion to substratum. The external surface of intact avian flexor tendon stained positively with antibody to cellular fibronectin. However, if the surface synovial cells were first removed with collagenase, no positive reaction with antifibronectin antibody was detected. Analysis of immunologically stained frozen sections of tendon also revealed fibronectin at the tendon synovium, but little was associated with cells internal in tendon. The staining pattern with isolated, cultured synovial cells and fibroblasts from the tendon interior substantiated the histological observations. Analysis of polyacrylamide gel profiles of /sup 35/S-methionine-labeled proteins synthesized by synovial cells and internal fibroblasts indicated that fibronectin was synthesized principally by synovial cells. Fibronectin at the tendon surface may play a role in cell attachment to prevent cell removal by the friction of gliding. Alternatively, fibronectin, with its binding sites for hyaluronic acid and collagen, may act as a complex for boundary lubrication.

  7. Fibronectin Inhibits Chronic Pain Development after Spinal Cord Injury

    PubMed Central

    Lee, Yu-Shang; Lin, Vernon W.; Silver, Jerry

    2012-01-01

    Abstract Chronic pain following spinal cord injury (SCI) is a highly prevalent clinical condition that is difficult to treat. Using both von Frey filaments and radiant infrared heat to assess mechanical allodynia and thermal hyperalgesia, respectively, we have demonstrated that a one-time injection of fibronectin (50 μg/mL) into the spinal dorsal column (1 μL/min each injection for a total of 5 μL) immediately after SCI inhibits the development of mechanical allodynia (but not thermal hyperalgesia) over an 8-month observation period following spinal cord dorsal column crush (DCC). DCC will only induce mechanical Allodynia, but not thermal hyperalgesia or overt motor deficits. By applying various fibronectin fragments as well as competitive inhibitors, these effects were shown to be dependent on the connecting segment-1 (CS-1) motif of fibronectin. Furthermore, we found that acute fibronectin treatment diminished inflammation and blood–spinal cord barrier permeability, which in turn leads to enhanced fiber sparing and sprouting. In particular, the reduction of serotonin (5-HT) in the superficial dorsal horn, an important descending brainstem system in the modulation of pain, was blocked with fibronectin treatment. We conclude that treatment of SCI with fibronectin preserves sensory regulation and prevents the development of chronic allodynia, providing a potential therapeutic intervention to treat chronic pain following SCI. PMID:22022865

  8. Integrin activation and focal complex formation in cardiac hypertrophy

    NASA Technical Reports Server (NTRS)

    Laser, M.; Willey, C. D.; Jiang, W.; Cooper, G. 4th; Menick, D. R.; Zile, M. R.; Kuppuswamy, D.

    2000-01-01

    Cardiac hypertrophy is characterized by both remodeling of the extracellular matrix (ECM) and hypertrophic growth of the cardiocytes. Here we show increased expression and cytoskeletal association of the ECM proteins fibronectin and vitronectin in pressure-overloaded feline myocardium. These changes are accompanied by cytoskeletal binding and phosphorylation of focal adhesion kinase (FAK) at Tyr-397 and Tyr-925, c-Src at Tyr-416, recruitment of the adapter proteins p130(Cas), Shc, and Nck, and activation of the extracellular-regulated kinases ERK1/2. A synthetic peptide containing the Arg-Gly-Asp (RGD) motif of fibronectin and vitronectin was used to stimulate adult feline cardiomyocytes cultured on laminin or within a type-I collagen matrix. Whereas cardiocytes under both conditions showed RGD-stimulated ERK1/2 activation, only collagen-embedded cells exhibited cytoskeletal assembly of FAK, c-Src, Nck, and Shc. In RGD-stimulated collagen-embedded cells, FAK was phosphorylated only at Tyr-397 and c-Src association occurred without Tyr-416 phosphorylation and p130(Cas) association. Therefore, c-Src activation is not required for its cytoskeletal binding but may be important for additional phosphorylation of FAK. Overall, our study suggests that multiple signaling pathways originate in pressure-overloaded heart following integrin engagement with ECM proteins, including focal complex formation and ERK1/2 activation, and many of these pathways can be activated in cardiomyocytes via RGD-stimulated integrin activation.

  9. Expression of fibronectin, fibronectin isoforms and integrin receptors in melanocytic lesions.

    PubMed Central

    Natali, P. G.; Nicotra, M. R.; Di Filippo, F.; Bigotti, A.

    1995-01-01

    In vitro studies have demonstrated that fibronectin (FN) can deliver a mitogenic signal to quiescent human melanoma cells and that the alpha 5/beta 1-integrin receptor mediates this stimulus. In view of this finding we have analysed the in vivo expression of FN, and of ED-A and ED-B FN isoforms, in benign and malignant lesions of melanocyte origin. In the same specimens the expression of fibronectin integrin receptors was evaluated. The results demonstrate that, while detection of FN does not correlate with transformation and tumour progression, the expression of the two isoforms is associated with transformation and that only the ED-A variant is found in metastases. Integrin phenotyping disclosed that alpha 3/beta 1 expression is associated with tumour progression, alpha v/beta 3 is a marker of transformation, alpha 4 is rarely expressed and alpha 5 is expressed by about 50% and 30% of the primary and metastatic lesions respectively. Taken together, the results of this study demonstrate that transformation and tumour progression of the melanocyte lineage are associated with modulation of expression of FN isoforms and FN integrin receptors. Furthermore, the expression of alpha 5-integrin in a considerable percentage of primary and metastatic lesions indicates that FN may deliver a proliferative stimulus to melanoma cells in vivo. Images Figure 1 PMID:7779718

  10. Partial (focal) seizure

    MedlinePlus

    ... Jacksonian seizure; Seizure - partial (focal); Temporal lobe seizure; Epilepsy - partial seizures ... Abou-Khalil BW, Gallagher MJ, Macdonald RL. Epilepsies. In: Daroff ... Practice . 7th ed. Philadelphia, PA: Elsevier; 2016:chap 101. ...

  11. Focal vibration in neurorehabilitation.

    PubMed

    Murillo, N; Valls-Sole, J; Vidal, J; Opisso, E; Medina, J; Kumru, H

    2014-04-01

    During the last decade, many studies have been carried out to understand the effects of focal vibratory stimuli at various levels of the central nervous system and to study pathophysiological mechanisms of neurological disorders as well as the therapeutic effects of focal vibration in neurorehabilitation. This review aimed to describe the effects of focal vibratory stimuli in neurorehabilitation including the neurological diseases or disorders like stroke, spinal cord injury, multiple sclerosis, Parkinson's' disease and dystonia. In conclusion, focal vibration stimulation is well tolerated, effective and easy to use, and it could be used to reduce spasticity, to promote motor activity and motor learning within a functional activity, even in gait training, independent from etiology of neurological pathology. Further studies are needed in the future well-designed trials with bigger sample size to determine the most effective frequency, amplitude and duration of vibration application in the neurorehabilitation. PMID:24842220

  12. Focal neurological deficits

    MedlinePlus

    A focal neurologic deficit is a problem with nerve, spinal cord, or brain function. It affects a specific ... of the back, neck, or head Electromyogram (EMG)/ nerve conduction velocities (NCV) MRI of the back, neck, or head Spinal tap

  13. Partial (focal) seizure

    MedlinePlus

    ... Jacksonian seizure; Seizure - partial (focal); Temporal lobe seizure; Epilepsy - partial seizures ... Abou-Khalil BW, Gallagher MJ, Macdonald RL. Epilepsies. In: Daroff RB, ... 6th ed. Philadelphia, PA: Elsevier Saunders; 2012:chap 67. ...

  14. Purification of a mycobacterial adhesin for fibronectin.

    PubMed Central

    Ratliff, T L; McCarthy, R; Telle, W B; Brown, E J

    1993-01-01

    Previous studies have demonstrated that mycobacteria attach to fibronectin (FN). The attachment of mycobacteria to FN is considered to be biologically important in Mycobacterium bovis BCG therapy for superficial bladder cancer, initiation of delayed hypersensitivity to mycobacterial antigens, and the phagocytosis of mycobacteria by epithelial cells. Therefore, we purified the mycobacterial receptor for FN. Culture supernatants from 3-week cultures of Mycobacterium vaccae, which contained proteins that bound FN and inhibited the attachment of both M. vaccae and BCG to FN, were used as a source of receptor. Lyophilized M. vaccae supernatants were reconstituted in 0.02 M bis-Tris (pH 6.0) and applied sequentially to an ACA 54 gel filtration column and a DEAE-Sephacel anion-exchange column. A purified inhibitory protein of 55 kDa (p55) was obtained. The purified p55 protein was observed to bind to FN and to inhibit 125I-FN binding to viable BCG in a dose-dependent manner. Polyclonal and monoclonal antibodies to the protein were generated. The resulting polyclonal antiserum blotted a single protein band at 55 kDa in crude M. vaccae supernatants, cross-reacted with a 55-kDa BCG protein by Western blot (immunoblot), and recognized a 55-kDa band that was associated with the BCG cell wall, which is consistent with its function as a FN receptor. A monoclonal immunoglobulin M(lambda) was isolated from mice immunized with purified M. vaccae p55 protein that was not functional in Western blots but inhibited the attachment of viable BCG to FN. These studies demonstrate that a protein or antigenically related proteins with M(r)s of 55,000 function as FN receptors for at least two distinct mycobacteria. Images PMID:8478078

  15. Expression of laminin and fibronectin in renal dysplasia.

    PubMed

    Menon, Santosh; Kakkar, Nandita; Radotra, B D

    2004-01-01

    The pathogenesis of renal dysplasia is a matter of debate. Recent theories have conceptualized the role of extracellular matrix proteins in the genesis of renal dysplasia. During normal nephrogenesis, collagen type I and III and fibronectins are lost and laminin and syndecan appear once proper induction has occurred. Any deviation from the normal pattern is said to lead to dysplasia. In this study, the expressions of adhesive glycoproteins, laminin, and fibronectin were studied immunohistochemically in 25 autopsy cases of renal dysplasia and normal age-matched control cases. These cases of renal dysplasia were categorized into 3 groups based on the period of gestation: 20 to 26 weeks, 27 to 33 weeks, and 34 to 40 weeks. The immunohistochemical findings were graded from 0 to 4+ based on the visual intensity. Chi-square analysis was used to calculate the difference in expressions of laminin and fibronectin in cases and controls as a whole and within and between age groups. Immunostaining for laminin in all age groups showed a significant difference in expression between dysplastic kidneys (less expression) and normal controls (greater expression). In the case of fibronectin expression, all but 1 group showed a significant difference, with dysplastic kidneys showing more and normal controls showing less expression. The inference derived is that laminin expression decreases and fibronectin expression increases in renal dysplasia compared with normal nephrogenesis. PMID:15630524

  16. Mesothelial cells promote early ovarian cancer metastasis through fibronectin secretion

    PubMed Central

    Kenny, Hilary A.; Chiang, Chun-Yi; White, Erin A.; Schryver, Elizabeth M.; Habis, Mohammed; Romero, Iris L.; Ladanyi, Andras; Penicka, Carla V.; George, Joshy; Matlin, Karl; Montag, Anthony; Wroblewski, Kristen; Yamada, S. Diane; Mazar, Andrew P.; Bowtell, David; Lengyel, Ernst

    2014-01-01

    Ovarian cancer (OvCa) metastasizes to organs in the abdominal cavity, such as the omentum, which are covered by a single layer of mesothelial cells. Mesothelial cells are generally thought to be “bystanders” to the metastatic process and simply displaced by OvCa cells to access the submesothelial extracellular matrix. Here, using organotypic 3D cultures, we found that primary human mesothelial cells secrete fibronectin in the presence of OvCa cells. Moreover, we evaluated the tumor stroma of 108 human omental metastases and determined that fibronectin was consistently overexpressed in these patients. Blocking fibronectin production in primary mesothelial cells in vitro or in murine models, either genetically (fibronectin 1 floxed mouse model) or via siRNA, decreased adhesion, invasion, proliferation, and metastasis of OvCa cells. Using a coculture model, we determined that OvCa cells secrete TGF-β1, which in turn activates a TGF-β receptor/RAC1/SMAD-dependent signaling pathway in the mesothelial cells that promotes a mesenchymal phenotype and transcriptional upregulation of fibronectin. Additionally, blocking α5 or β1 integrin function with antibodies reduced metastasis in an orthotopic preclinical model of OvCa metastasis. These findings indicate that cancer-associated mesothelial cells promote colonization during the initial steps of OvCa metastasis and suggest that mesothelial cells actively contribute to metastasis. PMID:25202979

  17. The dispersion-focalization theory of sound systems

    NASA Astrophysics Data System (ADS)

    Schwartz, Jean-Luc; Abry, Christian; Boë, Louis-Jean; Vallée, Nathalie; Ménard, Lucie

    2005-04-01

    The Dispersion-Focalization Theory states that sound systems in human languages are shaped by two major perceptual constraints: dispersion driving auditory contrast towards maximal or sufficient values [B. Lindblom, J. Phonetics 18, 135-152 (1990)] and focalization driving auditory spectra towards patterns with close neighboring formants. Dispersion is computed from the sum of the inverse squared inter-spectra distances in the (F1, F2, F3, F4) space, using a non-linear process based on the 3.5 Bark critical distance to estimate F2'. Focalization is based on the idea that close neighboring formants produce vowel spectra with marked peaks, easier to process and memorize in the auditory system. Evidence for increased stability of focal vowels in short-term memory was provided in a discrimination experiment on adult French subjects [J. L. Schwartz and P. Escudier, Speech Comm. 8, 235-259 (1989)]. A reanalysis of infant discrimination data shows that focalization could well be the responsible for recurrent discrimination asymmetries [J. L. Schwartz et al., Speech Comm. (in press)]. Recent data about children vowel production indicate that focalization seems to be part of the perceptual templates driving speech development. The Dispersion-Focalization Theory produces valid predictions for both vowel and consonant systems, in relation with available databases of human languages inventories.

  18. [A simple and effective step in the purification of bovine blood fibronectin].

    PubMed

    Zykova, T A; Zlatopol'skiĭ, A D; Mazurov, V I

    1983-01-01

    Preparations of fibronectin from bovine blood serum, obtained by means of affinity chromatography on collagen-Sepharose, contained immunoglobulins and other proteins, concentration of which constituted 48 +/- 5%. Differential salting out of fibronectin and other non-fibronectin proteins, using 0.8-2.0 M ammonium sulfate at pH 5.0, demonstrated that precipitation of fibronectin occurred more effectively as compared with non-fibronectin proteins at all the salt concentrations studied. If 0.8 M or 1.0 M ammonium sulfate concentrations were used, the fibronectin preparations contained less than 10% of other proteins and fibronectin loss was about 20%. Salting out of fibronectin is an effective additional step of its purification. PMID:6649521

  19. Fibronectin Binds to Some Bacteria but Does not Promote Their Uptake by Phagocytic Cells

    NASA Astrophysics Data System (ADS)

    van de Water, L.; Destree, A. T.; Hynes, R. O.

    1983-04-01

    The involvement of plasma fibronectin in phagocytosis of bacteria was investigated by testing the binding of fibronectin to several species of bacteria and by evaluating the ability of fibronectin to promote binding and endocytosis of two species of these bacteria by phagocytic cells. Fibronectin binds non-covalently to Gram-positive and Gram-negative bacteria and to yeast but did not appear to be necessary or sufficient for uptake of Staphylococcus aureus and Salmonella typhimurium by several different phagocytic cell types.

  20. Display of Cell Surface Sites for Fibronectin Assembly Is Modulated by Cell Adherence to 1F3 and C-Terminal Modules of Fibronectin

    PubMed Central

    Zhang, Qinghong; Annis, Douglas S.; Erickson, Harold P.; Mosher, Deane F.

    2009-01-01

    Background Fibronectin-null cells assemble soluble fibronectin shortly after adherence to a substrate coated with intact fibronectin but not when adherent to the cell-binding domain of fibronectin (modules 7F3-10F3). Interactions of adherent cells with regions of adsorbed fibronectin other than modules 7F3-10F3, therefore, are required for early display of the cell surface sites that initiate and direct fibronectin assembly. Methodology/Principal Findings To identify these regions, coatings of proteolytically derived or recombinant pieces of fibronectin containing modules in addition to 7F3-10F3 were tested for effects on fibronectin assembly by adherent fibronectin-null fibroblasts. Pieces as large as one comprising modules 2F3-14F3, which include the heparin-binding and cell adhesion domains, were not effective in supporting fibronectin assembly. Addition of module 1F3 or the C-terminal modules to modules 2F3-14F3 resulted in some activity, and addition of both 1F3 and the C-terminal modules resulted in a construct, 1F3-C, that best mimicked the activity of a coating of intact fibronectin. Constructs 1F3-C V0, 1F3-C V64, and 1F3-C Δ(V15F310F1) were all able to support fibronectin assembly, suggesting that 1F3 through 11F1 and/or 12F1 were important for activity. Coatings in which the active parts of 1F3-C were present in different proteins were much less active than intact 1F3-C. Conclusions These results suggest that 1F3 acts together with C-terminal modules to induce display of fibronectin assembly sites on adherent cells. PMID:19119318

  1. SNAP focal plane

    SciTech Connect

    Lampton, Michael L.; Kim, A.; Akerlof, C.W.; Aldering, G.; Amanullah, R.; Astier, P.; Barrelet, E.; Bebek, C.; Bergstrom, L.; Berkovitz, J.; Bernstein, G.; Bester, M.; Bonissent, A.; Bower, C.; Carithers Jr., W.C.; Commins, E.D.; Day, C.; Deustua, S.E.; DiGennaro,R.; Ealet, A.; Ellis, R.S.; Eriksson, M.; Fruchter, A.; Genat, J.-F.; Goldhaber, G.; Goobar, A.; Groom, D.; Harris, S.E.; Harvey, P.R.; Heetderks, H.D.; Holland, S.E.; Huterer, D.; Karcher, A.; Kolbe, W.; Krieger, B.; Lafever, R.; Lamoureux, J.; Levi, M.E.; Levin, D.S.; Linder,E.V.; Loken, S.C.; Malina, R.; Massey, R.; McKay, T.; McKee, S.P.; Miquel, R.; Mortsell, E.; Mostek, N.; Mufson, S.; Musser, J.; Nugent, P.; Oluseyi, H.; Pain, R.; Palaio, N.; Pankow, D.; Perlmutter, S.; Pratt, R.; Prieto, E.; Refregier, A.; Rhodes, J.; Robinson, K.; Roe, N.; Sholl, M.; Schubnell, M.; Smadja, G.; Smoot, G.; Spadafora, A.; Tarle, G.; Tomasch,A.; von der Lippe, H.; Vincent, R.; Walder, J.-P.; Wang, G.

    2002-07-29

    The proposed SuperNova/Acceleration Probe (SNAP) mission will have a two-meter class telescope delivering diffraction-limited images to an instrumented 0.7 square-degree field sensitive in the visible and near-infrared wavelength regime. We describe the requirements for the instrument suite and the evolution of the focal plane design to the present concept in which all the instrumentation--visible and near-infrared imagers, spectrograph, and star guiders--share one common focal plane.

  2. Selective Cell Growth on Fibronectin-Carbon Nanotube Hybrid Nanostructures

    NASA Astrophysics Data System (ADS)

    Namgung, Seon; Park, Sung Young; Lee, Byung Yang; Lee, Minbaek; Nam, Jwa-Min; Hong, Seunghun

    2008-03-01

    Carbon nanotubes (CNT) have been considered a promising material for biological applications including biosensors, therapeutic application, and nano-structured scaffolds. However, there are still controversies associated with toxicity and biocompatibility of CNTs on live cells. Here, we report general strategy to functionalize CNTs with cell adhesion molecules (fibronectins) for selective and stable adhesion of cells on CNTs. Interestingly, more fibronectins were adsorbed and activated on CNTs rather than on hydrophobic self assembled monolayers (SAMs) or bare substrates (SiO2). We demonstrate the functionality of fibronectins on CNTs with immunofluorescence and molecule-level force measurement study using atomic force microscopy (AFM). These fibronectin-CNT hybrid nanostructures were successfully applied to attract cells selectively onto predefined regions on the substrate. Our strategy was generally available on various cell types including mesenchymal stem cells, KB cells, and NIH3T3 fibroblast cells (Advanced Materials 19, 2530-2534 (2007)). We will also discuss about its impacts on cell biology combined with CNTs.

  3. Detection of fibronectin receptor in sera: its clinical significance as a parameter of hepatic fibrosis.

    PubMed

    Yamauchi, M; Nakajima, H; Ohata, M; Hirakawa, J; Mizuhara, Y; Nakahara, M; Kimura, K; Fujisawa, K; Kameda, H

    1991-08-01

    Pooled sera collected from cirrhotic patients was fractionated by affinity chromatography with a fibronectin receptor monoclonal antibody against the beta-subunit of fibronectin receptor. Eluates were assayed using Western immunoblotting. The relative mobility of the protein reactive with fibronectin receptor antibody was nearly identical to that of the beta-subunit of fibronectin receptor, confirming that fibronectin receptor is present in human serum. Serum levels of the beta-subunit of fibronectin receptor were analyzed by sandwich enzyme-linked immunosorbent assay in patients with various liver diseases. The serum level of fibronectin receptor (micrograms/ml) was significantly higher in patients with chronic hepatitis (inactive, 2.59 +/- 0.04; active, 3.45 +/- 0.13), cirrhosis (4.77 +/- 0.30), alcoholic liver disease (2.96 +/- 0.16) and hepatocellular carcinoma (4.71 +/- 0.49) than in normal subjects (2.11 +/- 0.08). Strong positive correlation was observed between serum levels of fibronectin receptor and histological findings, particularly in the degree of hepatic fibrosis. Immunohistochemical studies with fibronectin receptor antibody revealed that the beta-subunit of fibronectin receptor was present on the plasma membrane of hepatocytes and sinusoidal lining cells in the normal liver and was increased in fibrotic areas and on the plasma membrane of hepatocytes and sinusoidal lining cells of fibrotic liver. The serum level of fibronectin receptor in patients with chronic liver diseases may therefore be a useful marker of hepatic fibrosis. PMID:1830562

  4. Focal adhesions, stress fibers and mechanical tension

    PubMed Central

    Burridge, Keith; Guilluy, Christophe

    2016-01-01

    Stress fibers and focal adhesions are complex protein arrays that produce, transmit and sense mechanical tension. Evidence accumulated over many years led to the conclusion that mechanical tension generated within stress fibers contributes to the assembly of both stress fibers themselves and their associated focal adhesions. However, several lines of evidence have recently been presented against this model. Here we discuss the evidence for and against the role of mechanical tension in driving the assembly of these structures. We also consider how their assembly is influenced by the rigidity of the substratum to which cells are adhering. Finally, we discuss the recently identified connections between stress fibers and the nucleus, and the roles that these may play, both in cell migration and regulating nuclear function. PMID:26519907

  5. Allosteric Regulation of Fibronectin/α5β1 Interaction by Fibronectin-Binding MSCRAMMs

    PubMed Central

    Liang, Xiaowen; Garcia, Brandon L.; Visai, Livia; Prabhakaran, Sabitha; Meenan, Nicola A. G.; Potts, Jennifer R.; Humphries, Martin J.; Höök, Magnus

    2016-01-01

    Adherence of microbes to host tissues is a hallmark of infectious disease and is often mediated by a class of adhesins termed MSCRAMMs (Microbial Surface Components Recognizing Adhesive Matrix Molecules). Numerous pathogens express MSCRAMMs that specifically bind the heterodimeric human glycoprotein fibronectin (Fn). In addition to roles in adhesion, Fn-binding MSCRAMMs exploit physiological Fn functions. For example, several pathogens can invade host cells by a mechanism whereby MSCRAMM-bound Fn bridges interaction with α5β1 integrin. Here, we investigate two Fn-binding MSCRAMMs, FnBPA (Staphylococcus aureus) and BBK32 (Borrelia burgdorferi) to probe structure-activity relationships of MSCRAMM-induced Fn/α5β1integrin activation. Circular dichroism, fluorescence resonance energy transfer, and dynamic light scattering techniques uncover a conformational rearrangement of Fn involving domains distant from the MSCRAMM binding site. Surface plasmon resonance experiments demonstrate a significant enhancement of Fn/α5β1 integrin affinity in the presence of FnBPA or BBK32. Detailed kinetic analysis of these interactions reveal that this change in affinity can be attributed solely to an increase in the initial Fn/α5β1 on-rate and that this rate-enhancement is dependent on high-affinity Fn-binding by MSCRAMMs. These data implicate MSCRAMM-induced perturbation of specific intramolecular contacts within the Fn heterodimer resulting in activation by exposing previously cryptic α5β1 interaction motifs. By correlating structural changes in Fn to a direct measurement of increased Fn/α5β1 affinity, this work significantly advances our understanding of the structural basis for the modulation of integrin function by Fn-binding MSCRAMMs. PMID:27434228

  6. Requirement of focal adhesion kinase in branching tubulogenesis.

    PubMed

    Wei, Wei-Chun; Kopec, Anna K; Tang, Ming-Jer

    2009-01-01

    We previously demonstrated that alpha3beta1 integrins are essential to hepatocyte growth factor (HGF)-independent branching tubulogenesis in Mardin-Darby Canine Kidney (MDCK) cells. However, the involvement of integrin downstream signaling molecules remains unclear. In the present study, we successfully isolated cell lines possessing different tubulogenic potentials from the MDCK cells; cyst clones (CA4, CA6) forming cystic structures when cultured in 0.3% type I collagen gel and mass clones (M610, M611, M612) forming aggregated masses. Cyst clones maintained cystic structure in 0.1% collagen gel, whereas mass clones spontaneously developed into tubules. Both clones exhibited various morphologies when cultured on a dish: cyst clones formed aggregated islands, while mass clones were more scattered and exhibited higher migration capacity. Among several focal adhesion machinery proteins examined, only the expression and phosphorylation level of focal adhesion kinase (FAK) in mass clones was higher than in cyst clones, while other proteins showed no obvious differences. However, overexpression of wild type FAK in CA6 cells did not facilitate branching tubule formation in 0.1% collagen gel. Targeted decrease in the expression level of FAK in M610 cells with the application of antisense cDNA resulted in a marked reduction of branching tubule formation in 0.1% collagen gel and showed a down-regulation of fibronectin assembly, which is known to promote tubulogenesis. In contrast, overexpression of wild type FAK in CA6 cells had no effect on fibronectin assembly. Taken together, our data demonstrates that FAK is required, but not sufficient for HGF-independent branching tubulogenesis in MDCK cells. PMID:19272169

  7. Demonstration of fibronectin in human articular cartilage by an indirect immunoperoxidase technique.

    PubMed

    Clemmensen, I; Hølund, B; Johansen, N; Andersen, R B

    1982-01-01

    Fresh frozen tissue sections of human articular cartilage was treated without and with human testicular hyaluronidase (2 x 10(6) units/l) for 60 min at 37 degrees C and stained by the indirect immunoperoxidase technique with rabbit antihuman fibronectin. The rabbit antihuman fibronectin was purified by affinity chromatography on human fibronectin-Sepharose. Fibronectin was only found on the acellular surface of the articular cartilage in tissue sections not treated with hyaluronidase. In this surface layer, probably identical to "lamina splendens", the arrangement of fibronectin was as a membrane. No collagen was seen in this area by van Gieson staining. No staining for fibronectin was found in the cartilage matrix or in the chondrocytes. Treatment of the cartilage tissue with hyaluronidase resulted in visualization of high amount of fibronectin in the cartilage matrix, with the highest intensity around the chondrocytes. The staining of the acellular surface layer of the articular cartilage was identical with the results obtained without hyaluronidase treatment. These results indicate that articular cartilage is rich in fibronectin probably in complex with hyaluronic acid, and that the chondrocytes produce fibronectin in situ. It also demonstrates the steric hindrance of hyaluronic acid aggregates in diffusion of the antibody and the value of hyaluronidase treatment of tissue before demonstration of fibronectin. PMID:6757202

  8. Dexamethasone-Mediated Activation of Fibronectin Matrix Assembly Reduces Dispersal of Primary Human Glioblastoma Cells

    PubMed Central

    Shannon, Stephen; Vaca, Connan; Jia, Dongxuan; Entersz, Ildiko; Schaer, Andrew; Carcione, Jonathan; Weaver, Michael; Avidar, Yoav; Pettit, Ryan; Nair, Mohan; Khan, Atif; Foty, Ramsey A.

    2015-01-01

    Despite resection and adjuvant therapy, the 5-year survival for patients with Glioblastoma multiforme (GBM) is less than 10%. This poor outcome is largely attributed to rapid tumor growth and early dispersal of cells, factors that contribute to a high recurrence rate and poor prognosis. An understanding of the cellular and molecular machinery that drive growth and dispersal is essential if we are to impact long-term survival. Our previous studies utilizing a series of immortalized GBM cell lines established a functional causation between activation of fibronectin matrix assembly (FNMA), increased tumor cohesion, and decreased dispersal. Activation of FNMA was accomplished by treatment with Dexamethasone (Dex), a drug routinely used to treat brain tumor related edema. Here, we utilize a broad range of qualitative and quantitative assays and the use of a human GBM tissue microarray and freshly-isolated primary human GBM cells grown both as conventional 2D cultures and as 3D spheroids to explore the role of Dex and FNMA in modulating various parameters that can significantly influence tumor cell dispersal. We show that the expression and processing of fibronectin in a human GBM tissue-microarray is variable, with 90% of tumors displaying some abnormality or lack in capacity to secrete fibronectin or assemble it into a matrix. We also show that low-passage primary GBM cells vary in their capacity for FNMA and that Dex treatment reactivates this process. Activation of FNMA effectively “glues” cells together and prevents cells from detaching from the primary mass. Dex treatment also significantly increases the strength of cell-ECM adhesion and decreases motility. The combination of increased cohesion and decreased motility discourages in vitro and ex vivo dispersal. By increasing cell-cell cohesion, Dex also decreases growth rate of 3D spheroids. These effects could all be reversed by an inhibitor of FNMA and by the glucocorticoid receptor antagonist, RU-486. Our

  9. Fish Rhabdovirus Cell Entry Is Mediated by Fibronectin

    PubMed Central

    Bearzotti, Monique; Delmas, Bernard; Lamoureux, Annie; Loustau, Anne-Marie; Chilmonczyk, Stefan; Bremont, Michel

    1999-01-01

    Three monoclonal antibodies (MAbs) generated against rainbow trout gonad cells (RTG-2) have been selected for their ability to protect cells from the viral hemorrhagic septicemia virus (VHSV) infection, a salmonid rhabdovirus. Protection from infection was restricted to the salmonid-derived cell lines indicating species specificity of the blocking MAbs. Surprisingly, the blocking activity of these MAbs was also effective against other nonantigenically related fish rhabdoviruses. Indirect immunofluorescence and immunoelectron microscopy observations demonstrated that the three MAbs were all directed against an abundant cell plasma membrane component, and immunoprecipitation studies indicated that the target consisted of a heterodimeric complex with molecular masses of 200 and 44 kDa. Biochemical data provided the following evidence that fibronectin is part of this complex and that it could represent the main receptor for fish rhabdoviruses. (i) An antiserum generated against the 200-kDa protein reacted against the recombinant rainbow trout fibronectin expressed in Escherichia coli. (ii) The purified rainbow trout fibronectin was able to bind specifically to VHSV. To our knowledge, this is the first identification of a cellular component acting as a primary receptor for a virus replicating in lower vertebrates and, more interestingly, for viruses belonging to the Rhabdoviridae family. PMID:10438860

  10. Improved interaction of osteoblast-like cells with apatite-nanodiamond coatings depends on fibronectin.

    PubMed

    Hristova, K; Pecheva, E; Pramatarova, L; Altankov, G

    2011-08-01

    New apatite (AP)/nanodiamond (ND) coating has been developed to improve physical and biological properties of stainless steel (SS) versus single AP coating. Homogeneously electrodeposited AP-ND layer demonstrates increased mechanical strength, interlayer cohesion and ductility. In the absence of serum, osteoblast-like MG63 cells attach well but poorly spread on both AP and AP-ND substrata. Pre-adsorption with serum or fibronectin (FN) improves the cellular interaction-an effect that is better pronounced on the AP-ND coating. In single protein adsorption study fluorescein isothiocyanate-labeled FN (FITC-FN) shows enhanced deposition on the AP-ND layer consistent with the significantly improved cell adhesion, spreading and focal adhesions formation (in comparison to SS and AP), particularly at low FN adsorption concentrations (1 μg/ml). Higher FN concentrations (20 μg/ml) abolish this difference suggesting that the promoted cellular interaction of serum (where FN is low) is caused by the greater affinity for FN. Moreover, it is found that MG63 cells tend to rearrange both adsorbed and secreted FN on the AP-ND layer suggesting facilitated FN matrix formation. PMID:21706219

  11. Suberoylanilide hydroxamic acid (SAHA) at subtoxic concentrations increases the adhesivity of human leukemic cells to fibronectin.

    PubMed

    Kuzelová, Katerina; Pluskalová, Michaela; Brodská, Barbora; Otevrelová, Petra; Elknerová, Klára; Grebenová, Dana; Hrkal, Zbynek

    2010-01-01

    Suberoylanilide hydroxamic acid (SAHA) is an inhibitor of histone deacetylases (HDACs) which is being introduced into clinic for the treatment of hematological diseases. We studied the effect of this compound on six human hematopoietic cell lines (JURL-MK1, K562, CML-T1, Karpas-299, HL-60, and ML-2) as well as on normal human lymphocytes and on leukemic primary cells. SAHA induced dose-dependent and cell type-dependent cell death which displayed apoptotic features (caspase-3 activation and apoptotic DNA fragmentation) in most cell types including the normal lymphocytes. At subtoxic concentrations (0.5-1 microM), SAHA increased the cell adhesivity to fibronectin (FN) in all leukemia/lymphoma-derived cell lines but not in normal lymphocytes. This increase was accompanied by an enhanced expression of integrin beta1 and paxillin, an essential constituent of focal adhesion complexes, both at the protein and mRNA level. On the other hand, the inhibition of ROCK protein, an important regulator of cytoskeleton structure, had no consistent effect on SAHA-induced increase in the cell adhesivity. The promotion of cell adhesivity to FN seems to be specific for SAHA as we observed no such effects with other HDAC inhibitors (trichostatin A and sodium butyrate). PMID:19911379

  12. Evolutionary connections between coding and splicing regulatory regions in the fibronectin EDA exon.

    PubMed

    Zago, Paola; Buratti, Emanuele; Stuani, Cristiana; Baralle, Francisco E

    2011-08-01

    Research on exonic coding sequences has demonstrated that many substitutions at the amino acid level may also reflect profound changes at the level of splicing regulatory regions. These results have revealed that, for many alternatively spliced exons, there is considerable pressure to strike a balance between two different and sometimes conflicting forces: the drive to improve the quality and production efficiency of proteins and the maintenance of proper exon recognition by the splicing machinery. Up to now, the systems used to investigate these connections have mostly focused on short alternatively spliced exons that contain a high density of splicing regulatory elements. Although this is obviously a desirable feature in order to maximize the chances of spotting connections, it also complicates the process of drawing straightforward evolutionary pathways between different species (because of the numerous alternative pathways through which the same end point can be achieved). The alternatively spliced fibronectin extra domain A exon (also referred to as EDI or EIIIA) does not have these limitations, as its inclusion is already known to depend on a single exonic splicing enhancer element within its sequence. In this study, we have compared the rat and human fibronectin EDA exons with regard to RNA structure, exonic splicing enhancer strengths, and SR protein occupancy. The results gained from these analyses have then been used to perform an accurate evaluation of EDA sequences observed in a wide range of animal species. This comparison strongly suggests the existence of an evolutionary connection between changes at the nucleotide levels and the need to maintain efficient EDA recognition in different species. PMID:21663748

  13. Focal adhesions in osteoneogenesis

    PubMed Central

    Biggs, M.J.P; Dalby, M.J

    2010-01-01

    As materials technology and the field of tissue engineering advances, the role of cellular adhesive mechanisms, in particular the interactions with implantable devices, becomes more relevant in both research and clinical practice. A key tenet of medical device technology is to use the exquisite ability of biological systems to respond to the material surface or chemical stimuli in order to help develop next-generation biomaterials. The focus of this review is on recent studies and developments concerning focal adhesion formation in osteoneogenesis, with an emphasis on the influence of synthetic constructs on integrin mediated cellular adhesion and function. PMID:21287830

  14. Nuclear transport of paxillin depends on focal adhesion dynamics and FAT domains

    PubMed Central

    Sathe, Aneesh R.; Shivashankar, G. V.; Sheetz, Michael P.

    2016-01-01

    ABSTRACT The nuclear transport of paxillin appears to be crucial for paxillin function but the mechanism of transport remains unclear. Here, we show that the nuclear transport of paxillin is regulated by focal adhesion turnover and the presence of FAT domains. Focal adhesion turnover was controlled using triangular or circular fibronectin islands. Circular islands caused higher focal adhesion turnover and increased the nuclear transport of paxillin relative to triangular islands. Mutating several residues of paxillin had no effect on its nuclear transport, suggesting that the process is controlled by multiple domains. Knocking out FAK (also known as PTK2) and vinculin caused an increase in nuclear paxillin. This could be reversed by rescue with wild-type FAK but not by FAK with a mutated FAT domain, which inhibits paxillin binding. Expressing just the FAT domain of FAK not only brought down nuclear levels of paxillin but also caused a large immobile fraction of paxillin to be present at focal adhesions, as demonstrated by fluorescence recovery after photobleaching (FRAP) studies. Taken together, focal adhesion turnover and FAT domains regulate the nuclear localization of paxillin, suggesting a possible role for transcriptional control, through paxillin, by focal adhesions. PMID:27068537

  15. Self-assembly of fibronectin mimetic peptide-amphiphile nanofibers

    NASA Astrophysics Data System (ADS)

    Rexeisen, Emilie Lynn

    Many therapeutic strategies incorporate peptides into their designs to mimic the natural protein ligands found in vivo. A few examples are the short peptide sequences RGD and PHSRN that mimic the primary and synergy-binding domains of the extracellular matrix protein, fibronectin, which is recognized by the cell surface receptor, alpha5beta 1 integrin. Even though scaffold modification with biomimetic peptides remains one of the most promising approaches for tissue engineering, the use of these peptides in therapeutic tissue-engineered products and drug delivery systems available on the commercial market is limited because the peptides are not easily able to mimic the natural protein. The design of a peptide that can effectively target the alpha5beta1 integrin would greatly increase biomimetic scaffold therapeutic potential. A novel peptide containing both the RGD primary binding domain and PHSRN synergy-binding domain for fibronectin joined with the appropriate linker should bind alpha 5beta1 integrin more efficiently and lead to greater cell adhesion over RGD alone. Several fibronectin mimetic peptides were designed and coupled to dialkyl hydrocarbon tails to make peptide-amphiphiles. The peptides contained different linkers connecting the two binding domains and different spacers separating the hydrophobic tails from the hydrophilic headgroups. The peptide-amphiphiles were deposited on mica substrates using the Langmuir-Blodgett technique. Langmuir isotherms indicated that the peptide-amphiphiles that contained higher numbers of serine residues formed a more tightly packed monolayer, but the increased number of serines also made transferring the amphiphiles to the mica substrate more difficult. Atomic force microscopy (AFM) images of the bilayers showed that the headgroups might be bent, forming small divots in the surface. These divots may help expose the PHSRN synergy-binding domain. Parallel studies undertaken by fellow group members showed that human

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

    PubMed Central

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

    2011-01-01

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

  17. iDriving (Intelligent Driving)

    SciTech Connect

    Malikopoulos, Andreas

    2012-09-17

    iDriving identifies the driving style factors that have a major impact on fuel economy. An optimization framework is used with the aim of optimizing a driving style with respect to these driving factors. A set of polynomial metamodels is constructed to reflect the responses produced in fuel economy by changing the driving factors. The optimization framework is used to develop a real-time feedback system, including visual instructions, to enable drivers to alter their driving styles in responses to actual driving conditions to improve fuel efficiency.

  18. iDriving (Intelligent Driving)

    Energy Science and Technology Software Center (ESTSC)

    2012-09-17

    iDriving identifies the driving style factors that have a major impact on fuel economy. An optimization framework is used with the aim of optimizing a driving style with respect to these driving factors. A set of polynomial metamodels is constructed to reflect the responses produced in fuel economy by changing the driving factors. The optimization framework is used to develop a real-time feedback system, including visual instructions, to enable drivers to alter their driving stylesmore » in responses to actual driving conditions to improve fuel efficiency.« less

  19. Mosaic Focal Plane Development

    NASA Astrophysics Data System (ADS)

    Mason, David L.; Horner, Scott D.; Aamodt, Earl K.

    2002-12-01

    Advances in systems engineering, applied sciences, and manufacturing technologies have enabled the development of large ground based and spaced based astronomical instruments having a large Field of View (FOV) to capture a large portion of the universe in a single image. A larger FOV can be accomplished using light weighted optical elements, improved support structures, and the development of mosaic Focal Plane Assemblies (mFPA). A mFPA designed for astronomy can use multiple Charged Coupled Devices (CCD) mounted onto a single camera baseplate integrated at the instrument plane of focus. Examples of current, or proposed, missions utilizing mFPA technology include FAME, GEST, Kepler, GAIA, LSST, and SNAP. The development of a mFPA mandates tighter control on the design trades, component development, CCD characterization, component integration, and performance verification testing. This paper addresses the capability Lockheed Martin Space Systems Company's (LMSSC) Advanced Technology Center (ATC) has developed to perform CCD characterization, mFPA assembly and alignment, and mFPA system level testing.

  20. Mosaic Focal Plane Development

    NASA Astrophysics Data System (ADS)

    Mason, D.; Horner, S.; Aamodt, E.

    Advances in manufacturing and applied sciences have enabled the development of large ground and spaced based astronomical instruments having a Field of View (FOV) large enough to capture a large portion of the universe in a single image. A large FOV can be accomplished using light weighted optics, improved structures, and the development of mosaic Focal Plane Assemblies (mFPAs). A mFPA comprises multiple Charged Coupled Devices (CCD) mounted onto a single baseplate integrated at the focus plane of the instrument. Examples of current, or proposed, missions utilizing mFPA technology include FAME, GEST, Kepler, GAIA, LSST, and SNAP. The development of a mFPA mandates tight control on the design trades of component development, CCD definition and characterization, component integration, and performance verification testing. This paper addresses the results of the Lockheed Martin Space Systems Company (LMSSC), Advanced Technology Center (ATC) developed mFPA. The design trades and performance characterization are services provided by the LMSSC ATC but not detailed in this paper.

  1. Stretching Fibroblasts Remodels Fibronectin and Alters Cancer Cell Migration

    NASA Astrophysics Data System (ADS)

    Ao, Mingfang; Brewer, Bryson M.; Yang, Lijie; Franco Coronel, Omar E.; Hayward, Simon W.; Webb, Donna J.; Li, Deyu

    2015-02-01

    Most investigations of cancer-stroma interactions have focused on biochemical signaling effects, with much less attention being paid to biophysical factors. In this study, we investigated the role of mechanical stimuli on human prostatic fibroblasts using a microfluidic platform that was adapted for our experiments and further developed for both repeatable performance among multiple assays and for compatibility with high-resolution confocal microscopy. Results show that mechanical stretching of normal tissue-associated fibroblasts (NAFs) alters the structure of secreted fibronectin. Specifically, unstretched NAFs deposit and assemble fibronectin in a random, mesh-like arrangement, while stretched NAFs produce matrix with a more organized, linearly aligned structure. Moreover, the stretched NAFs exhibited an enhanced capability for directing co-cultured cancer cell migration in a persistent manner. Furthermore, we show that stretching NAFs triggers complex biochemical signaling events through the observation of increased expression of platelet derived growth factor receptor α (PDGFRα). A comparison of these behaviors with those of cancer-associated fibroblasts (CAFs) indicates that the observed phenotypes of stretched NAFs are similar to those associated with CAFs, suggesting that mechanical stress is a critical factor in NAF activation and CAF genesis.

  2. In vitro splicing of fibronectin pre-mRNAs.

    PubMed Central

    Norton, P A; Hynes, R O

    1990-01-01

    We have investigated the alternative splicing of the EIIIB exon of the rat fibronectin gene. Mini-gene constructs containing this exon and portions of adjacent introns and exons, when transfected into HeLa cells, are transcribed and spliced, but omit the EIIIB exon. In vitro, HeLa nuclear extracts similarly splice out (skip) the EIIIB exon from similarly structured transcripts. Therefore, the HeLa splicing apparatus recognizes as atypical the EIIIB exon and its flanking intron sequences, both in vivo and in vitro. We also report that alterations in the ionic conditions of the in vitro splicing reaction can promote the initiation of EIIIB exon inclusion, as reflected by the formation of intermediate and product RNAs related to the removal of the intron upstream of EIIIB. Processing of this intron correlates with the formation of complexes resembling intermediates in spliceosome assembly. The branch sites involved in this alternative processing pathway are rather distant from the EIIIB 3' splice site, and lie within a region which is well conserved in the fibronectin genes of other species. Thus, the intron upstream of EIIIB shows singular structure and behavior which probably have a bearing on the regulated alternative splicing of this exon. Images PMID:2377454

  3. Fibronectin and stem cell differentiation – lessons from chondrogenesis

    PubMed Central

    Singh, Purva; Schwarzbauer, Jean E.

    2012-01-01

    Summary The extracellular matrix (ECM) is an intricate network of proteins that surrounds cells and has a central role in establishing an environment that is conducive to tissue-specific cell functions. In the case of stem cells, this environment is the stem cell niche, where ECM signals participate in cell fate decisions. In this Commentary, we describe how changes in ECM composition and mechanical properties can affect cell shape and stem cell differentiation. Using chondrogenic differentiation as a model, we examine the changes in the ECM that occur before and during mesenchymal stem cell differentiation. In particular, we focus on the main ECM protein fibronectin, its temporal expression pattern during chondrogenic differentiation, its potential effects on functions of differentiating chondrocytes, and how its interactions with other ECM components might affect cartilage development. Finally, we discuss data that support the possibility that the fibronectin matrix has an instructive role in directing cells through the condensation, proliferation and/or differentiation stages of cartilage formation. PMID:22976308

  4. Stretching Fibroblasts Remodels Fibronectin and Alters Cancer Cell Migration

    PubMed Central

    Ao, Mingfang; Brewer, Bryson M.; Yang, Lijie; Franco Coronel, Omar E.; Hayward, Simon W.; Webb, Donna J.; Li, Deyu

    2015-01-01

    Most investigations of cancer-stroma interactions have focused on biochemical signaling effects, with much less attention being paid to biophysical factors. In this study, we investigated the role of mechanical stimuli on human prostatic fibroblasts using a microfluidic platform that was adapted for our experiments and further developed for both repeatable performance among multiple assays and for compatibility with high-resolution confocal microscopy. Results show that mechanical stretching of normal tissue-associated fibroblasts (NAFs) alters the structure of secreted fibronectin. Specifically, unstretched NAFs deposit and assemble fibronectin in a random, mesh-like arrangement, while stretched NAFs produce matrix with a more organized, linearly aligned structure. Moreover, the stretched NAFs exhibited an enhanced capability for directing co-cultured cancer cell migration in a persistent manner. Furthermore, we show that stretching NAFs triggers complex biochemical signaling events through the observation of increased expression of platelet derived growth factor receptor α (PDGFRα). A comparison of these behaviors with those of cancer-associated fibroblasts (CAFs) indicates that the observed phenotypes of stretched NAFs are similar to those associated with CAFs, suggesting that mechanical stress is a critical factor in NAF activation and CAF genesis. PMID:25660754

  5. Adhesion and migration of avian neural crest cells on fibronectin require the cooperating activities of multiple integrins of the (beta)1 and (beta)3 families.

    PubMed

    Testaz, S; Delannet, M; Duband, J

    1999-12-01

    Based on genetic, functional and histological studies, the extracellular matrix molecule fibronectin has been proposed to play a key role in the migration of neural crest cells in the vertebrate embryo. In the present study, we have analyzed in vitro the repertoire and function of integrin receptors involved in the adhesive and locomotory responses of avian truncal neural crest cells to fibronectin. Immunoprecipitation experiments showed that neural crest cells express multiple integrins, namely (alpha)3(beta)1, (alpha)4(beta)1, (alpha)5(beta)1, (alpha)8(beta)1, (alpha)v(beta)1, (alpha)v(beta)3 and a (beta)8 integrin, as potential fibronectin receptors, and flow cytometry analyses revealed no major heterogeneity among the cell population for expression of integrin subunits. In addition, the integrin repertoire expressed by neural crest cells was found not to change dramatically during migration. At the cellular level, only (alpha)v(beta)1 and (alpha)v(beta)3 were concentrated in focal adhesion sites in connection with the actin microfilaments, whereas the other integrins were predominantly diffuse over the cell surface. In inhibition assays with function-perturbing antibodies, it appeared that complete abolition of cell spreading and migration could be achieved only by blocking multiple integrins of the (beta)1 and (beta)3 families, suggesting possible functional compensations between different integrins. In addition, these studies provided evidence for functional partitioning of integrins in cell adhesion and migration. While spreading was essentially mediated by (alpha)v(beta)1 and (alpha)8(beta)1, migration involved primarily (alpha)4(beta)1, (alpha)v(beta)3 and (alpha)8(beta)1 and, more indirectly, (alpha)3(beta)1. (alpha)5(beta)1 and the (beta)8 integrin were not found to play any major role in either adhesion or migration. Finally, consistent with the results of inhibition experiments, recruitment of (alpha)4(beta)1 and (alpha)v(beta)3, individually or in

  6. Fibronectin receptor functions in embryonic cells deficient in alpha 5 beta 1 integrin can be replaced by alpha V integrins.

    PubMed Central

    Yang, J T; Hynes, R O

    1996-01-01

    alpha 5 beta 1 integrin mediates cell adhesion to extracellular matrix by interacting with fibronectin (FN). Mouse lines carrying null mutations in genes encoding either the alpha 5 integrin subunit or FN have been generated previously. Both mutations are embryonic lethal with overlapping defects, but the defects of alpha 5-null embryos are less severe. Primary embryonic cells lacking alpha 5 beta 1 are able to adhere to FN, form focal contacts, migrate on FN, and assemble FN matrix. These results suggest the involvement of (an)other FN receptors(s). In this study, we examined functions of alpha 4 beta 1 and alpha V integrins in embryonic cells lacking alpha 5 beta 1. Our analysis of cells lacking both alpha 4 beta 1 and alpha 5 beta 1 showed that alpha 4 beta 1 is also not required for these FN-dependent functions. Using alpha V-specific blocking reagents, we showed that alpha V integrins are required for alpha 5-null cells, but not wild-type cells, to adhere and spread on FN. Our data also showed that, although the expression levels of alpha V integrins on the wild-type and alpha 5-null cells are similar, there is an increase in recruitment of alpha V integrins into focal contacts in alpha 5-null cells plated on FN, indicating that alpha V integrins can compensate functionally for the loss of alpha 5 beta 1 in focal contacts of alpha 5-null cells. Finally, our data suggested possible roles for alpha V integrins in replacing the role of alpha 5 beta 1 in FN matrix assembly in vitro and in FN-dependent embryonic functions in vivo. Images PMID:8930896

  7. Impaired Driving

    MedlinePlus

    ... Risk Factors BAC Effects Prevention Additional Resources How big is the problem? In 2014, 9,967 people ... Driving: A Threat to Everyone (October 2011) Additional Data Drunk Driving State Data and Maps Motor Vehicle ...

  8. Drugged Driving

    MedlinePlus

    ... Infographics » Drugged Driving Drugged Driving Email Facebook Twitter Text Description of Infographic Top Right Figure : In 2009, ... crash than those who don't smoke. Bottom Text: Develop Social Strategies Offer to be a designated ...

  9. Plakoglobin regulates cell motility through Rho- and fibronectin-dependent Src signaling

    PubMed Central

    Todorović, Viktor; Desai, Bhushan V.; Patterson, Melanie J. Schroeder; Amargo, Evangeline V.; Dubash, Adi D.; Yin, Taofei; Jones, Jonathan C. R.; Green, Kathleen J.

    2010-01-01

    We previously showed that the cell–cell junction protein plakoglobin (PG) not only suppresses motility of keratinocytes in contact with each other, but also, unexpectedly, of single cells. Here we show that PG deficiency results in extracellular matrix (ECM)-dependent disruption of mature focal adhesions and cortical actin organization. Plating PG−/− cells onto ECM deposited by PG+/− cells partially restored normal cell morphology and inhibited PG−/− cell motility. In over 70 adhesion molecules whose expression we previously showed to be altered in PG−/− cells, a substantial decrease in fibronectin (FN) in PG−/− cells stood out. Re-introduction of PG into PG−/− cells restored FN expression, and keratinocyte motility was reversed by plating PG−/− cells onto FN. Somewhat surprisingly, based on previously reported roles for PG in regulating gene transcription, PG-null cells exhibited an increase, not a decrease, in FN promoter activity. Instead, PG was required for maintenance of FN mRNA stability. PG−/− cells exhibited an increase in activated Src, one of the kinases controlled by FN, a phenotype reversed by plating PG−/− cells on ECM deposited by PG+/− keratinocytes. PG−/− cells also exhibited Src-independent activation of the small GTPases Rac1 and RhoA. Both Src and RhoA inhibition attenuated PG−/− keratinocyte motility. We propose a novel role for PG in regulating cell motility through distinct ECM–Src and RhoGTPase-dependent pathways, influenced in part by PG-dependent regulation of FN mRNA stability. PMID:20876660

  10. Combinatorial Fibronectin and Laminin Signaling Promote Highly Efficient Cardiac Differentiation of Human Embryonic Stem Cells

    PubMed Central

    Sa, Silin; Wong, Lian

    2014-01-01

    Abstract Cardiomyocytes (CMs) differentiated from human embryonic stem cells (hESCs) are a promising and potentially unlimited cell source for myocardial repair and regeneration. Recently, multiple methodologies—primarily based on the optimization of growth factors—have been described for efficient cardiac differentiation of hESCs. However, the role of extracellular matrix (ECM) signaling in CM differentiation has not yet been explored fully. This study examined the role of ECM signaling in the efficient generation of CMs from both H7 and H9 ESCs. The hESCs were differentiated on ECM substrates composed of a range of fibronectin (FN) and laminin (LN) ratios and gelatin and evaluated by the fluorescence activated cell scanning (FACS) analysis on day 14. Of the ECM substrates examined, the 70:30 FN:LN reproducibly generated the greatest numbers of CMs from both hESC lines. Moreover, the LN receptor integrin β4 (ITGB4) and FN receptor integrin β5 (ITGB5) genes, jointly with increased phosphorylated focal adhension kinase and phosphorylated extracellular signal-regulated kinases (p-ERKs), were up-regulated over 13-fold in H7 and H9 cultured on 70:30 FN:LN compared with gelatin. Blocking studies confirmed the role of all these molecules in CM specification, suggesting that the 70:30 FN:LN ECM promotes highly efficient differentiation of CMs through the integrin-mediated MEK/ERK signaling pathway. Lastly, the data suggest that FN:LN-induced signaling utilizes direct cell-to-cell signaling from distinct ITGB4+ and ITGB5+ cells. PMID:25126479

  11. WISE focal plane module lessons learned in light of success

    NASA Astrophysics Data System (ADS)

    Masterjohn, S.; Hogue, H.; Muzilla, M.; Rector, S.; Mattson, R.

    2010-08-01

    DRS Sensors & Targeting Systems, under contract to the Space Dynamics Laboratory of Utah State University, provided the focal plane detector system for NASA's Wide-field Infrared Survey Explorer (WISE). The focal plane detector system consists of two mercury cadmium telluride (MCT) focal plane module assemblies (FPMAs), two arsenic doped silicon (Si:As) Blocked Impurity Band (BIB) FPMAs, electronics to drive the FPMAs and report digital data from them, and the cryogenic and ambient temperature cabling that connect the FPMAs and electronics. The WISE Satellite was launched in late 2009 and has been a very rewarding success. In light of the recent success on orbit, there were many challenges and hurdles the DRS team had to overcome in order to guarantee the ultimate success of the instrument. This report highlights a few of the challenges that the team overcame in hopes that the information can be made available to the astronomy community for future use.

  12. Statistical Earthquake Focal Mechanism Forecasts

    NASA Astrophysics Data System (ADS)

    Kagan, Y. Y.; Jackson, D. D.

    2013-12-01

    The new whole Earth focal mechanism forecast, based on the GCMT catalog, has been created. In the present forecast, the sum of normalized seismic moment tensors within 1000 km radius is calculated and the P- and T-axes for the focal mechanism are evaluated on the basis of the sum. Simultaneously we calculate an average rotation angle between the forecasted mechanism and all the surrounding mechanisms. This average angle shows tectonic complexity of a region and indicates the accuracy of the prediction. The method was originally proposed by Kagan and Jackson (1994, JGR). Recent interest by CSEP and GEM has motivated some improvements, particularly to extend the previous forecast to polar and near-polar regions. The major problem in extending the forecast is the focal mechanism calculation on a spherical surface. In the previous forecast as our average focal mechanism was computed, it was assumed that longitude lines are approximately parallel within 1000 km radius. This is largely accurate in the equatorial and near-equatorial areas. However, when one approaches the 75 degree latitude, the longitude lines are no longer parallel: the bearing (azimuthal) difference at points separated by 1000 km reach about 35 degrees. In most situations a forecast point where we calculate an average focal mechanism is surrounded by earthquakes, so a bias should not be strong due to the difference effect cancellation. But if we move into polar regions, the bearing difference could approach 180 degrees. In a modified program focal mechanisms have been projected on a plane tangent to a sphere at a forecast point. New longitude axes which are parallel in the tangent plane are corrected for the bearing difference. A comparison with the old 75S-75N forecast shows that in equatorial regions the forecasted focal mechanisms are almost the same, and the difference in the forecasted focal mechanisms rotation angle is close to zero. However, though the forecasted focal mechanisms are similar

  13. Fibronectin tetrapeptide is target for syphilis spirochete cytadherence

    SciTech Connect

    Thomas, D.D.; Baseman, J.B.; Alderete, J.F.

    1985-11-01

    The syphilis bacterium, Treponema pallidum, parasitizes host cells through recognition of fibronectin (Fn) on cell surfaces. The active site of the Fn molecule has been identified as a four-amino acid sequence, arg-gly-asp-ser (RGDS), located on each monomer of the cell-binding domain. The synthetic heptapeptide gly-arg-gly-asp-ser-pro-cys (GRGDSPC), with the active site sequence RGDS, specifically competed with SVI-labeled cell-binding domain acquisition by T. pallidum. Additionally, the same heptapeptide with the RGDS sequence diminished treponemal attachment to HEp-2 and HT1080 cell monolayers. Related heptapeptides altered in one key amino acid within the RGDS sequence failed to inhibit Fn cell-binding domain acquisition or parasitism of host cells by T. pallidum. The data support the view that T. pallidum cytadherence of host cells is through recognition of the RGDS sequence also important for eukaryotic cell-Fn binding.

  14. Conformational changes of fibronectin induced by polystyrene derivatives with a heparin-like function

    SciTech Connect

    Stanislawski, L. ); Serne, H.; Jozefowicz, M. ); Stanislawski, M. )

    1993-05-01

    It was previously reported that polystyrene substituted with the sulfonate group, PSSO[sub 3], which has anticoagulant heparin-like properties, and then coated with fibronectin supports the growth of human umbilical vein endothelial cells. On the other hand, polystyrene substituted with the amino acid sulfamide group, PSSO[sub 2]-Asp, which has a higher anticoagulant activity, and then coated with fibronectin no longer supported the growth of endothelial cells. The authors report here that, while the affinity of fibronectin to either polymer is of the same order of magnitude, fibronectin is adsorbed onto the PSSO[sub 2]-Asp polymer in a different conformation compared to the PSSO[sub 3] polymer. This was shown by a higher binding of polyclonal antifibronectin antibodies to fibronectin-coated PSSO[sub 2]-Asp polymer, and by a decreased susceptibility of the coated fibronectin to proteolysis by thermolysin. This study provides evidence that a solid phase substrate with a strong heparin-like function may influence the conformation and biological properties of fibronectin.

  15. A fibronectin receptor on Candida albicans mediates adherence of the fungus to extracellular matrix

    SciTech Connect

    Klotz, S.A.; Smith, R.L. )

    1991-03-01

    Binding of fibronectin, an extracellular matrix (ECM) protein, to Candida albicans was measured, and adherence of the fungus to immobilized ECM proteins, fibronectin, laminin, types I and IV collagen, and subendothelial ECM was studied. 125I-labeled fibronectin was inhibited from binding to the fungus by unlabeled human plasma fibronectin and by Arg-Gly-Asp (RGD), Gly-Arg-Gly-Glu-Ser-Pro (GRGESP), and Gly-Arg-Gly-Asp-Thr-Pro (GRGDTP), but binding was not inhibited by Gly-Arg-Gly-Asp-Ser-Pro. Soluble fibronectin, RGD, GRGESP, and GRGDTP also inhibited fungal adherence to the individual immobilized ECM proteins in a complex pattern, but only soluble fibronectin (10(-7) M) inhibited fungal adherence to subendothelial ECM. Thus, C. albicans possesses at least one type of cell surface receptor for binding soluble fibronectin that can be inhibited with peptides. This receptor apparently is used to bind the fungus to immobilized ECM proteins and to subendothelial ECM and may play a role in the initiation of disseminated disease by bloodborne fungi by providing for adherence of the microorganisms to ECM proteins.

  16. Fibronectin-Containing Extracellular Vesicles Protect Melanocytes against Ultraviolet Radiation-Induced Cytotoxicity.

    PubMed

    Bin, Bum-Ho; Kim, Dae-Kyum; Kim, Nan-Hyung; Choi, Eun-Jeong; Bhin, Jinhyuk; Kim, Sung Tae; Gho, Yong Song; Lee, Ai-Young; Lee, Tae Ryong; Cho, Eun-Gyung

    2016-05-01

    Skin melanocytes are activated by exposure to UV radiation to secrete melanin-containing melanosomes to protect the skin from UV-induced damage. Despite the continuous renewal of the epidermis, the turnover rate of melanocytes is very slow, and they survive for long periods. However, the mechanisms underlying the survival of melanocytes exposed to UV radiation are not known. Here, we investigated the role of melanocyte-derived extracellular vesicles in melanocyte survival. Network analysis of the melanocyte extracellular vesicle proteome identified the extracellular matrix component fibronectin at a central node, and the release of fibronectin-containing extracellular vesicles was increased after exposure of melanocytes to UVB radiation. Using an anti-fibronectin neutralizing antibody and specific inhibitors of extracellular vesicle secretion, we demonstrated that extracellular vesicles enriched in fibronectin were involved in melanocyte survival after UVB radiation. Furthermore, we observed that in the hyperpigmented lesions of patients with melasma, the extracellular space around melanocytes contained more fibronectin compared with normal skin, suggesting that fibronectin is involved in maintaining melanocytes in pathological conditions. Collectively, our findings suggest that melanocytes secrete fibronectin-containing extracellular vesicles to increase their survival after UVB radiation. These data provide important insight into how constantly stimulated melanocytes can be maintained in pathological conditions such as melasma. PMID:26854492

  17. Fibronectin in the ruptured human Achilles tendon and its paratenon. An immunoperoxidase study.

    PubMed

    Lehto, M; Jozsa, L; Kvist, M; Järvinen, M; Balint, B J; Reffy, A

    1990-01-01

    The purpose of the present study was to examine the role and distribution of fibronectin--a major connective tissue protein--in normal and ruptured Achilles tendon and its paratenon using peroxidase antiperoxidase (PAP) method and conventional histology. Samples were taken intraoperatively 6 to 48 hours after rupture from 30 patients, and 4 male cadavers served as controls. In controls fibronectin could not be detected in the tendinous connective tissue or in the areolar tissue of the paratenon. In the ruptured tendons fibronectin was detected around the tenocytes and in the collagen fibres in the necrotic and mucinous degenerated areas, but also in the macroscopically healthy areas of the tendon. In the oedematous paratenon vascular walls were rich in fibronectin and fat cells were outlined by fibronectin. Areas of homogenous deposition of fibronectin were also frequently seen in the paratenon. A vast amount of fibronectin was observed also at the site of rupture. The present results indicate that there are striking changes in the composition of connective tissue in degenerated tendon and paratenon. These changes seem to have increased susceptibility of the tendon to rupture. PMID:2201247

  18. Role of Fibronectin in the Adhesion of Acinetobacter baumannii to Host Cells

    PubMed Central

    Smani, Younes; McConnell, Michael J.; Pachón, Jerónimo

    2012-01-01

    Adhesion to host cells is an initial and important step in Acinetobacter baumannii pathogenesis. However, there is relatively little information on the mechanisms by which A. baumannii binds to and interacts with host cells. Adherence to extracellular matrix proteins, such as fibronectin, affords pathogens with a mechanism to invade epithelial cells. Here, we found that A. baumannii adheres more avidly to immobilized fibronectin than to control protein. Free fibronectin used as a competitor resulted in dose-dependent decreased binding of A. baumannii to fibronectin. Three outer membrane preparations (OMPs) were identified as fibronectin binding proteins (FBPs): OMPA, TonB-dependent copper receptor, and 34 kDa OMP. Moreover, we demonstrated that fibronectin inhibition and neutralization by specific antibody prevented significantly the adhesion of A. baumannii to human lung epithelial cells (A549 cells). Similarly, A. baumannii OMPA neutralization by specific antibody decreased significantly the adhesion of A. baumannii to A549 cells. These data indicate that FBPs are key adhesins that mediate binding of A. baumannii to human lung epithelial cells through interaction with fibronectin on the surface of these host cells. PMID:22514602

  19. Defects in MAP1S-mediated autophagy turnover of fibronectin cause renal fibrosis

    PubMed Central

    He, Yongzhong; Li, Xun; Zhao, Haibo; Su, Zhengming; Jiang, Xianhan; Li, Wenjiao; Zou, Jing; Chen, Qi; Liu, Leyuan

    2016-01-01

    Excessive deposition of extracellular matrix proteins in renal tissues causes renal fibrosis and renal function failure. Mammalian cells primarily use the autophagy-lysosome system to degrade misfolded/aggregated proteins and dysfunctional organelles. MAP1S is an autophagy activator and promotes the biogenesis and degradation of autophagosomes. Previously, we reported that MAP1S suppresses hepatocellular carcinogenesis in a mouse model and predicts a better prognosis in patients suffering from clear cell renal cell carcinomas. Furthermore, we have characterized that MAP1S enhances the turnover of fibronectin, and mice overexpressing LC3 but with MAP1S deleted accumulate fibronectin and develop liver fibrosis because of the synergistic impact of LC3-induced over-synthesis of fibronectin and MAP1S depletion-caused impairment of fibronectin degradation. Here we show that a suppression of MAP1S in renal cells caused an impairment of autophagy clearance of fibronectin and an activation of pyroptosis. Depletion of MAP1S in mice leads to an accumulation of fibrosis-related proteins and the development of renal fibrosis in aged mice. The levels of MAP1S were dramatically reduced and levels of fibronectin were greatly elevated in renal fibrotic tissues from patients diagnosed as renal atrophy and renal failure. Therefore, MAP1S deficiency may cause the accumulation of fibronectin and the development of renal fibrosis. PMID:27236336

  20. Plasma fibronectin promotes lung metastasis by contributions to fibrin clots and tumor cell invasion.

    PubMed

    Malik, Gunjan; Knowles, Lynn M; Dhir, Rajiv; Xu, Shuping; Yang, Shuting; Ruoslahti, Erkki; Pilch, Jan

    2010-06-01

    The attachment of circulating tumor cells to the blood vessels of distant organs is an important step in metastasis. We show here that experimental lung metastasis by two cell lines, B16F1 melanoma and 3LL lung carcinoma, is greatly reduced in transgenic mice that lack plasma fibronectin. This multifunctional adhesive glycoprotein becomes cross-linked to fibrin during clotting. Here, we report that eliminating plasma fibronectin from the blood circulation reverses the prometastatic effects of blood clotting and tumor cell integrin alphavbeta3. In vitro studies showed that fibrin-fibronectin complexes, but not purified fibrin, supported tumor cell attachment and invasion. These functions correlate with the ability of fibrin-fibronectin complexes to induce the activation of integrin alphavbeta3. Our findings reveal an important contribution of plasma fibronectin in lung metastasis. Furthermore, they suggest that the previously noted effects of blood clotting on lung metastasis might be mediated in part by a fibronectin-alphavbeta3 integrin axis, in which plasma fibronectin has to be incorporated into the blood clot. PMID:20501851

  1. Design of large aperture focal plane shutter

    NASA Astrophysics Data System (ADS)

    Hu, Jia-wen; Ma, Wen-li; Huang, Jin-long

    2012-09-01

    To satisfy the requirement of large telescope, a large aperture focal plane shutter with aperture size of φ200mm was researched and designed to realize, which could be started and stopped in a relative short time with precise position, and also the blades could open and close at the same time at any orientation. Timing-belts and stepper motors were adopted as the drive mechanism. Velocity and position of the stepper motors were controlled by the PWM pulse generated by DSP. Exponential curve is applied to control the velocity of the stepper motors to make the shutter start and stop in a short time. The closing/open time of shutter is 0.2s, which meets the performance requirements of large telescope properly.

  2. Drug discovery in focal and segmental glomerulosclerosis.

    PubMed

    Pullen, Nick; Fornoni, Alessia

    2016-06-01

    Despite the high medical burden experienced by patients with focal segmental glomerulosclerosis, the etiology of the condition remains largely unknown. Focal segmental glomerulosclerosis is highly heterogeneous in clinical and morphologic manifestations. While this presents challenges for the development of new treatments, research investments over the last 2 decades have yielded a surfeit of potential avenues for therapeutic intervention. The development of many of those ideas and concepts into new therapies, however, has been very disappointing. Here, we describe some of the factors that have potentially contributed to the poor translational performance from this research investment, including the confidence we ascribe to a target, the conduct of experimental studies, and the availability of selective reagents to test hypotheses. We will discuss the significance of genetic and systems traits as well as other methods for reducing bias. We will analyze the limitations of a successful drug development. We will use specific examples hoping that these will guide a consensus for investment and drive greater translational quality. We hope that this substrate will serve to exemplify the tremendous opportunity for intervention as well as facilitate greater collaborative effort between industry, academia, and private foundations in promoting appropriate validation of these targets. Only then will we have achieved our goal for curative therapies for this devastating disease. PMID:27165834

  3. The Focal Surface of the JEM-EUSO Instrument

    SciTech Connect

    Kawasaki, Y.; Casolino, M.; Gorodetzky, P.; Santangelo, A.; Ricci, M.; Kajino, F.; Ebisuzaki, T.

    2011-09-22

    The Extreme Universe Space Observatory on JEM/EF (JEM-EUSO) is a space mission to study extremely high-energy cosmic rays. The JEM-EUSO instrument is a wide-angle refractive telescope in the near-ultraviolet wavelength region which will be mounted to the International Space Station. Its goal is to measure time-resolved fluorescence images of extensive air showers in the atmosphere. In this paper we describe in detail the main features and technological aspects of the focal surface of the instrument. The JEM-EUSO focal surface is a spherically curved surface, with an area of about 4.5m{sup 2}. The focal surface detector is made of more than 5,000 multi-anode photomultipliers (MAPMTs). Current baseline is Hamamatsu R11265-03-M64. The approach to the focal surface detector is highly modular. Photo-Detector-Modules (PDM) are the basic units that drive the mechanical structure and data acquisition. Each PDM consists of 9 Elementary Cells (ECs). The EC, which is the basic unit of the MAPMT support structure and of the front-end electronics, contains 4 units of MAPMTs. In total, about 1,200 ECs or about 150 PDMs are arranged on the whole of the focal surface of JEM-EUSO.

  4. Fibronectin binding by Streptococcus milleri group strains and partial characterisation of the fibronectin receptor of Streptococcus anginosus F4.

    PubMed

    Willcox, M D; Loo, C Y; Harty, D W; Knox, K W

    1995-09-01

    The Streptococcus milleri group were shown to bind fibronectin (Fn) to their cell-surface and this binding increased the adhesion of cells to hydroxyapatite. The binding of Fn to Streptococcus anginosus F4 was studied in more detail. Fn binding to bacterial cells increased the association of the bacteria with the polymorphonuclear leukocytes obtained from the peritoneal cavity of rats but did not increase killing of the bacteria. The cell-surface receptor was a protein of M(r) 14,000 which was released from cells after mutanolysin digestion. The binding was specific, with cells having a maximum number of binding sites per cell of 770. Electron microscopy, using gold-labelled Fn, localised the receptor to areas between daughter cells. PMID:8559042

  5. Precise Measurement of Effective Focal Length

    NASA Technical Reports Server (NTRS)

    Wise, T. D.; Young, J. B.

    1983-01-01

    Computerized instrument measures effective focal lengths to 0.01 percent accuracy. Laser interferometers measure mirror angle and stage coordinate y in instrument for accurate measurment of focal properties of optical systems. Operates under computer control to measure effective focal length, focal surface shape, modulation transfer function, and astigmatism.

  6. Pile Driving

    NASA Technical Reports Server (NTRS)

    1987-01-01

    Machine-oriented structural engineering firm TERA, Inc. is engaged in a project to evaluate the reliability of offshore pile driving prediction methods to eventually predict the best pile driving technique for each new offshore oil platform. Phase I Pile driving records of 48 offshore platforms including such information as blow counts, soil composition and pertinent construction details were digitized. In Phase II, pile driving records were statistically compared with current methods of prediction. Result was development of modular software, the CRIPS80 Software Design Analyzer System, that companies can use to evaluate other prediction procedures or other data bases.

  7. Continuously variable focal length lens

    DOEpatents

    Adams, Bernhard W; Chollet, Matthieu C

    2013-12-17

    A material preferably in crystal form having a low atomic number such as beryllium (Z=4) provides for the focusing of x-rays in a continuously variable manner. The material is provided with plural spaced curvilinear, optically matched slots and/or recesses through which an x-ray beam is directed. The focal length of the material may be decreased or increased by increasing or decreasing, respectively, the number of slots (or recesses) through which the x-ray beam is directed, while fine tuning of the focal length is accomplished by rotation of the material so as to change the path length of the x-ray beam through the aligned cylindrical slows. X-ray analysis of a fixed point in a solid material may be performed by scanning the energy of the x-ray beam while rotating the material to maintain the beam's focal point at a fixed point in the specimen undergoing analysis.

  8. Sex prevalence of focal dystonias.

    PubMed Central

    Soland, V L; Bhatia, K P; Marsden, C D

    1996-01-01

    The sex prevalence of idiopathic focal dystonia is reported from a data base review of all patients seen at the National Hospital of Neurology, Queen Square and King's College, London up to 1993. There was a higher prevalence of females to males in all categories of focal dystonia involving the craniocervical region. The female to male ratio for cranial dystonia was 1.92:1 (P < 0.01) and 1.6:1 (P < 0.001) for spasmodic torticollis. On the other hand, twice as many men than women had writer's cramp (M:F = 2.0:1, P < 0.01). At present, there is no clear explanation to account for this differences in the sex prevalence of different types of focal dystonia. PMID:8708656

  9. Fibronectin is a binding partner for the myelin-associated glycoprotein (siglec-4a).

    PubMed

    Strenge, K; Brossmer, R; Ihrig, P; Schauer, R; Kelm, S

    2001-06-22

    The myelin-associated glycoprotein (MAG) mediates cell-cell interactions between myelinating glial cells and neurons. Here we describe the extracellular matrix glycoprotein fibronectin as a binding partner of MAG. It has been identified by affinity precipitation with MAG-Fc from NG108-15 cells and by microsequencing of two peptides derived from a 210-kDa protein band. Western blot analysis showed that fibronectin is also present in MAG binding partners isolated from N(2)A (murine neuroblastoma) cells, rat brain and rat spinal cord. Different fibronectin isoforms have been isolated from brains of young and adult rats, indicating that the expression of MAG binding fibronectin changes during development. PMID:11423128

  10. Anastellin, an FN3 Fragment with Fibronectin Polymerization Activity, Resembles Amyloid Fibril Precursors

    SciTech Connect

    Briknarova, Klara; Akermann, Maria; Hoyt, David W. ); Ruoslahti, Erkki; Ely, Kathryn R.

    2003-08-01

    Anastellin is a carboxy-terminal fragment of the 1st FN3 domain from human fibronectin. It is capable of polymerizing fibronectin in vitro, and it displays anti-tumor, antimetastatic and anti-angiogenic properties in vivo. We have determined the structure of anastellin using nuclear magnetic resonance spectroscopy and identified residues critical for its activity. Anastellin exhibits dynamic fluctuations and conformational exchange in solution. Its overall topology is very similar to the corresponding region of full-length FN3 domains. However, its hydrophobic core becomes solvent accessible and some of its -strands lose their protection against hydrogen bonding to -strands from other molecules. These features seem to be relevant for the fibronectin polymerization activity of anastellin and resemble the characteristics of amyloid fibril precursors. We suggest that this analogy is not random and may reflect similarities between fibronectin and amyloid fibril formation.

  11. Effects of class I heparin binding growth factor and fibronectin on platelet adhesion and aggregation

    SciTech Connect

    Greisler, H.P.; Klosak, J.J.; Steinam, S.J.; Lam, T.M.; Burgess, W.H.; Kim, D.U. )

    1990-05-01

    Fibronectin and heparin binding growth factor-type 1 have been affixed to vascular graft surfaces to enhance the attachment and the proliferation of transplanted endothelial cells, respectively. The current study examines the effect of fibronectin and heparin binding growth factor-type 1 on platelet adhesion and activation in vivo and on platelet aggregation in vitro. Expanded polytetrafluoroethylene prostheses (5 cm x 4 mm internal diameter) were treated either with fibronectin (n = 9), fibronectin/heparin/heparin binding growth factor-type 1/heparin (n = 12), or neither (n = 13) and were interposed into canine aortoiliac systems bilaterally. Autogenous radiolabeled (Indium 111 oxine, 650 microCi) platelets were injected intravenously before reestablishment of circulation. Perfusion was maintained for 30 minutes, and prostheses were removed with segments of native aorta and distal iliac arteries bilaterally. Specimens were examined for thrombus-free surface area, by gamma well counting for adherent radiolabeled platelets, and by light microscopy and transmission and scanning electron microscopic techniques. Results showed that both the fibronectin and fibronectin/heparin/heparin binding growth factor-type 1/heparin pretreated prostheses contained significantly greater numbers of platelets and adherent radioactivity than did control graft segments when normalized to their ipsilateral iliac arteries. Fibronectin/heparin/heparin binding growth factor-type 1/heparin pretreated prostheses contained 27 +/- 16 times more radioactivity per square millimeter than ipsilateral iliac arteries, fibronectin pretreated prostheses had 13 +/- 8 times more radioactivity per square millimeter than ipsilateral iliac arteries, and untreated expanded polytetrafluoroethylene had 4 +/- 3 times more radioactivity per square millimeter than ipsilateral iliac arteries.

  12. Interaction between Fibronectin and β1 Integrin Is Essential for Tooth Development

    PubMed Central

    Yamada, Aya; Yuasa, Kenji; Yoshizaki, Keigo; Iwamoto, Tsutomu; Saito, Masahiro; Nakamura, Takashi; Fukumoto, Satoshi

    2015-01-01

    The dental epithelium and extracellular matrix interact to ensure that cell growth and differentiation lead to the formation of teeth of appropriate size and quality. To determine the role of fibronectin in differentiation of the dental epithelium and tooth formation, we analyzed its expression in developing incisors. Fibronectin mRNA was expressed during the presecretory stage in developing dental epithelium, decreased in the secretory and early maturation stages, and then reappeared during the late maturation stage. The binding of dental epithelial cells derived from postnatal day-1 molars to a fibronectin-coated dish was inhibited by the RGD but not RAD peptide, and by a β1 integrin-neutralizing antibody, suggesting that fibronectin-β1 integrin interactions contribute to dental epithelial-cell binding. Because fibronectin and β1 integrin are highly expressed in the dental mesenchyme, it is difficult to determine precisely how their interactions influence dental epithelial differentiation in vivo. Therefore, we analyzed β1 integrin conditional knockout mice (Intβ1lox-/lox-/K14-Cre) and found that they exhibited partial enamel hypoplasia, and delayed eruption of molars and differentiation of ameloblasts, but not of odontoblasts. Furthermore, a cyst-like structure was observed during late ameloblast maturation. Dental epithelial cells from knockout mice did not bind to fibronectin, and induction of ameloblastin expression in these cells by neurotrophic factor-4 was inhibited by treatment with RGD peptide or a fibronectin siRNA, suggesting that the epithelial interaction between fibronectin and β1 integrin is important for ameloblast differentiation and enamel formation. PMID:25830530

  13. Renal proximal tubular cell fibronectin accumulation in response to glucose is polyol pathway dependent

    PubMed

    Morrisey; Steadman; Williams; Phillips

    1999-06-01

    Thickening and reduplication of the tubular basement membrane has been reported as an early event in diabetic nephropathy. In the current study we have examined the polar requirements of proximal tubular cells for the D-glucose stimulated accumulation of fibronectin. We also examined the mechanism by which glucose led to accumulation of fibronectin, with particular emphasis on the polyol pathway. Incubation of confluent monolayers of LLC-PK1 cells grown on tissue culture inserts with 25 mM D-glucose on either their apical or basolateral aspect, led to fibronectin accumulation in the basolateral compartment. This reached statistical significance 24 h following apical addition of glucose (2.7 fold increase compared to 5 mM D-glucose, p = 0.007, n = 6), and 12 h after the basolateral addition of glucose (2.54 fold increase compared to 5 mM D-glucose, p = 0.02, n = 6). The increase in fibronectin concentration in response to glucose was inhibited by the aldose reductase inhibitor sorbinil. At a dose of 100&mgr;M sorbinil there was 59% inhibition of fibronectin accumulation in response to glucose, 48 h after the addition of the inhibitor (4.76 +/- 1.4 vs 11.53 +/- 1.41, mean +/- SD, p = 0.01, n = 3). Exposure of cells to glucose at either their apical or basolateral aspect lead to accumulation of intracellular glucose and polyol pathway activation, as assessed by sorbitol accumulation. Accumulation of intracellular glucose and hence subsequent polyol pathway activation occurred independently of transport of glucose by either apical sodium linked glucose transporter (SLGT) or basolateral GLUT 1. The data demonstrate that fibronectin generation in response to glucose was non-polar in terms application of glucose, but polar in terms of fibronectin accumulation. Furthermore modulation of fibronectin was mediated by polyol pathway activation, and more specifically related to the metabolism of sorbitol to fructose. PMID:10354307

  14. α-Actinin-4 Enhances Colorectal Cancer Cell Invasion by Suppressing Focal Adhesion Maturation

    PubMed Central

    Yamada, Tesshi; Takenawa, Tadaomi

    2015-01-01

    α-Actinins (ACTNs) are known to crosslink actin filaments at focal adhesions in migrating cells. Among the four isoforms of mammalian ACTNs, ACTN1 and ACTN4 are ubiquitously expressed. Recently, ACTN4 was reported to enhance cancer cell motility, invasion, and metastasis. However, the mechanism by which ACTN4 drives these malignant phenotypes remains unclear. Here, we show that ACTN4, but not ACTN1, induces the formation of immature focal adhesions in DLD-1 cells, leading to the rapid turnover of focal adhesions. Interestingly, zyxin (ZYX) assembly to focal adhesions was markedly decreased in ACTN4-expressing DLD-1 cells, while the recruitment of paxillin (PAX) occurred normally. On the other hand, in ACTN1-expressing DLD-1 cells, PAX and ZYX were normally recruited to focal adhesions, suggesting that ACTN4 specifically impairs focal adhesion maturation by inhibiting the recruitment of ZYX to focal complexes. Using purified recombinant proteins, we found that ZYX binding to ACTN4 was defective under conditions where ZYX binding to ACTN1 was observed. Furthermore, Matrigel invasion of SW480 cells that express high endogenous levels of ACTN4 protein was inhibited by ectopic expression of ACTN1. Altogether, our results suggest that ZYX defective binding to ACTN4, which occupies focal adhesions instead of ACTN1, induces the formation of immature focal adhesions, resulting in the enhancement of cell motility and invasion. PMID:25860875

  15. Transforming growth factor beta increases cell surface binding and assembly of exogenous (plasma) fibronectin by normal human fibroblasts.

    PubMed Central

    Allen-Hoffmann, B L; Crankshaw, C L; Mosher, D F

    1988-01-01

    Transforming growth factor beta (TGF-beta) enhances the cell surface binding of 125I-fibronectin by cultured human fibroblasts. The effect of TGF-beta on cell surface binding was maximal after 2 h of exposure to TFG-beta and did not require epidermal growth factor or protein synthesis. The enhancement was dose dependent and was found with the 125I-labeled 70-kilodalton amino-terminal fragment of fibronectin as well as with 125I-fibronectin. Treatment of cultures with TGF-beta for 6 h resulted in a threefold increase in the estimated number of fibronectin binding sites. The increase in number of binding sites was accompanied by an increased accumulation of labeled fibronectin in detergent-insoluble extracellular matrix. The effect of TGF-beta was biphasic; after 6 h of exposure, less labeled fibronectin bound to treated cultures than to control cultures. Exposure of cells to TGF-beta for greater than 6 h caused a two- to threefold increase in the accumulation of cellular fibronectin in culture medium as detected by a quantitative enzyme-linked immunosorbent assay. The second phase of the biphasic effect and the increase in soluble cellular fibronectin were blocked by cycloheximide. Immunofluorescence staining of fibroblast cultures with antifibronectin revealed that TGF-beta caused a striking increase in fibronectin fibrils. The 70-kilodalton amino-terminal fragment of fibronectin, which blocks incorporation of fibronectin into extracellular matrix, blocked anchorage-independent growth of NRK-49F cells in the presence of epidermal growth factor. Our results show that an increase in the binding and rate of assembly of exogenous fibronectin is an early event preceding the increase in expression of extracellular matrix proteins. Such an early increase in cell surface binding of exogenous fibronectin may be a mechanism whereby TGF-beta can modify extracellular matrix characteristics rapidly after tissue injury or during embryonic morphogenesis. Images PMID:3054513

  16. Expression of α5 integrin rescues fibronectin responsiveness in NT2N CNS neuronal cells

    PubMed Central

    Meland, Marit N.; Herndon, Mary E.; Stipp, Christopher S.

    2010-01-01

    The extracellular matrix protein fibronectin is implicated in neuronal regeneration in the peripheral nervous system. In the central nervous system (CNS), fibronectin is upregulated at sites of penetrating injuries and stroke; however, CNS neurons downregulate the fibronectin receptor, α5β1 integrin, during differentiation and generally respond poorly to fibronectin. NT2N CNS neuron-like cells (derived from NT2 precursor cells) have been used in pre-clinical and clinical studies for treatment of stroke and a variety of CNS injury and disease models. Here we show that, like primary CNS neurons, NT2N cells downregulate α5β1 integrin during differentiation and respond poorly to fibronectin. The poor neurite outgrowth by NT2N cells on fibronectin can be rescued by transducing NT2 precursors with a retroviral vector expressing α5 integrin under the control of the Murine Stem Cell Virus 5′ long terminal repeat. Sustained α5 integrin expression is compatible with the CNS-like neuronal differentiation of NT2N cells and does not prevent robust neurite outgrowth on other integrin ligands. Thus, α5 integrin expression in CNS neuronal precursor cells may provide a strategy for enhancing the outgrowth and survival of implanted cells in cell replacement therapies for CNS injury and disease. PMID:19598247

  17. Fibronectin and asialoglyprotein receptor mediate hepatitis B surface antigen binding to the cell surface.

    PubMed

    Yang, Jing; Wang, Feng; Tian, Linlin; Su, Jing; Zhu, Xiangqian; Lin, Li; Ding, Xiaoran; Wang, Xuejun; Wang, Shengqi

    2010-06-01

    Both fibronectin and the asialoglycoprotein receptor (ASGPR) have been identified by some investigators as partners for hepatitis B virus (HBV) envelope proteins. Because fibronectin is a natural ligand for ASGPR, we speculated that HBV might attach to ASGPR expressed on the hepatocyte surface via fibronectin. To test this hypothesis, we first confirmed by co-immunoprecipitation that ASGPR, fibronectin and HBsAg bind to each other in HepG2.2.15 cells, and possible binding domains were identified by GST pull-down. In addition, by measuring binding of HBsAg to cells, we found that ASGPR and fibronectin enhanced the binding capability of HBsAg to HepG2 cells, and even to 293T and CHO cells, which normally do not bind HBV. In conclusion, our findings suggest that both fibronectin and ASGPR mediate HBsAg binding to the cell surface, which provides further evidence for the potential roles of these two proteins in mediating HBV binding to liver cells. PMID:20364278

  18. Cryptic activity within the Type III1 domain of fibronectin regulates tissue inflammation and angiogenesis

    PubMed Central

    Cho, Christina; Kelsh-Lasher, Rhiannon; Ambesi, Anthony; McKeown-Longo, Paula J.

    2016-01-01

    The fibronectin matrix provides mechanical and biochemical information to regulate homeostatic and pathological processes within tissues. Fibronectin consists of independently-folded modules termed Types I, II and III. In response to cellular contractile force, Type III domains unfold to initiate a series of homophilic binding events which result in the assembly of a complex network of intertwining fibrils. The unfolding of Type III modules provides elasticity to the assembled fibronectin matrix allowing it to function as a dynamic scaffold which provides binding sites for cellular receptors, growth factors and other matrix molecules. Access to bioactive sites within the fibronectin matrix is under complex regulation and controlled through a combination of mechanical and proteolytic activity. Mechanical unfolding of Type III modules and limited proteolysis can alter the topographical display of bioactive sites within the fibronectin fibrils by exposing previously cryptic sites and disrupting functional sites. In this review we will discuss cryptic activity found within the first Type III module of fibronectin and its impact on tissue angiogenesis and inflammation.

  19. Binding of tenascin-C to soluble fibronectin and matrix fibrils.

    PubMed

    Chung, C Y; Zardi, L; Erickson, H P

    1995-12-01

    The small splice variant of tenascin-C (TN) has eight fibronectin type III (FN3) domains. The major large splice variant has three (in chicken) or seven (in human) additional FN3 domains inserted between domains five and six. Chiquet-Ehrismann et al. (Chiquet-Ehrismann, R., Matsuoka, Y., Hofer, U., Spring, J., Bernasconi, C., and Chiquet, M. (1991, Cell Regul. 2, 927-938) demonstrated that the small variant bound preferentially to fibronectin in enzyme-linked immunosorbent assay, and only the small variant was incorporated into the matrix by cultures of chicken fibroblasts. Here we have studied human TN, and confirmed that the small variant binds preferentially to purified fibronectin and to fibronectin-containing extracellular matrix. Thus this differential binding appears to be conserved across vertebrate species. Using bacterial expression proteins, we mapped the major binding site to the third FN3 domain of TN. Consistent with this mapping, a monoclonal antibody against an epitope in this domain did not stain TN segments bound to cell culture matrix fibrils. The enhanced binding of the small TN variant suggests the existence of another, weak binding site probably in FN3 domains 6-8, which is only positioned to bind fibronectin in the small splice variant. This binding of domains 6-8 may involve a third molecule present in matrix fibrils, as the enhanced binding of small TN was much more prominent to matrix fibrils than to purified fibronectin. PMID:7499434

  20. Mapping the Ligand-Binding Region of Borrelia hermsii Fibronectin-Binding Protein

    PubMed Central

    Brenner, Christiane; Bomans, Katharina; Habicht, Jüri; Simon, Markus M.; Wallich, Reinhard

    2013-01-01

    Many pathogenic microorganisms express fibronectin-binding molecules that facilitate their adherence to the extracellular matrix and/or entry into mammalian cells. We have previously described a Borrelia recurrentis gene, cihC that encodes a 40-kDa surface receptor for both, fibronectin and the complement inhibitors C4bp and C1-Inh. We now provide evidence for the expression of a group of highly homologues surface proteins, termed FbpA, in three B. hermsii isolates and two tick-borne relapsing fever spirochetes, B. parkeri and B. turicatae. When expressed in Escherichia coli or B. burgdorferi, four out of five proteins were shown to selectively bind fibronectin, whereas none of five proteins were able to bind the human complement regulators, C4bp and C1-Inh. By applying deletion mutants of the B. hermsii fibronectin-binding proteins a putative high-affinity binding site for fibronectin was mapped to its central region. In addition, the fibronectin-binding proteins of B. hermsii were found to share sequence homology with BBK32 of the Lyme disease spirochete B. burgdorferi with similar function suggesting its involvement in persistence and/or virulence of relapsing fever spirochetes. PMID:23658828

  1. Fibronectin-like protein in Porifera: its role in cell aggregation.

    PubMed Central

    Labat-Robert, J; Robert, L; Auger, C; Lethias, C; Garrone, R

    1981-01-01

    Experiments were carried out on a freshwater sponge (Ephydatia mulleri) in order to demonstrate the presence of fibronectin in Porifera. By using antibodies to highly purified human plasma fibronectin, the presence of a similar or identical protein could be demonstrated in the membranes of E. mulleri cells such as epithelial cells, fibroblast-like cells, and choanocytes. The reaction was specific, could be abolished by the addition of excess fibronectin, and was not observed with nonimmune rabbit serum. The immune fluorescent reaction became stronger when the sponge cells were pretreated with acetone and could also be observed, although with a less intense staining, on the intercellular matrix. This shows the predominant presence of a sponge fibronectin-like protein in the cell membranes and also its presence to a lesser extent in the intercellular matrix. When dissociated sponge cells were led to reassociate under the microscope, reassociation could be completely inhibited by anti-human fibronectin antiserum up to a dilution of 1:120 and partially inhibited up to a dilution of 1:240. The reassociation of dissociated sponge cells could also be inhibited by the addition of purified gelatin but not with serum albumin or with a normal, nonimmune rabbit serum. These results clearly indicate that a sponge cell fibronectin-like protein may play an important role as the (or one of the) recognition site(s) of the aggregation factor(s) and can therefore be directly involved in cell association, morphogenesis, and differentiation. Images PMID:7031644

  2. Loss of fibronectin from the aged stem cell niche affects the regenerative capacity of skeletal muscle in mice.

    PubMed

    Lukjanenko, Laura; Jung, M Juliane; Hegde, Nagabhooshan; Perruisseau-Carrier, Claire; Migliavacca, Eugenia; Rozo, Michelle; Karaz, Sonia; Jacot, Guillaume; Schmidt, Manuel; Li, Liangji; Metairon, Sylviane; Raymond, Frederic; Lee, Umji; Sizzano, Federico; Wilson, David H; Dumont, Nicolas A; Palini, Alessio; Fässler, Reinhard; Steiner, Pascal; Descombes, Patrick; Rudnicki, Michael A; Fan, Chen-Ming; von Maltzahn, Julia; Feige, Jerome N; Bentzinger, C Florian

    2016-08-01

    Age-related changes in the niche have long been postulated to impair the function of somatic stem cells. Here we demonstrate that the aged stem cell niche in skeletal muscle contains substantially reduced levels of fibronectin (FN), leading to detrimental consequences for the function and maintenance of muscle stem cells (MuSCs). Deletion of the gene encoding FN from young regenerating muscles replicates the aging phenotype and leads to a loss of MuSC numbers. By using an extracellular matrix (ECM) library screen and pathway profiling, we characterize FN as a preferred adhesion substrate for MuSCs and demonstrate that integrin-mediated signaling through focal adhesion kinase and the p38 mitogen-activated protein kinase pathway is strongly de-regulated in MuSCs from aged mice because of insufficient attachment to the niche. Reconstitution of FN levels in the aged niche remobilizes stem cells and restores youth-like muscle regeneration. Taken together, we identify the loss of stem cell adhesion to FN in the niche ECM as a previously unknown aging mechanism. PMID:27376579

  3. Focal weakness following herpes zoster.

    PubMed Central

    Cockerell, O C; Ormerod, I E

    1993-01-01

    Three patients presented with focal weakness of an arm which followed segmental herpes zoster affecting the same limb. Neurophysiological investigations suggest that the site of the lesion lay at the root, plexus, or peripheral nerve level. This reflects the various ways in which the virus may affect the peripheral nervous system. PMID:8410022

  4. FibronectinEDA Promotes Chronic Cutaneous Fibrosis Through Toll-like Receptor Signaling

    PubMed Central

    Bhattacharyya, Swati; Tamaki, Zenshiro; Wang, Wenxia; Hinchcliff, Monique; Hoover, Paul; Getsios, Spiro; White, Eric S.; Varga, John

    2015-01-01

    Scleroderma is a progressive autoimmune disease affecting multiple organs. Fibrosis, the hallmark of scleroderma, represents transformation of self-limited wound healing into a deregulated self-sustaining process. The factors responsible for maintaining persistent fibroblast activation in scleroderma and other conditions with chronic fibrosis are not well understood. Toll-like receptor 4 (TLR4) and its damage-associated endogenous ligands are implicated in immune and fibrotic responses. We now show that fibronectin extra domain A (FnEDA) is an endogenous TLR4 ligand markedly elevated in the circulation and lesional skin biopsies from patients with scleroderma, as well as in mice with experimentally induced cutaneous fibrosis. Synthesis of FnEDA was preferentially stimulated by transforming growth factor–β in normal fibroblasts and was constitutively up-regulated in scleroderma fibroblasts. Exogenous FnEDA was a potent stimulus for collagen production, myofibroblast differentiation, and wound healing in vitro and increased the mechanical stiffness of human organotypic skin equivalents. Each of these profibrotic FnEDA responses was abrogated by genetic, RNA interference, or pharmacological disruption of TLR4 signaling. Moreover, either genetic loss of FnEDA or TLR4 blockade using a small molecule mitigated experimentally induced cutaneous fibrosis in mice. These observations implicate the FnEDA-TLR4 axis in cutaneous fibrosis and suggest a paradigm in which aberrant FnEDA accumulation in the fibrotic milieu drives sustained fibroblast activation via TLR4. This model explains how a damage-associated endogenous TLR4 ligand might contribute to converting self-limited tissue repair responses into intractable fibrogenesis in chronic conditions such as scleroderma. Disrupting sustained TLR4 signaling therefore represents a potential strategy for the treatment of fibrosis in scleroderma. PMID:24739758

  5. Fibronectin contributes to pathological cardiac hypertrophy but not physiological growth

    PubMed Central

    Konstandin, Mathias H.; Völkers, Mirko; Collins, Brett; Quijada, Pearl; Quintana, Mercedes; De La Torre, Andrea; Ormachea, Lucy; Din, Shabana; Gude, Natalie; Toko, Haruhiro; Sussman, Mark A.

    2013-01-01

    Background Ability of the heart to undergo pathological or physiological hypertrophy upon increased wall stress is critical for long-term compensatory function in response to increased workload demand. While substantial information has been published on the nature of the fundamental molecular signaling involved in hypertrophy, the role of extracellular matrix (ECM) protein Fibronectin (Fn) in hypertrophic signaling is unclear. Objective Delineate the role of Fn during pressure overload-induced pathological cardiac hypertrophy and physiological growth prompted by exercise. Methods and Results Genetic conditional ablation of Fn in adulthood blunts cardiomyocyte hypertrophy upon pressure overload via attenuated activation of Nuclear Factor of Activated T cells (NFAT). Loss of Fn delays development of heart failure and improves survival. In contrast, genetic deletion of Fn has no impact on physiological cardiac growth induced by voluntary wheel running. Down regulation of the transcription factor c/EBPβ (Ccaat-enhanced binding protein β), which is essential for induction of the physiological growth program, is unaffected by Fn deletion. Nuclear NFAT translocation is triggered by Fn in conjunction with up-regulation of the fetal gene program and hypertrophy of cardiomyocytes in vitro. Furthermore, activation of the physiological gene program induced by Insulin stimulation in vitro is attenuated by Fn, whereas Insulin had no impact on Fn-induced pathological growth program. Conclusion Fn contributes to pathological cardiomyocyte hypertrophy in vitro and in vivo via NFAT activation. Fn is dispensable for physiological growth in vivo, and Fn attenuates the activation of the physiological growth program in vitro. PMID:23912225

  6. Exposure to Biomass Smoke Extract Enhances Fibronectin Release from Fibroblasts

    PubMed Central

    Krimmer, David; Ichimaru, Yukikazu; Burgess, Janette; Black, Judith; Oliver, Brian

    2013-01-01

    COPD induced following biomass smoke exposure has been reported to be associated with a more fibrotic phenotype than cigarette smoke induced COPD. This study aimed to investigate if biomass smoke induced extracellular matrix (ECM) protein production from primary human lung fibroblasts in vitro. Primary human lung fibroblasts (n = 5–10) were stimulated in vitro for up to 72 hours with increasing concentrations of biomass smoke extract (BME) or cigarette smoke extract (CSE) prior to being assessed for deposition of ECM proteins, cytokine release, and activation of intracellular signalling molecules. Deposition of the ECM proteins perlecan and fibronectin was upregulated by both CSE (p<0.05) and BME (p<0.05). The release of the neutrophilic chemokine IL-8 was also enhanced by BME. ERK1/2 phosphorylation was significantly upregulated by BME (p<0.05). Chemical inhibition of ERK signalling molecules partially attenuated these effects (p<0.05). Stimulation with endotoxin had no effect. This study demonstrated that BME had similar effects to CSE in vitro and had the capacity to directly induce fibrosis by upregulating production of ECM proteins. The mechanisms by which both biomass and cigarette smoke exposure cause lung damage may be similar. PMID:24386310

  7. Fibronectin-Grafted Titanium Dental Implants: An In Vivo Study.

    PubMed

    Chang, Yu-Chi; Ho, Kuo-Ning; Feng, Sheng-Wei; Huang, Haw-Ming; Chang, Chia-Hsun; Lin, Che-Tong; Teng, Nai-Chia; Pan, Yu Hwa; Chang, Wei-Jen

    2016-01-01

    Modification of the physiochemical properties of titanium surfaces using glow discharge plasma (GDP) and fibronectin coating has been shown to enhance the surface hydrophilicity, surface roughness, cell adhesion, migration, and proliferation. This in vivo study aimed to evaluate the bone integration efficacy of a biologically modified implant surface. Two different surface-modified implants (Ar-GDP and GDP-fib) were placed in the mandibular premolar area of six beagle dogs for 2-8 weeks. Three techniques [histologic evaluation, resonance frequency analysis (RFA), and microcomputed tomography (micro-CT) evaluation] were used to detect the implant stability and bone-implant contact. The implant stability quotient values of GDP-fib implants were significantly greater than the Ar-GDP implants at 2 and 4 weeks (P < 0.01). The bone volume/total volume ratio of GDP-fib implants was greater than the Ar-GDP implants in micro-CT evaluation. A high positive correlation was observed between RFA and micro-CT measurements. At 2 weeks, osteoblasts were seen to line the implant surface, and multinuclear osteoclasts could be seen on the surface of old parent bone. After 8 weeks, a majority of the space in the wound chamber appeared to be replaced by bone. Enhancement of the stability of biologically modified implants was proved by the results of RFA, micro-CT, and histological analysis. This enhanced stability may help fasten treatment and be clinically beneficial. PMID:27366739

  8. Increased plasma fibronectin in patients with systemic lupus erythematosus.

    PubMed

    Nishinarita, S; Yamamoto, M; Takizawa, T; Hayakawa, J; Karasaki, M; Sawada, S

    1990-06-01

    To add to our knowledge of collagen diseases, plasma fibronectin (FN) in patients with systemic lupus erythematosus (SLE) has been measured, and it was determined that the plasma FN value in those with SLE was 454 +/- 36 micrograms/ml, is significantly higher than the FN value in normal subjects (234 +/- 21 micrograms/ml) than that of patients with FN value of patients with active SLE was significantly higher (591 +/- 46 micrograms/ml) that of the patients with non-active SLE (287 +/- 31 micrograms/ml). The plasma FN value of SLE patients was also seen to be associated with the peripheral blood platelet count and with the dose level of the corticosteroid hormone administered to patients. In active SLE patients, it was similarly found that the plasma FN value had a significant correlation with the peripheral blood lymphocyte count and with the dose level of the corticosteroid hormone given to patients. Since the plasma FN value is known to be high in untreated SLE patients, it was felt that the increase of the FN value in SLE patients is not due to the effect of the corticosteroid but to the disease itself. PMID:2202543

  9. Human macrophage differentiation involves an interaction between integrins and fibronectin

    SciTech Connect

    Laouar, A.; Chubb, C.B.H.; Collart, F.; Huberman, E.

    1997-03-14

    The authors have examined the role of integrins and extracellular matrix (ECM) proteins in macrophage differentiation of (1) human HL-60 myeloid leukemia cells induced by phorbol 12-myristate 13-acetate (PMA) and (2) human peripheral blood monocytes induced by either PMA or macrophage-colony stimulating factor (M-CSF). Increased {beta}{sub 1} integrin and fibronectin (FN) gene expression was observed in PMA-treated HL-60 cells and PMA- or M-CSF-treated monocytes, even at a time preceding the manifestation of macrophage markers. Treated HL-60 cells and monocytes also released and deposited FN on the culture dishes. An HL-60 cell variant, HL-525, which is deficient in protein kinase C {beta} (PKC{beta}) and resistant to PMA-induced differentiation, failed to express FN after PMA treatment. Restoration of PKC{beta} resulted in PMA-induced FN gene expression and macrophage differentiation. The macrophage phenotype induced in HL-60 cells or monocytes was attenuated by anti-{beta}{sub 1} integrin or anti-FN MAbs. The authors suggest that macrophage differentiation involves activation of PKC and expression of specific integrins and ECM proteins. The stimulated cells, through their integrins, attach and spread on these substrates by binding to the deposited ECM proteins. This attachment and spreading in turn, through integrin signaling, leads to the macrophage phenotype.

  10. EDA-containing fibronectin increases proliferation of embryonic stem cells.

    PubMed

    Losino, Noelia; Waisman, Ariel; Solari, Claudia; Luzzani, Carlos; Espinosa, Darío Fernández; Sassone, Alina; Muro, Andrés F; Miriuka, Santiago; Sevlever, Gustavo; Barañao, Lino; Guberman, Alejandra

    2013-01-01

    Embryonic stem cells (ESC) need a set of specific factors to be propagated. They can also grow in conditioned medium (CM) derived from a bovine granulosa cell line BGC (BGC-CM), a medium that not only preserves their main features but also increases ESC´s proliferation rate. The mitogenic properties of this medium were previously reported, ascribing this effect to an alternative spliced generated fibronectin isoform that contains the extra domain A (FN EDA(+)). Here, we investigated if the FN EDA(+) isoform increased proliferation of mouse and human ES cells. We analyzed cell proliferation using conditioned media produced by different mouse embryonic fibroblast (MEF) lines genetically engineered to express FN constitutively including or excluding the EDA domain (FN EDA(-)), and in media supplemented with recombinant peptides containing or not the EDA. We found that the presence of EDA in the medium increased mouse and human ESC's proliferation rate. Here we showed for the first time that this FN isoform enhances ESC's proliferation. These findings suggest a possible conserved behavior for regulation of ES cells proliferation by this FN isoform and could contribute to improve their culturing conditions both for research and cell therapy. PMID:24244705

  11. EDA-Containing Fibronectin Increases Proliferation of Embryonic Stem Cells

    PubMed Central

    Losino, Noelia; Waisman, Ariel; Solari, Claudia; Luzzani, Carlos; Espinosa, Darío Fernández; Sassone, Alina; Muro, Andrés F.; Miriuka, Santiago; Sevlever, Gustavo; Barañao, Lino; Guberman, Alejandra

    2013-01-01

    Embryonic stem cells (ESC) need a set of specific factors to be propagated. They can also grow in conditioned medium (CM) derived from a bovine granulosa cell line BGC (BGC-CM), a medium that not only preserves their main features but also increases ESC´s proliferation rate. The mitogenic properties of this medium were previously reported, ascribing this effect to an alternative spliced generated fibronectin isoform that contains the extra domain A (FN EDA+). Here, we investigated if the FN EDA+ isoform increased proliferation of mouse and human ES cells. We analyzed cell proliferation using conditioned media produced by different mouse embryonic fibroblast (MEF) lines genetically engineered to express FN constitutively including or excluding the EDA domain (FN EDA-), and in media supplemented with recombinant peptides containing or not the EDA. We found that the presence of EDA in the medium increased mouse and human ESC’s proliferation rate. Here we showed for the first time that this FN isoform enhances ESC’s proliferation. These findings suggest a possible conserved behavior for regulation of ES cells proliferation by this FN isoform and could contribute to improve their culturing conditions both for research and cell therapy. PMID:24244705

  12. Fibronectin fragments modulate monocyte VLA-5 expression and monocyte migration.

    PubMed

    Trial, J; Baughn, R E; Wygant, J N; McIntyre, B W; Birdsall, H H; Youker, K A; Evans, A; Entman, M L; Rossen, R D

    1999-08-01

    To identify the mechanisms that cause monocyte localization in infarcted myocardium, we studied the impact of ischemia-reperfusion injury on the surface expression and function of the monocyte fibronectin (FN) receptor VLA-5 (alpha(5)beta(1) integrin, CD49e/CD29). Myocardial infarction was associated with the release of FN fragments into cardiac extracellular fluids. Incubating monocytes with postreperfusion cardiac lymph that contained these FN fragments selectively reduced expression of VLA-5, an effect suppressed by specific immunoadsorption of the fragments. Treating monocytes with purified, 120-kDa cell-binding FN fragments (FN120) likewise decreased VLA-5 expression, and did so by inducing a serine proteinase-dependent proteolysis of this beta(1) integrin. We postulated that changes in VLA-5 expression, which were induced by interactions with cell-binding FN fragments, may alter monocyte migration into tissue FN, a prominent component of the cardiac extracellular matrix. Support for this hypothesis came from experiments showing that FN120 treatment significantly reduced both spontaneous and MCP-1-induced monocyte migration on an FN-impregnated collagen matrix. In vivo, it is likely that contact with cell-binding FN fragments also modulates VLA-5/FN adhesive interactions, and this causes monocytes to accumulate at sites where the fragment concentration is sufficient to ensure proteolytic degradation of VLA-5. PMID:10449434

  13. Plasma fibronectin promotes thrombus growth and stability in injured arterioles

    PubMed Central

    Ni, Heyu; Yuen, Peter S. T.; Papalia, Jessie M.; Trevithick, Jane E.; Sakai, Takao; Fässler, Reinhard; Hynes, Richard O.; Wagner, Denisa D.

    2003-01-01

    Mice lacking both of the best-known platelet ligands, von Willebrand factor and fibrinogen, can still form occlusive thrombi in injured arterioles. The platelets of these animals accumulate excessive amounts of fibronectin (FN). These observations led us to examine the contribution of plasma FN (pFN) to thrombus formation. Inactivation of the FN gene in FN conditional knockout mice reduced pFN levels to <2% and platelet FN to ≈20% of the levels in similarly treated control mice. The mice were then observed in a model of arterial injury to evaluate their capacity to form thrombi. The deficiency of pFN did not affect the initial platelet adhesion, but a delay of several minutes in thrombus formation was observed in the arterioles of pFN-deficient mice as compared with control mice. The thrombi that formed in the absence of pFN were stably anchored to the vessel wall but continuously shed platelets or small platelet clumps, thus slowing their growth significantly; the platelet/platelet cohesion was apparently diminished. Consequently the occlusion of pFN-deficient vessels was delayed, with the majority of vessels remaining patent at the end of the 40-min observation period. We conclude that, in addition to von Willebrand factor and fibrinogen, FN plays a significant role in thrombus initiation, growth, and stability at arterial shear rates and that deficiency in each of the three platelet ligands has its own specific impact on platelet plug formation. PMID:12606706

  14. Fibronectin Fiber Extension Decreases Cell Spreading and Migration.

    PubMed

    Hubbard, Brant; Buczek-Thomas, Jo Ann; Nugent, Matthew A; Smith, Michael L

    2016-08-01

    The extracellular matrix (ECM) is present in a range of molecular conformations and intermolecular arrangements. Fibronectin (Fn) molecules that constitute fibers within the ECM can exist in a variety of conformations that result from both mechanical stress and chemical factors such as allosteric binding partners. The long-standing hypothesis that conformational changes regulate the binding of cells to Fn fibers has only been tested for mutated molecules of Fn and has yet to be fully evaluated with Fn fibers. Using time-lapse microscopy we examined how mechanical extension of single fibers of Fn affects the adhesion and migration of endothelial cells. Using this single fiber adhesion technique, we show that high levels of mechanical strain applied to Fn fibers decreases the rates of both cell spreading and cell migration. These data indicate a fundamental cellular response to mechanical strain in the ECM that might have important implications for understanding how cells are recruited during tissue development and repair. J. Cell. Physiol. 231: 1728-1736, 2016. © 2015 Wiley Periodicals, Inc. PMID:26621030

  15. Fibronectin-Grafted Titanium Dental Implants: An In Vivo Study

    PubMed Central

    Chang, Yu-Chi; Ho, Kuo-Ning; Feng, Sheng-Wei; Huang, Haw-Ming; Chang, Chia-Hsun; Lin, Che-Tong; Teng, Nai-Chia; Pan, Yu Hwa; Chang, Wei-Jen

    2016-01-01

    Modification of the physiochemical properties of titanium surfaces using glow discharge plasma (GDP) and fibronectin coating has been shown to enhance the surface hydrophilicity, surface roughness, cell adhesion, migration, and proliferation. This in vivo study aimed to evaluate the bone integration efficacy of a biologically modified implant surface. Two different surface-modified implants (Ar-GDP and GDP-fib) were placed in the mandibular premolar area of six beagle dogs for 2–8 weeks. Three techniques [histologic evaluation, resonance frequency analysis (RFA), and microcomputed tomography (micro-CT) evaluation] were used to detect the implant stability and bone-implant contact. The implant stability quotient values of GDP-fib implants were significantly greater than the Ar-GDP implants at 2 and 4 weeks (P < 0.01). The bone volume/total volume ratio of GDP-fib implants was greater than the Ar-GDP implants in micro-CT evaluation. A high positive correlation was observed between RFA and micro-CT measurements. At 2 weeks, osteoblasts were seen to line the implant surface, and multinuclear osteoclasts could be seen on the surface of old parent bone. After 8 weeks, a majority of the space in the wound chamber appeared to be replaced by bone. Enhancement of the stability of biologically modified implants was proved by the results of RFA, micro-CT, and histological analysis. This enhanced stability may help fasten treatment and be clinically beneficial. PMID:27366739

  16. Statistical earthquake focal mechanism forecasts

    NASA Astrophysics Data System (ADS)

    Kagan, Yan Y.; Jackson, David D.

    2014-04-01

    Forecasts of the focal mechanisms of future shallow (depth 0-70 km) earthquakes are important for seismic hazard estimates and Coulomb stress, and other models of earthquake occurrence. Here we report on a high-resolution global forecast of earthquake rate density as a function of location, magnitude and focal mechanism. In previous publications we reported forecasts of 0.5° spatial resolution, covering the latitude range from -75° to +75°, based on the Global Central Moment Tensor earthquake catalogue. In the new forecasts we have improved the spatial resolution to 0.1° and the latitude range from pole to pole. Our focal mechanism estimates require distance-weighted combinations of observed focal mechanisms within 1000 km of each gridpoint. Simultaneously, we calculate an average rotation angle between the forecasted mechanism and all the surrounding mechanisms, using the method of Kagan & Jackson proposed in 1994. This average angle reveals the level of tectonic complexity of a region and indicates the accuracy of the prediction. The procedure becomes problematical where longitude lines are not approximately parallel, and where shallow earthquakes are so sparse that an adequate sample spans very large distances. North or south of 75°, the azimuths of points 1000 km away may vary by about 35°. We solved this problem by calculating focal mechanisms on a plane tangent to the Earth's surface at each forecast point, correcting for the rotation of the longitude lines at the locations of earthquakes included in the averaging. The corrections are negligible between -30° and +30° latitude, but outside that band uncorrected rotations can be significantly off. Improved forecasts at 0.5° and 0.1° resolution are posted at http://eq.ess.ucla.edu/kagan/glob_gcmt_index.html.

  17. Interactions between integrin receptors and fibronectin are required for calvarial osteoblast differentiation in vitro

    NASA Technical Reports Server (NTRS)

    Moursi, A. M.; Globus, R. K.; Damsky, C. H.

    1997-01-01

    We previously showed that anti-fibronectin antibodies or soluble fibronectin fragments containing the central cell-binding domain inhibit formation of mineralized nodules by fetal calvarial osteoblasts in vitro. These findings suggest a critical role for fibronectin in osteoblast differentiation and morphogenesis. In this study we tested the hypothesis that fibronectin's effects on osteogenesis are mediated via direct interactions with integrin receptors for fibronectin on osteoblasts. Immunocytochemical analysis identified the integrin fibronectin receptor alpha5ss1 in fetal rat calvarial tissue and in cultured osteoblasts at all stages of differentiation. Three other integrins, alpha3ss1, alpha8ss1 and alphavss3, which can bind fibronectin, as well as other matrix components, were also identified in tissue and at all stages of cell culture. Immunoprecipitation data showed that alpha5ss1 levels are constant throughout osteoblast differentiation whereas levels of alpha3ss1 and alpha8ss1 decline in mature mineralized cultures. To determine whether integrin fibronectin receptors are required for osteoblast formation of mineralized nodules, we examined the extent of nodule formation in the presence and absence of function-perturbing anti-integrin antibodies. The antibodies were present continuously in cultures beginning at confluence (day 3), and nodule formation was measured at days 10 and 20. An anti-alpha5 integrin subunit antibody reduced nodule formation to less than 5% of control values at both time points. Inhibition of nodule formation was reversible and did not affect cell attachment and viability. Function-perturbing antibodies against alpha3ss1 and alpha8ss1 also reduced nodule formation, to less than 20% of control values. In contrast, function-perturbing antibodies to alphavss3 and alphavss5 did not affect nodule formation, indicating that the inhibitions noted were indeed specific. To determine the effect of antibody treatment on gene expression, steady

  18. Quantification of fibronectin as a method to assess ex vivo extracellular matrix remodeling.

    PubMed

    Bager, C L; Gudmann, N; Willumsen, N; Leeming, D J; Karsdal, M A; Bay-Jensen, A C; Høgdall, E; Balslev, I; He, Y

    2016-09-16

    Altered architecture, composition and quality of the extracellular matrix (ECM) are pathological hallmarks of several inflammatory and fibro-proliferative pathological processes such as osteoarthritis (OA), rheumatoid arthritis (RA), fibrosis and cancer. One of the most important components of the ECM is fibronectin. Fibronectin serves as an adhesion molecule anchoring cells to the underlying basement membrane through direct interaction with integrin receptors. Fibronectin hereby modulates the properties of the ECM and affects cellular processes. Quantification of fibronectin remodeling could therefore be used to assess the changes in the ECM that occur during progression of fibro-proliferative pathologies. Ex vivo models are becoming state-of-the-art tools to study ECM remodeling as the cellular composition and the organization of the ECM are preserved. Ex vivo models may therefore be a valuable tool to study the ECM remodeling that occurs during progression of fibro-proliferative pathologies. The aim of this study was to quantify fibronectin remodeling in ex vivo models of cartilage and cancer. A competitive The enzyme-linked immunosorbent assay (ELISA) against the C-terminus of fibronectin was developed (FBN-C). The assay was evaluated in relation to specificity, technical performance and as a marker for quantification of fibronectin in cartilage and cancer ex vivo models. The ELISA was specific and technically stable. Cleavage of tumor tissue with MMP-2 released significantly higher levels of FBN-C compared to tissue with buffer only and western blot analysis revealed that FBN-C recognizes both full length and degraded fibronectin. When ex vivo cartilage cultures were stimulated with the anabolic factor TGFβ and catabolic factors TNF-α and OSM, significantly higher levels of FBN-C were found in the conditioned media. Lastly, FBN-C was released from a cancer ex vivo model. In conclusion, we were able to quantify fibronectin remodeling in ex vivo models

  19. Distracted driving

    MedlinePlus

    ... stay safe with a cell phone in the car. ... for Disease Control and Prevention Injury Prevention & Control. Motor Vehicle Safety. www.cdc.gov/motorvehiclesafety/distracted_driving . Accessed May ...

  20. Driving Safely

    MedlinePlus

    ... drivers’ flexibility and coordination, and reduced driving errors. S l Hand grip strengthening to help you hold on to the steering wheel l Shoulder and upper arm flexibility exercises to make ...

  1. Distracted Driving

    MedlinePlus

    ... combines all three types of distraction. 3 How big is the problem? Deaths In 2013, 3,154 ... European countries. More A CDC study analyzed 2011 data on distracted driving, including talking on a cell ...

  2. Fibronectin fibrillogenesis facilitates mechano-dependent cell spreading, force generation, and nuclear size in human embryonic fibroblasts.

    PubMed

    Scott, Lewis E; Mair, Devin B; Narang, Jiten D; Feleke, Kirubel; Lemmon, Christopher A

    2015-11-01

    Cells respond to mechanical cues from the substrate to which they are attached. These mechanical cues drive cell migration, proliferation, differentiation, and survival. Previous studies have highlighted three specific mechanisms through which substrate stiffness directly alters cell function: increasing stiffness drives (1) larger contractile forces; (2) increased cell spreading and size; and (3) altered nuclear deformation. While studies have shown that substrate mechanics are an important cue, the role of the extracellular matrix (ECM) has largely been ignored. The ECM is a crucial component of the mechanosensing system for two reasons: (1) many ECM fibrils are assembled by application of cell-generated forces, and (2) ECM proteins have unique mechanical properties that will undoubtedly alter the local stiffness sensed by a cell. We specifically focused on the role of the ECM protein fibronectin (FN), which plays a critical role in de novo tissue production. In this study, we first measured the effects of substrate stiffness on human embryonic fibroblasts by plating cells onto microfabricated pillar arrays (MPAs) of varying stiffness. Cells responded to increasing substrate stiffness by generating larger forces, spreading to larger sizes, and altering nuclear geometry. These cells also assembled FN fibrils across all stiffnesses, with optimal assembly occurring at approximately 6 kPa. We then inhibited FN assembly, which resulted in dramatic reductions in contractile force generation, cell spreading, and nuclear geometry across all stiffnesses. These findings suggest that FN fibrils play a critical role in facilitating cellular responses to substrate stiffness. PMID:26412391

  3. Analysis of Soluble Molecular Fibronectin-Fibrin Complexes and EDA-Fibronectin Concentration in Plasma of Patients with Atherosclerosis.

    PubMed

    Lemańska-Perek, Anna; Krzyżanowska-Gołąb, Dorota; Pupek, Małgorzata; Klimeczek, Piotr; Witkiewicz, Wojciech; Kątnik-Prastowska, Iwona

    2016-06-01

    Atherosclerosis, a chronic vascular disease, leads to molecular events bound with interplaying processes of inflammation and coagulation. In the present study, fibronectin (FN), FN containing extra domain A (EDA-FN), frequency of occurrence, and relative amounts of soluble plasma FN-fibrin complexes were analyzed in 80 plasma samples of patients suspected of coronary artery disease based on clinical evaluation and changes in arteries found by computed tomographic coronary angiography. The study showed that in the plasma of the patients' group with high risk of coronary artery disease EDA-FN concentration was significantly higher (3.5 ± 2.5 mg/L; P < 0.025) and the molecular FN-fibrin complexes of 1000 kDa and higher occurred more often than in the groups of patients with mild risk of coronary artery disease and the normal age-matched. The increased level of EDA-FN and occurrence of FN-fibrin complexes could have a potential diagnostic value in the diagnosis and management of patients with coronary artery disease. PMID:27022744

  4. Fibronectin Regulation by Vitamin C Treatment in Kidneys of Nicotinic Mice Offspring

    PubMed Central

    Pahang, Hasan; Nikravesh, Mohammad Reza; Jalali, Mehdi; Ebrahimzadeh Bideskan, Alireza; Zargari, Peyman; Sadr Nabavi, Ariane

    2014-01-01

    Background: Maternal cigarette smoking causes health risks and developmental defects in the offspring. So far, many studies have been conducted to suppress the effects of nicotine. However, the effects of coadministration of vitamin C and nicotine on extracellular matrix have not gained enough attention. Objectives: This study decided to investigate the effects of vitamin C on fibronectin expression in kidneys of mice offspring, treated with nicotine. Materials and Methods: Eighteen female pregnant BALB/c mice were selected; six mice in the experimental group 1 (exp 1) received nicotine (3 mg/kg/day), six mice in the experimental group 2 (exp 2) received 3 mg/kg/day nicotine and 9 mg/kg/day vitamin C simultaneously, and six were used as the control group and received 3 mL/kg/day normal saline via intraperitoneal (IP) injection parallel to other groups, since the 6th day of gestation to the end of prenatal period. In the first days of delivery, fibronectin content of neonatal kidneys was studied by immunohistochemistry (IHC) assay and gene expression was studied by the real-time PCR. Results: IHC results showed that fibronectin reaction significantly increased in proximal convoluted tubules of exp 1 compared with the control offspring; on the other hand, fibronectin reaction decreased in the mice offspring of exp 2. Gene expression results showed that fibronectin expression in the exp 1 offspring significantly increased compared with the control ones and fibronectin expression decreased in the mice offspring of exp 2. Conclusions: This study revealed that vitamin C could reduce the fibronectin accumulation effects of nicotine on kidney. PMID:25237577

  5. Trop-2 is up-regulated in invasive prostate cancer and displaces FAK from focal contacts.

    PubMed

    Trerotola, Marco; Ganguly, Kirat K; Fazli, Ladan; Fedele, Carmine; Lu, Huimin; Dutta, Anindita; Liu, Qin; De Angelis, Tiziana; Riddell, Luke W; Riobo, Natalia A; Gleave, Martin E; Zoubeidi, Amina; Pestell, Richard G; Altieri, Dario C; Languino, Lucia R

    2015-06-10

    In this study, we show that the transmembrane glycoprotein Trop-2 is up-regulated in human prostate cancer (PCa) with extracapsular extension (stages pT3/pT4) as compared to organ-confined (stage pT2) PCa. Consistent with this evidence, Trop-2 expression is found to be increased in metastatic prostate tumors of Transgenic Adenocarcinoma of Mouse Prostate mice and to strongly correlate with α5β1 integrin levels. Using PCa cells, we show that Trop-2 specifically associates with the α5 integrin subunit, as binding to α3 is not observed, and that Trop-2 displaces focal adhesion kinase from focal contacts. In support of the role of Trop-2 as a promoter of PCa metastatic phenotype, we observe high expression of this molecule in exosomes purified from Trop-2-positive PCa cells. These vesicles are then found to promote migration of Trop-2-negative PCa cells on fibronectin, an α5β1 integrin/focal adhesion kinase substrate, thus suggesting that the biological function of Trop-2 may be propagated to recipient cells. In summary, our findings show that Trop-2 promotes an α5β1 integrin-dependent pro-metastatic signaling pathway in PCa cells and that the altered expression of Trop-2 may be utilized for early identification of capsule-invading PCa. PMID:26015409

  6. MRI of Focal Liver Lesions.

    PubMed

    Albiin, Nils

    2012-05-01

    Magnetic resonance imaging, MRI has more advantages than ultrasound, computed tomography, CT, positron emission tomography, PET, or any other imaging modality in diagnosing focal hepatic masses. With a combination of basic T1 and T2 weighted sequences, diffusion weighted imaging, DWI, and hepatobiliary gadolinium contrast agents, that is gadobenate dimeglumine (Gd-BOPTA) and gadoxetic acid (Gd-EOB), most liver lesions can be adequately diagnosed. Benign lesions, as cyst, hemangioma, focal nodular hyperplasia, FNH or adenoma, can be distinguished from malignant lesions. In a non-cirrhotic liver, the most common malignant lesions are metastases which may be hypovascular or hypervascular. In the cirrhotic liver hepatocellular carcinoma, HCC, is of considerable importance. Besides, intrahepatic cholangiocarcinoma and other less common malignancies has to be assessed. In this review, the techniques and typical MRI features are presented as well as the new algorithm issued by American Association for the Study of the Liver Diseases (AASLD). PMID:23049491

  7. Variable focal length deformable mirror

    DOEpatents

    Headley, Daniel; Ramsey, Marc; Schwarz, Jens

    2007-06-12

    A variable focal length deformable mirror has an inner ring and an outer ring that simply support and push axially on opposite sides of a mirror plate. The resulting variable clamping force deforms the mirror plate to provide a parabolic mirror shape. The rings are parallel planar sections of a single paraboloid and can provide an on-axis focus, if the rings are circular, or an off-axis focus, if the rings are elliptical. The focal length of the deformable mirror can be varied by changing the variable clamping force. The deformable mirror can generally be used in any application requiring the focusing or defocusing of light, including with both coherent and incoherent light sources.

  8. Human macrophage differentiation involves an interaction between integrins and fibronectin

    SciTech Connect

    Laouar, A.; Chubb, C.B.H.; Collart, F.; Huberman, E.

    1996-11-15

    The authors have examined the role of the {beta}{sub 1} integrin family of adhesion receptors (VLA) and the extracellular matrix protein fibronectin (FN) in macrophage differentiation of (1) human HL-60 myeloid leukemia cells induced by phorbol 12-myristate 13-acetate (PMA) and (2) human peripheral blood monocytes induced by either PMA or macrophage-colony stimulating factor (M=CSF). Increased VLA and FN gene expression was observed as early as 4 h after PMA treatment of HL-60 cells and PMA- or M-CSF-treatment of monocytes, and it preceded the manifestation of macrophage markers. Treated HL-60 cells and monocytes also released and deposited FN on the surface of the tissue culture dishes. An HL-60 cell variant, HL-525, which is deficient in protein kinase C {beta} and resistant to PMA-induced differentiation, exhibited elevated levels of the VLA antigen but failed to express the FN gene. Incubation of HL-525 cells on dishes precoated with exogenous FN resulted in a macrophage differentiation. The macrophage phenotype induced in HL-60 cells, HL-525 cells, or monocytes was attenuated to various degrees by anti-VLA or anti-FN MAbs or by exogenous RGDS, a VLA-binding motif on FN. The authors suggest that macrophage differentiation is initiated by the activation of protein kinase C, which leads to the expression of the integrin, FN and related genes. The integrins mediate cell attachment and spreading on appropriate substrates by binding to deposited extracellular proteins such as FN. This attachment and spreading, in turn, leads to the expression of genes that code for the macrophage functions.

  9. Novel Mycobacteria Antigen 85 Complex Binding Motif on Fibronectin*

    PubMed Central

    Kuo, Chih-Jung; Bell, Hannah; Hsieh, Ching-Lin; Ptak, Christopher P.; Chang, Yung-Fu

    2012-01-01

    The members of the antigen 85 protein family (Ag85), consisting of members Ag85A, Ag85B, and Ag85C, are the predominantly secreted proteins of mycobacteria and possess the ability to specifically interact with fibronectin (Fn). Because Fn-binding proteins are likely to be important virulence factors of Mycobacterium spp., Ag85 may contribute to the adherence, invasion, and dissemination of organisms in host tissue. In this study, we reported the Fn binding affinity of Ag85A, Ag85B, and Ag85C from Mycobacterium avium subsp. paratuberculosis (MAP) (KD values were determined from 33.6 to 68.4 nm) and mapped the Ag85-binding motifs of Fn. Fn14, a type III module located on the heparin-binding domain II (Hep-2) of Fn, was discovered to interact with Ag85 from MAP. The peptide inhibition assay subsequently demonstrated that a peptide consisting of residues 17–26 from Fn14 (17SLLVSWQPPR26, termed P17–26) could interfere with Ag85B binding to Fn (73.3% reduction). In addition, single alanine substitutions along the sequence of P17–26 revealed that the key residues involved in Ag85-Fn binding likely contribute through hydrophobic and charge interactions. Moreover, binding of Ag85 on Fn siRNA-transfected Caco2 cells was dramatically reduced (44.6%), implying the physiological significance of the Ag85-Fn interaction between mycobacteria and host cells during infection. Our results indicate that Ag85 binds to Fn at a novel motif and plays a critical role in mycobacteria adherence to host cells by initiating infection. Ag85 might serve as an important colonization factor potentially contributing to mycobacterial virulence. PMID:22128161

  10. Modulation of endothelial cell adhesion to synthetic vascular grafts using biotinylated fibronectin in a dual ligand protein system

    NASA Astrophysics Data System (ADS)

    Anamelechi, Charles Chibuzor

    Over half a million coronary artery bypass operations are performed annually in the US yielding an annual health care cost of over 16 billion dollars. Only five percent of bypasses are repeat operations in spite of the procedures prevalence. Patients facing repeat coronary artery bypass operations often lack transplantable autologous arteries or veins, necessitating the use of substitutes. Unfortunately, synthetic small diameter vascular grafts have unacceptable patency rates, primarily due to lumenal thrombus formation and intimal thickening. Endothelial cells (EC) mediate the anti-thrombotic activity in healthy blood vessels, and due to the scarcity of suitable autologous vascular replacement, EC-seeded small diameter synthetic vascular grafts represent a clear, immediate, and practical solution. The fundamental goal of this project was to optimize the dual ligand (DL) system on synthetic vascular graft (SVG) surrogates to show enhanced cell adhesion, retention, and native functionality compared to fibronectin alone. Initially, two SVG surrogates were identified through characterization by x-ray photoelectron spectroscopy (XPS), atomic force microscopy (AFM), and 125I radiolabeling. The first modification to the DL system involved direct biotinylation of fibronectin (bFN) as a replacement for co-adsorption of FN with biotinylated bovine serum albumin (bBSA). This was analyzed with a Langmuir model using surface plasmon resonance (SPR) spectroscopy to verify the binding affinity of bFN and ELISA to detect the availability of the RGD binding motif post biotinylation. The second major change in this project examined cell binding and formation of focal adhesion after shifting from direct incubation of HUVECs with RGD-SA to sequentially adsorbing bFN(9) and RGD-SA prior to introducing unmodified HUVECs. These experiments were conducted under static seeding conditions. Next, dynamic cell seeding onto the sequentially adsorbed protein surface was examined as a function

  11. [Focal liver lesion, incidental finding].

    PubMed

    Dietrich, C F; Jenssen, C

    2012-10-01

    The differential diagnosis of incidentally found Focal Liver Lesions (FLL) is complex. Screening procedures so far are only defined for patients with liver cirrhosis. Characterization of a FLL begins as soon as it is detected. Taking patients history and thorough clinical examination are essential. An imaging procedure that is used to detect liver masses should also allow the examiner to determine whether the lesion is benign or malignant. Conventional B-mode US and colour Doppler imaging are effective at detecting and characterizing typical liver cysts and calcifications. Laboratory data, computed tomography, magnetic resonance imaging and imaging guided liver biopsy are complementary methods.Contrast Enhanced Ultrasound (CEUS) is a well established diagnostic imaging technique for a variety of indications and applications. One of the most important applications is in the liver where it is frequently a first-line technique for the detection and diagnosis (characterization) of focal liver lesions (FLL). In this setting the accurate differentiation of benign from malignant lesions is critical to ensure the patient undergoes the appropriate therapeutic option. This has been documented in recently published guidelines, in particular in terms of the enhancement patterns of the most common FLL hemangioma, focal nodular hyperplasia hepatocellular adenoma and their differentiation from malignant lesions. In this article the role of CEUS in the characterization of incidentally found FLL is described. PMID:23033169

  12. Persistent Focal Behavior and Physical Activity Performance

    ERIC Educational Resources Information Center

    Erfle, Stephen E.

    2014-01-01

    This article examines the proclivity and performance attributes of focal students across time and activities using data from 9,345 students. Three systematic focal behavior partitions are examined: Across activities, across time, and across activities and time. A student's performance is focal if it ends in 0 or 5 for push-ups and 0 for…

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

    PubMed Central

    Wasil, L R; Shair, K H Y

    2015-01-01

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

  14. Fibronectin and Cyclic Strain Improve Cardiac Progenitor Cell Regenerative Potential In Vitro

    PubMed Central

    Ghosh-Choudhary, Shohini; Fierro, Marcos J.; Christman, Karen L.; Taylor, W. Robert

    2016-01-01

    Cardiac progenitor cells (CPCs) have rapidly advanced to clinical trials, yet little is known regarding their interaction with the microenvironment. Signaling cues present in the microenvironment change with development and disease. This work aims to assess the influence of two distinct signaling moieties on CPCs: cyclic biaxial strain and extracellular matrix. We evaluate four endpoints for improving CPC therapy: paracrine signaling, proliferation, connexin43 expression, and alignment. Vascular endothelial growth factor A (about 900 pg/mL) was secreted by CPCs cultured on fibronectin and collagen I. The application of mechanical strain increased vascular endothelial growth factor A secretion 2–4-fold for CPCs cultured on poly-L-lysine, laminin, or a naturally derived cardiac extracellular matrix. CPC proliferation was at least 25% higher on fibronectin than that on other matrices, especially for lower strain magnitudes. At 5% strain, connexin43 expression was highest on fibronectin. With increasing strain magnitude, connexin43 expression decreased by as much as 60% in CPCs cultured on collagen I and a naturally derived cardiac extracellular matrix. Cyclic mechanical strain induced the strongest CPC alignment when cultured on fibronectin or collagen I. This study demonstrates that culturing CPCs on fibronectin with 5% strain magnitude is optimal for their vascular endothelial growth factor A secretion, proliferation, connexin43 expression, and alignment. PMID:27610140

  15. Polymorphisms in Fibronectin Binding Protein A of Staphylococcus Aureus are Associated with Infection of Cardiovascular Devices

    SciTech Connect

    Lower, Steven; Lamlertthon, Supaporn; Casillas-Ituarte, Nadia; Lins, Roberto D.; Yongsunthon, Ruchirej; Taylor, Eric S.; DiBartola, Alex; Edmondson, Catherine; McIntyre, Lauren M.; Reller, L. Barth; Que, Yok-Ai; Ros, Robert; Lower, Brian; Fowler, Vance

    2011-11-08

    Medical implants, like cardiovascular devices, improve the quality of life for countless individuals but may become infected with bacteria like Staphylococcus aureus. Such infections take the form of a bio-film, a structured community of bacterial cells adherent to the surface of a solid substrate. Every bio-film begins with an attractive force or bond between bacterium and substratum. We used atomic force microscopy to probe experimentally forces between a fibronectin-coated surface (i.e., proxy for an implanted cardiac device) and fibronectin-binding receptors on the surface of individual living bacteria from each of 80 clinical isolates of S. aureus. These isolates originated from humans with infected cardiac devices (CDI; n = 26), uninfected cardiac devices (n = 20), and the anterior nares of asymptomatic subjects (n = 34). CDI isolates exhibited a distinct bindingforce signature and had speci!c single amino acid polymorphisms in fibronectin-binding protein A corresponding to E652D, H782Q, and K786N. In silico molecular dynamics simulations demonstrate that residues D652, Q782, and N786 in fibronectin-binding protein A form extra hydrogen bonds with fibronectin, complementing the higher binding force and energy measured by atomic force microscopy for the CDI isolates. This study is significant, because it links pathogenic bacteria biofilms from the length scale of bonds acting across a nanometer-scale space to the clinical presentation of disease at the human dimension.

  16. Fibronectin and Cyclic Strain Improve Cardiac Progenitor Cell Regenerative Potential In Vitro.

    PubMed

    French, Kristin M; Maxwell, Joshua T; Bhutani, Srishti; Ghosh-Choudhary, Shohini; Fierro, Marcos J; Johnson, Todd D; Christman, Karen L; Taylor, W Robert; Davis, Michael E

    2016-01-01

    Cardiac progenitor cells (CPCs) have rapidly advanced to clinical trials, yet little is known regarding their interaction with the microenvironment. Signaling cues present in the microenvironment change with development and disease. This work aims to assess the influence of two distinct signaling moieties on CPCs: cyclic biaxial strain and extracellular matrix. We evaluate four endpoints for improving CPC therapy: paracrine signaling, proliferation, connexin43 expression, and alignment. Vascular endothelial growth factor A (about 900 pg/mL) was secreted by CPCs cultured on fibronectin and collagen I. The application of mechanical strain increased vascular endothelial growth factor A secretion 2-4-fold for CPCs cultured on poly-L-lysine, laminin, or a naturally derived cardiac extracellular matrix. CPC proliferation was at least 25% higher on fibronectin than that on other matrices, especially for lower strain magnitudes. At 5% strain, connexin43 expression was highest on fibronectin. With increasing strain magnitude, connexin43 expression decreased by as much as 60% in CPCs cultured on collagen I and a naturally derived cardiac extracellular matrix. Cyclic mechanical strain induced the strongest CPC alignment when cultured on fibronectin or collagen I. This study demonstrates that culturing CPCs on fibronectin with 5% strain magnitude is optimal for their vascular endothelial growth factor A secretion, proliferation, connexin43 expression, and alignment. PMID:27610140

  17. The glycation of fibronectin by glycolaldehyde and methylglyoxal as a model for aging in Bruch's membrane.

    PubMed

    Thao, Mai T; Gaillard, Elizabeth R

    2016-07-01

    The purpose of the study is to identify the sites of modification when fibronectin reacts with glycolaldehyde or methylglyoxal as a model system for aging of Bruch's membrane. A synthetic peptide consisting of the α5β1 integrin binding region of fibronectin was incubated with glycolaldehyde for 12 h or with methylglyoxal for 1 h at 37 °C. After tryptic digestion, the samples were analyzed with liquid chromatography-mass spectrometry (LC/MS). Tandem MS was used to determine the sites of modification. The adducts, aldoamine and N (ε)-carboxymethyl-lysine, attached preferably at lysine residues when the fibronectin peptide reacted with glycolaldehyde. When the fibronectin peptide reacted with methylglyoxal, modifications occurred at lysine and arginine residues. At lysine residues, N (ε)-carboxyethyl-lysine adducts were present. At arginine residues, hydroimidazolone and tetrapyrimidine adducts were present. Several advanced glycation endproducts were generated when fibronectin was glycated via glycolaldehyde and methylglyoxal. These results can help explain the structural changes Bruch's membrane undergoes during aging. PMID:27084712

  18. Matrix fibronectin disruption and altered endothelial cell adhesion induced by activated leukocytes

    SciTech Connect

    Vincent, P.; Richards, P.; Saba, T.; DelVecchio, P.

    1986-03-01

    Sequestration of activated leukocytes (PMN) within the lung may contribute to pulmonary vascular injury following trauma, sepsis, or intravascular coagulation. Monolayers of cultured rat endothelial cells were utilized to evaluate the effect of activated PMNs on endothelial cell attachment and the extracellular fibronectin matrix over a 4 hr incubation interval. Rat endothelial cells were identified by immunofluorescent staining of Factor VIII R:Ag. Endothelial cells were labeled with /sup 51/Cr in order to establish a cell injury assay in which the release of pelletable (cell associated) or non-pelletable activity was measured in the media. PMN activation was verified by chemiluminescence activity. Following phorbol myristate acetate (PMA) the leukocytes aggregated, chemiluminesced, and caused detachment of /sup 51/Cr endothelial cells. Endothelial detachment increased as a function of time with a plateau by 3 hrs. Immunofluorescent analysis of extracellular fibronectin in endothelial cell cultures revealed disruption of the fibrillar matrix fibronectin in association with endothelial cell disadhesion. Matrix fibronectin disruption was not seen with PMNs or PMA alone. Thus, disruption of the fibronectin matrix by released proteases may contribute to endothelial cell detachment.

  19. Lipoprotein(a) binds to fibronectin and has serine proteinase activity capable of cleaving it.

    PubMed Central

    Salonen, E M; Jauhiainen, M; Zardi, L; Vaheri, A; Ehnholm, C

    1989-01-01

    The plasma concentration of human lipoprotein(a) [Lp(a)] is correlated with the risk of heart disease. A distinct feature of the Lp(a) particle is the apolipoprotein (a) [apo(a)], which is associated with apoB-100, the main protein component of low-density lipoprotein. We now report that apo(a), which has extensive homology to plasminogen, binds to immobilized fibronectin. The binding of Lp(a) was localized to the C-terminal heparin-binding domain of fibronectin. Incubation of Lp(a) with fibronectin resulted in fragmentation of fibronectin. The cleavage pattern, as visualized by gel electrophoresis and immunoblotting, was reproducibly obtained with Lp(a) purified from five different individuals and was distinct from that obtained upon proteolysis of fibronectin by plasmin or kallikrein. The use of synthetic peptide substrates demonstrated that the amino acid specificity for Lp(a) was arginine rather than lysine. The proteolytic activity of Lp(a) was localized to apo(a) and experiments with inhibitors indicated that the proteolytic activity was of serine proteinase-type. Images PMID:2531657

  20. Polymorphisms in fibronectin binding protein A of Staphylococcus aureus are associated with infection of cardiovascular devices

    PubMed Central

    Lower, Steven K.; Lamlertthon, Supaporn; Casillas-Ituarte, Nadia N.; Lins, Roberto D.; Yongsunthon, Ruchirej; Taylor, Eric S.; DiBartola, Alex C.; Edmonson, Catherine; McIntyre, Lauren M.; Reller, L. Barth; Que, Yok-Ai; Ros, Robert; Lower, Brian H.; Fowler, Vance G.

    2011-01-01

    Medical implants, like cardiovascular devices, improve the quality of life for countless individuals but may become infected with bacteria like Staphylococcus aureus. Such infections take the form of a biofilm, a structured community of bacterial cells adherent to the surface of a solid substrate. Every biofilm begins with an attractive force or bond between bacterium and substratum. We used atomic force microscopy to probe experimentally forces between a fibronectin-coated surface (i.e., proxy for an implanted cardiac device) and fibronectin-binding receptors on the surface of individual living bacteria from each of 80 clinical isolates of S. aureus. These isolates originated from humans with infected cardiac devices (CDI; n = 26), uninfected cardiac devices (n = 20), and the anterior nares of asymptomatic subjects (n = 34). CDI isolates exhibited a distinct binding-force signature and had specific single amino acid polymorphisms in fibronectin-binding protein A corresponding to E652D, H782Q, and K786N. In silico molecular dynamics simulations demonstrate that residues D652, Q782, and N786 in fibronectin-binding protein A form extra hydrogen bonds with fibronectin, complementing the higher binding force and energy measured by atomic force microscopy for the CDI isolates. This study is significant, because it links pathogenic bacteria biofilms from the length scale of bonds acting across a nanometer-scale space to the clinical presentation of disease at the human dimension. PMID:22025727

  1. Fibronectin Binding Protein BBK32 of the Lyme Disease Spirochete Promotes Bacterial Attachment to Glycosaminoglycans

    PubMed Central

    Fischer, Joshua R.; LeBlanc, Kimberly T.; Leong, John M.

    2006-01-01

    Borrelia burgdorferi, the agent of Lyme disease, causes a multisystemic illness that can affect the skin, heart, joints, and nervous system and is capable of attachment to diverse cell types. Among the host components recognized by this spirochete are fibronectin and glycosaminoglycans (GAGs). Three surface-localized GAG-binding bacterial ligands, Bgp, DbpA, and DbpB, have been previously identified, but recent studies suggested that at least one additional GAG-binding ligand is expressed on the spirochetal surface when the spirochete is adapted to the mammalian host environment. BBK32 is a surface lipoprotein that is produced during infection and that has been shown to bind to fibronectin. In this study, we show that, when BBK32 was produced from a shuttle vector in an otherwise nonadherent high-passage B. burgdorferi strain, the protein localized on the bacterial surface and conferred attachment to fibronectin and to mammalian cell monolayers. In addition, the high-passage strain producing BBK32 bound to purified preparations of the GAGs dermatan sulfate and heparin, as well as to these GAGs on the surfaces of cultured mammalian cells. Recombinant BBK32 recognized purified heparin, indicating that the bacterial attachment to GAGs was due to direct binding by BBK32. This GAG-binding activity of BBK32 is apparently independent of fibronectin recognition, because exogenous heparin had no effect on BBK32-mediated bacterial binding to fibronectin. PMID:16368999

  2. Impact of fibronectin fragments on the transendothelial migration of HIV-infected leukocytes and the development of subendothelial foci of infectious leukocytes.

    PubMed

    Birdsall, Holly H; Porter, Wendy J; Green, David M; Rubio, Jose; Trial, JoAnn; Rossen, Roger D

    2004-08-15

    Leukocyte infiltrates that can serve as viral reservoirs, and sites for viral replication are found in many organs of HIV-1-infected patients. Patients whose blood leukocytes migrate across confluent endothelial monolayers ex vivo and transmit infectious virus to mononuclear leukocytes (MNLs) lodged beneath this endothelial barrier have a worse prognosis. We evaluated the ability of 110- to 120-kDa fibronectin fragments (FNf), which are found in the blood of >60% of HIV-1-infected patients, to stimulate transendothelial migration and drive productively infected MNLs into a potential perivascular space. FNf induced MNLs to release TNF-alpha in a dose-dependent fashion; the resulting increase in lymphocyte and monocyte transendothelial migration could be blocked with soluble TNF receptor I. Rather than penetrate deeply into the subendothelial matrix, as is seen with untreated controls, FNf-treated MNLs clustered just below the endothelial monolayer. Treatment with FNf during migration increased subsequent recovery of HIV-infected cells from the subendothelial compartment. FNf treatment also significantly increased the numbers of HLA-DR(bright), dendritic-type cells that reverse-migrated from the subendothelial depot to the apical endothelial surface 48 h after migration. Fibronectin fragments can be produced by viral and host proteases in the course of inflammatory conditions. The ability of FNf to stimulate transendothelial migration of HIV-1-infected MNLs may help to explain the dissemination of this infection into cardiac, renal, and CNS tissues. PMID:15294993

  3. Single-molecule dynamic force spectroscopy of the fibronectin-heparin interaction

    SciTech Connect

    Mitchell, Gabriel; Lamontagne, Charles-Antoine; Lebel, Rejean; Grandbois, Michel Malouin, Francois

    2007-12-21

    The integrity of cohesive tissues strongly depends on the presence of the extracellular matrix, which provides support and anchorage for cells. The fibronectin protein and the heparin-like glycosaminoglycans are key components of this dynamic structural network. In this report, atomic force spectroscopy was used to gain insight into the compliance and the resistance of the fibronectin-heparin interaction. We found that this interaction can be described by an energetic barrier width of 3.1 {+-} 0.2 A and an off-rate of 0.2 {+-} 0.1 s{sup -1}. These dissociation parameters are similar to those of other carbohydrate-protein interactions and to off-rate values reported for more complex interactions between cells and extracellular matrix components. Our results indicate that the function of the fibronectin-heparin interaction is supported by its capacity to sustain significant deformations and considerable external mechanical forces.

  4. Clinical studies on plasma fibronectin and factor XIII; with special reference to hyperlipoproteinemia.

    PubMed

    Cucuianu, M; Rus, H G; Cristea, A; Niculescu, F; Bedeleanu, D; Poruţiu, D; Roman, S

    1985-04-30

    When compared to age-matched normal weight normolipidemic control subjects, plasma factor XIII, plasma fibronectin and serum cholinesterase levels were found to be markedly decreased in patients with decompensated cirrhosis of the liver, not significantly changed in hyperlipoproteinemia type IIa (heterozygous subjects) and increased in hypertriglyceridemic subjects (type IIb and IV) as well as in hyperlipidemic nephrotic patients. A possible accelerated hepatic synthesis of certain plasma proteins including factor XIII and fibronectin in patients with the nephrotic syndrome as well as in endogenous hypertriglyceridemia is envisaged. It is also considered that mural thrombi, richer in factor XIII and fibronectin, would be more resistant to fibrinolysis and more readily attached to subendothelial structures. PMID:3922652

  5. Nucleotide sequence analysis of the DNA binding region of the chicken fibronectin gene.

    PubMed

    Karasaki, Y; Gotoh, S; Kubomura, S; Higashi, K; Hirano, H

    1988-12-01

    We have determined the nucleotide sequence of 2.0 kb EcoRI segment from the clone lambda FC32 of the genomic chicken fibronectin gene, which is called DNA binding domain. This segment overlapped another clone lambda FC36 and contained three exons which were 16, 17 and 18. They were classified as Type III repeat as originally shown in bovine plasma fibronectin. The average homologies of these three exons among the chicken, rat and human fibronectins in amino acid level are very high (87-98%) compared with that (79-88%) of the exons in the cell binding domain, indicating that this region is highly conservative during the evolution. PMID:3212295

  6. klf2a couples mechanotransduction and zebrafish valve morphogenesis through fibronectin synthesis

    PubMed Central

    Steed, Emily; Faggianelli, Nathalie; Roth, Stéphane; Ramspacher, Caroline; Concordet, Jean-Paul; Vermot, Julien

    2016-01-01

    The heartbeat and blood flow signal to endocardial cell progenitors through mechanosensitive proteins that modulate the genetic program controlling heart valve morphogenesis. To date, the mechanism by which mechanical forces coordinate tissue morphogenesis is poorly understood. Here we use high-resolution imaging to uncover the coordinated cell behaviours leading to heart valve formation. We find that heart valves originate from progenitors located in the ventricle and atrium that generate the valve leaflets through a coordinated set of endocardial tissue movements. Gene profiling analyses and live imaging reveal that this reorganization is dependent on extracellular matrix proteins, in particular on the expression of fibronectin1b. We show that blood flow and klf2a, a major endocardial flow-responsive gene, control these cell behaviours and fibronectin1b synthesis. Our results uncover a unique multicellular layering process leading to leaflet formation and demonstrate that endocardial mechanotransduction and valve morphogenesis are coupled via cellular rearrangements mediated by fibronectin synthesis. PMID:27221222

  7. Large format focal plane array integration with precision alignment, metrology and accuracy capabilities

    NASA Astrophysics Data System (ADS)

    Neumann, Jay; Parlato, Russell; Tracy, Gregory; Randolph, Max

    2015-09-01

    Focal plane alignment for large format arrays and faster optical systems require enhanced precision methodology and stability over temperature. The increase in focal plane array size continues to drive the alignment capability. Depending on the optical system, the focal plane flatness of less than 25μm (.001") is required over transition temperatures from ambient to cooled operating temperatures. The focal plane flatness requirement must also be maintained in airborne or launch vibration environments. This paper addresses the challenge of the detector integration into the focal plane module and housing assemblies, the methodology to reduce error terms during integration and the evaluation of thermal effects. The driving factors influencing the alignment accuracy include: datum transfers, material effects over temperature, alignment stability over test, adjustment precision and traceability to NIST standard. The FPA module design and alignment methodology reduces the error terms by minimizing the measurement transfers to the housing. In the design, the proper material selection requires matched coefficient of expansion materials minimizes both the physical shift over temperature as well as lowering the stress induced into the detector. When required, the co-registration of focal planes and filters can achieve submicron relative positioning by applying precision equipment, interferometry and piezoelectric positioning stages. All measurements and characterizations maintain traceability to NIST standards. The metrology characterizes the equipment's accuracy, repeatability and precision of the measurements.

  8. Potential regulation of cartilage metabolism in osteoarthritis by fibronectin fragments.

    PubMed

    Homandberg, G A

    1999-10-15

    There are few candidates for biochemical pathways that either initiate or amplify catabolic processes involved in osteoarthritis (OA). Perhaps, one of the most likely sources for such pathways may be within the extracellular matrix itself. This review focuses on an example of how specific degradation products of the extracellular matrix of cartilage, produced during proteolytic damage, have the potential to enhance OA-like processes. In this example, these products can induce or activate other factors, such as catabolic cytokines, that amplify the damage. The damage, in turn, enhances levels of the degradation products themselves, as in a positive feedback loop. Since these products are derived from the cartilage matrix, they could be considered barometers of the health of the cartilage that signal to the chondrocyte, through outside to inside signaling, the health or status of the surrounding matrix. The best example and most characterized system is that of fragments of the matrix protein, fibronectin (Fn), although as discussed later, other recently discovered fragment systems may also have the potential to regulate cartilage metabolism. In the case of Fn fragments (Fn-fs), the Fn-fs enhance levels of catabolic cytokines as in OA and, thus, are potentially earlier damage mediators than catabolic cytokines. The Fn-fs up-regulate matrix metalloproteinase (MMP) expression, significantly enhance degradation and loss of proteoglycan (PG) from cartilage and temporarily suppress PG synthesis, all events observed in OA. However, this Fn-f system may be involved in normal cartilage homeostasis as well. For example, low concentrations of Fn-fs enhance anabolic activities and could play a role in normal homeostasis. This system may also be involved in not only amplifying damage but also coupling damage to repair. For example, high concentrations of Fn-fs that might arise in OA temporarily offset the anabolic response of lower Fn-f concentrations and cause short

  9. Fibronectin Terminated Multilayer Films: Protein Adsorption and Cell Attachment Studies

    PubMed Central

    Wittmer, Corinne R.; Phelps, Jennifer A.; Saltzman, W. Mark; Van Tassel, Paul R.

    2006-01-01

    Electrostatically driven layer-by-layer (LbL) assembly is a simple and robust method for producing structurally tailored thin film biomaterials, of thickness ca. 10 nanometers, containing biofunctional ligands. We investigate the LbL formation of multilayer films composed of polymers of biological origin (poly(L-lysine) (PLL) and dextran sulfate (DS)), the adsorption of fibronectin (Fn) - a matrix protein known to promote cell adhesion - onto these films, and the subsequent spreading behavior of human umbilical vein endothelial cells (HUVEC). We employ optical waveguide lightmode spectroscopy (OWLS) and quartz crystal microgravimetry with dissipation (QCMD) to characterize multilayer assembly in situ, and find adsorbed Fn mass on PLL terminated films to exceed that on DS terminated films by 40%, correlating with the positive charge and lower degree of hydration of PLL terminated films. The extent and initial rate of Fn adsorption to both PLL and DS terminated films exceed those onto the bare substrate, indicating the important role of electrostatic complexation between negatively charged protein and positively charged PLL at or near the film surface. We use phase contrast optical microscopy to investigate the time dependent morphological changes of HUVEC as a function of layer number, charge of terminal layer, and the presence of Fn. We observe HUVEC to attach, spread, and lose circularity on all surfaces. (Positively charged) PLL terminated films exhibit a greater extent of cell spreading than do (negatively charged) DS terminated films, and spreading is enhanced while circularity loss is suppressed by the presence of adsorbed Fn. The number of layers plays a significant role only for DS terminated films with Fn, where spreading on a bilayer greatly exceeds that on a multilayer, and PLL terminated films without Fn, where initial spreading is significantly higher on a monolayer. We observe initial cell spreading to be followed by retraction (i.e. decreased cell

  10. Cellular fibronectin response to supervised moderate aerobic training in patients with type 2 diabetes

    PubMed Central

    Alghadir, Ahmad H.; Gabr, Sami A.; Al-Eisa, Einas

    2016-01-01

    [Purpose] Physical activity is one of the most pivotal targets for the prevention and management of vascular complications, especially endothelial dysfunctions. Cellular fibronectin is an endothelium-derived protein involved in subendothelial matrix assembly. Its plasma levels reflect matrix alterations and vessel wall destruction in patients with type II diabetes. This study investigated the influence of 12 weeks of supervised aerobic training on cellular fibronectin and its relationship with insulin resistance and body weight in type II diabetic subjects. [Subjects and Methods] This study included 50 men with type II diabetes who had a mean age of 48.8 ± 14.6 years and were randomly divided into two groups: an aerobic exercise group (12 weeks, three 50 minutes sessions per week) and control group. To examine changes in cellular fibronectin, glycosylated hemoglobin, insulin resistance, fasting insulin, fasting blood sugar, and lipid profile, 5 ml of blood was taken from the brachial vein of patients before and 48 hours after completion of the exercise period and after 12 hours of fasting at rest. Data analysis was performed using the SPSS-16 software with the independent and paired t-tests. [Results] A significant decrease was observed in body mass index and body fat percentage in the experimental group. Compared with the control group, the aerobic exercise group showed a significant decrease in cellular fibronectin, glycosylated hemoglobin, insulin resistance, fasting insulin, fasting blood sugar, and lipid profile after 12 weeks of aerobic exercise. The change in cellular fibronectin showed positive significant correlation with body mass index, diabetic biomarkers, and physical activity level. [Conclusion] The results showed that supervised aerobic exercise as a stimulus can change the levels of cellular fibronectin as matrix metalloproteinase protein a long with improvement of insulin sensitivity and glycosylated hemoglobin in order to prevent

  11. Cellular fibronectin response to supervised moderate aerobic training in patients with type 2 diabetes.

    PubMed

    Alghadir, Ahmad H; Gabr, Sami A; Al-Eisa, Einas

    2016-04-01

    [Purpose] Physical activity is one of the most pivotal targets for the prevention and management of vascular complications, especially endothelial dysfunctions. Cellular fibronectin is an endothelium-derived protein involved in subendothelial matrix assembly. Its plasma levels reflect matrix alterations and vessel wall destruction in patients with type II diabetes. This study investigated the influence of 12 weeks of supervised aerobic training on cellular fibronectin and its relationship with insulin resistance and body weight in type II diabetic subjects. [Subjects and Methods] This study included 50 men with type II diabetes who had a mean age of 48.8 ± 14.6 years and were randomly divided into two groups: an aerobic exercise group (12 weeks, three 50 minutes sessions per week) and control group. To examine changes in cellular fibronectin, glycosylated hemoglobin, insulin resistance, fasting insulin, fasting blood sugar, and lipid profile, 5 ml of blood was taken from the brachial vein of patients before and 48 hours after completion of the exercise period and after 12 hours of fasting at rest. Data analysis was performed using the SPSS-16 software with the independent and paired t-tests. [Results] A significant decrease was observed in body mass index and body fat percentage in the experimental group. Compared with the control group, the aerobic exercise group showed a significant decrease in cellular fibronectin, glycosylated hemoglobin, insulin resistance, fasting insulin, fasting blood sugar, and lipid profile after 12 weeks of aerobic exercise. The change in cellular fibronectin showed positive significant correlation with body mass index, diabetic biomarkers, and physical activity level. [Conclusion] The results showed that supervised aerobic exercise as a stimulus can change the levels of cellular fibronectin as matrix metalloproteinase protein a long with improvement of insulin sensitivity and glycosylated hemoglobin in order to prevent

  12. Heterologously Expressed Staphylococcus aureus Fibronectin-Binding Proteins Are Sufficient for Invasion of Host Cells

    PubMed Central

    Sinha, Bhanu; Francois, Patrice; Que, Yok-Ai; Hussain, Muzaffar; Heilmann, Christine; Moreillon, Philippe; Lew, Daniel; Krause, Karl-Heinz; Peters, Georg; Herrmann, Mathias

    2000-01-01

    Staphylococcus aureus invasion of mammalian cells, including epithelial, endothelial, and fibroblastic cells, critically depends on fibronectin bridging between S. aureus fibronectin-binding proteins (FnBPs) and the host fibronectin receptor integrin α5β1 (B. Sinha et al., Cell. Microbiol. 1:101–117, 1999). However, it is unknown whether this mechanism is sufficient for S. aureus invasion. To address this question, various S. aureus adhesins (FnBPA, FnBPB, and clumping factor [ClfA]) were expressed in Staphylococcus carnosus and Lactococcus lactis subsp. cremoris. Both noninvasive gram-positive microorganisms are genetically distinct from S. aureus, lack any known S. aureus surface protein, and do not bind fibronectin. Transformants of S. carnosus and L. lactis harboring plasmids coding for various S. aureus surface proteins (FnBPA, FnBPB, and ClfA) functionally expressed adhesins (as determined by bacterial clumping in plasma, specific latex agglutination, Western ligand blotting, and binding to immobilized and soluble fibronectin). FnBPA or FnBPB but not of ClfA conferred invasiveness to S. carnosus and L. lactis. Invasion of 293 cells by transformants was comparable to that of strongly invasive S. aureus strain Cowan 1. Binding of soluble and immobilized fibronectin paralleled invasiveness, demonstrating that the amount of accessible surface FnBPs is rate limiting. Thus, S. aureus FnBPs confer invasiveness to noninvasive, apathogenic gram-positive cocci. Furthermore, FnBP-coated polystyrene beads were internalized by 293 cells, demonstrating that FnBPs are sufficient for invasion of host cells without the need for (S. aureus-specific) coreceptors. PMID:11083807

  13. Lectins as probes for assessing the accessibility of N-linked glycans in relation to the conformational changes of fibronectin.

    PubMed

    Agniel, Rémy; Vendrely, Charlotte; Poulouin, Laurent; Bascetin, Rümeyza; Benachour, Hamanou; Gallet, Olivier; Leroy-Dudal, Johanne

    2015-12-01

    Fibronectin, a ≈ 450-kDa protein with 4-9% (w/w) glycosylation, is a key component of extracellular matrices and has a high conformational lability regarding its functions. However, the accessibility and the role of glycosylated moieties associated with the conformational changes of fibronectin are poorly understood. Using lectins as probes, we developed an approach comprising dynamic light scattering, turbidimetry measurements, and isothermal titration calorimetry to assess the accessibility of glycosylated moieties of fibronectin undergoing thermal-induced conformational changes. Among a set of 14 lectins, fibronectin mainly reacted with mannose-binding lectins, specifically concanavalin A. When temperature was raised from 25 to 50 °C, fibronectin underwent progressive unfolding, but the conformation of concanavalin A was unaffected. Dynamic light scattering, turbidimetry measurements, and isothermal titration calorimetry showed increased concanavalin A binding to fibronectin during progressive thermal-induced unfolding of the protein core. Such data suggest that mannosylated residues are progressively exposed as fibronectin unfolds. Because oligosaccharide moieties can be differently exposed to cells, and the cell's responses could be modified physiologically or pathologically, modulation of fibronectin sugar chains could be relevant to its biological functions. Thus, lectins might be useful tools to probe the glycosylation accessibility accompanying changes in protein core folding, for which a better understanding would be of value for biological and biomedical research. PMID:26148749

  14. REGULATION OF p38 MAP KINASE BY ANASTELLIN IS INDEPENDENT OF ANASTELLIN’S EFFECT ON MATRIX FIBRONECTIN

    PubMed Central

    You, Ran; Klein, R. Matthew; Zheng, Mingzhe; McKeown-Longo, Paula J.

    2009-01-01

    Anastellin is an angiogenesis inhibitor derived from the first type III repeat of fibronectin (FN). Anastellin binds to fibronectin and promotes the polymerization of soluble fibronectin into a highly polymerized form termed superfibronectin. In addition, anastellin also causes remodeling of pre-existing fibronectin matrix and modulates cell signaling pathways in both endothelial cells and fibroblasts. In the present study, we address the relationship of anastellin’s effects on fibronectin matrix to its effects on p38 MAP kinase (MAPK) activation. Using a mutant form of anastellin which binds to fibronectin matrix, but does not stimulate formation of superfibronectin, we demonstrate that the activation of p38 MAPK by anastellin is not dependent on the formation of superfibronectin. The mutant form of anastellin does stimulate matrix remodeling, but experiments using FN−/− cells show that the effect of anastellin on p38-MAPK activation is completely independent of fibronectin. Anastellin was able to activate p38 MAPK on cells in suspension as well as on cells null for β1 integrins, suggesting that anastellin activity did not require ligation of integrins. These data suggest that the activation of p38 MAPK by anastellin is independent of anastellin’s effects on fibronectin matrix organization. PMID:19379667

  15. Mislocalized Scaffolding by the Na-H Exchanger NHE1 Dominantly Inhibits Fibronectin Production and TGF-β Activation

    PubMed Central

    Karydis, Anastasios; Jimenez-Vidal, Maite; Denker, Sheryl P.

    2009-01-01

    Secretion and assembly of the extracellular matrix protein fibronectin regulates a number of normal cell and tissue functions and is dysregulated in disease states such as fibrosis, diabetes, and cancer. We found that mislocalized scaffolding by the plasma membrane Na-H exchanger NHE1 suppresses fibronectin expression, secretion, and assembly. In fibroblasts, wild-type NHE1 localizes to the distal margin of membrane protrusions or lamellipodia but a mutant NHE1-KRA2 lacking binding sites for PI(4,5)P2 and the ERM proteins ezrin, radixin, and moesin is mislocalized and found uniformly along the plasma membrane. Although NHE1 regulates intracellular pH homeostasis, fibronectin production is not regulated by changes in intracellular pH, nor is it attenuated in NHE1-deficient cells, indicating fibronectin expression is independent of NHE1 activity. However, fibronectin production is nearly absent in cells expressing NHE1-KRA2 because scaffolding by NHE1 is mislocalized. Additionally, secretion of active but not latent TGF-β is reduced and exogenous TGF-β restores fibronectin secretion and assembly. Our data indicate that scaffolding by NHE1-KRA2 dominantly suppresses fibronectin synthesis and TGF-β activation, and they suggest that NHE1-KRA2 can be used for obtaining a mechanistic understanding of how fibronectin production is regulated and speculatively for therapeutic control of dysregulated production in pathological conditions. PMID:19225158

  16. SNAP Satellite Focal Plane Development

    SciTech Connect

    Bebek, C.; Akerlof, C.; Aldering, G.; Amanullah, R.; Astier, P.; Baltay, C.; Barrelet, E.; Basa, S.; Bercovitz, J.; Bergstrom, L.; Berstein, G.P.; Bester, M.; Bohlin, R.; Bonissent, A.; Bower, C.; Campbell, M.; Carithers, W.; Commins, E.; Day, C.; Deustua, S.; DiGennaro, R.; Ealet, A.; Ellis, R.; Emmett, W.; Eriksson, M.; Fouchez,D.; Fruchter, A.; Genat, J-F.; Goldhaber, G.; Goobar, A.; Groom, D.; Heetderks, H.; Holland, S.; Huterer, D.; Johnson, W.; Kadel, R.; Karcher,A.; Kim, A.; Kolbe, W.; Lafever, R.; Lamoureaux, J.; Lampton, M.; Lefevre, O.; Levi, M.; Levin, D.; Linder, E.; Loken, S.; Malina, R.; Mazure, A.; McKay, T.; McKee, S.; Miquel, R.; Morgan, N.; Mortsell, E.; Mostek, N.; Mufson, S.; Musser, J.; Roe, N.; Nugent, P.; Oluseyi, H.; Pain, R.; Palaio, N.; Pankow, D.; Perlmutter, S.; Prieto, E.; Rabinowitz,D.; Refregier, A.; Rhodes, J.; Schubnell, M.; Sholl, M.; Smadja, G.; Smith, R.; Smoot, G.; Snyder, J.; Spadafora, A.; Szymkowiak, A.; Tarle,G.; Taylor, K.; Tilquin, A.; Tomasch, A.; Vincent, D.; von der Lippe, H.; Walder, J-P.; Wang, G.

    2003-07-07

    The proposed SuperNova/Acceleration Probe (SNAP) mission will have a two-meter class telescope delivering diffraction-limited images to an instrumented 0.7 square degree field in the visible and near-infrared wavelength regime. The requirements for the instrument suite and the present configuration of the focal plane concept are presented. A two year R&D phase, largely supported by the Department of Energy, is just beginning. We describe the development activities that are taking place to advance our preparedness for mission proposal in the areas of detectors and electronics.

  17. Design of an Osteoinductive Extracellular Fibronectin Matrix Protein for Bone Tissue Engineering

    PubMed Central

    Lee, Sujin; Lee, Dong-Sung; Choi, Ilsan; Pham, Le B. Hang; Jang, Jun-Hyeog

    2015-01-01

    Integrin-mediated cell-matrix interactions play an important role in osteogenesis. Here, we constructed a novel osteoinductive fibronectin matrix protein (oFN) for bone tissue engineering, designed to combine the integrin-binding modules from fibronectin (iFN) and a strong osteoinductive growth factor, bone morphogenetic protein-2. Compared with iFN, the purified oFN matrix protein caused a significant increase in cell adhesion and osteogenic differentiation of pre-osteoblast MC3T3-E1 cells (p < 0.05). PMID:25853265

  18. Frcp1 and Frcp2, two novel fibronectin type III repeat containing genes.

    PubMed

    Teufel, Andreas; Malik, Nasir; Mukhopadhyay, Mahua; Westphal, Heiner

    2002-09-01

    The fibronectin type III (FNIII) repeat is one of three structural motifs originally identified in the fibronectin protein and has been well characterized in recent years. The consensus sequence has since been found in many different proteins including receptors and cell adhesion molecules. We report the cloning and expression analysis of Frcp1 and Frcp2, two members of a new FNIII repeat containing gene family. During embryonic development both genes are primarily expressed in the brain. In adult tissues, Frcp1 is strongly expressed in the liver and Frcp2 in the heart. PMID:12384288

  19. Expression of DFak56, a Drosophila homolog of vertebrate focal adhesion kinase, supports a role in cell migration in vivo

    PubMed Central

    Fox, George L.; Rebay, Ilaria; Hynes, Richard O.

    1999-01-01

    Focal adhesion kinase (FAK) is a highly conserved, cytoplasmic tyrosine kinase that has been implicated in promoting cell migration and transmission of antiapoptotic signals in vertebrate cells. In cultured cells, integrin engagement with the extracellular matrix promotes the recruitment of FAK to focal contacts and increases in its phosphotyrosine content and kinase activity, suggesting FAK is an intracellular mediator of integrin signaling. We have identified a Drosophila FAK homolog, DFak56, that is 33% identical to vertebrate FAK, with the highest degree of homology in domains critical for FAK function, including the kinase and focal adhesion targeting domains, and several protein–protein interaction motifs. Furthermore, when expressed in NIH 3T3 cells, DFak56 both localizes to focal contacts and displays the characteristic elevation of phosphotyrosine content in response to plating the cells on fibronectin. During embryogenesis, DFak56 is broadly expressed, and it becomes elevated in the gut and central nervous system at later stages. Consistent with a role in cell migration, we also observe that DFak56 is abundant in the border cells of developing egg chambers before the onset of, and during, their migration. PMID:10611323

  20. Supporting prostate cancer focal therapy: a multidisciplinary International Consensus of Experts ("ICE").

    PubMed

    Reis, Leonardo O; Billis, Athanase; Zequi, Stenio C; Tobias-Machado, Marcos; Viana, Publio; Cerqueira, Michael; Ward, John F

    2014-06-01

    Prostate cancer is a common malignancy among men, and the current screening, imaging and sampling approaches aim to detect early-stage, organ-confined disease. In such scenario, focal prostate cancer therapy currently relies on the index lesion concept as the dominant lesion that drives the disease natural history. Focal therapy demands the essential imaging and sampling techniques to strategically locate and qualify the disease, but, despite advances in technology, prostate imaging and biopsy have several limitations that need to be overcome if focal therapy is to be developed further. The I Prostate Cancer Focal Treatment International Symposium was convened to foster discussion on this topic that sits at the crossroads of multiple disciplines (Urology, Pathology, Radiology, Radiation Oncology and Medical Oncology) all of which were represented for this comprehensive multidisciplinary review of the current literature. PMID:24597940

  1. Focal plane detectors for the WISE 12- and 23-μm bands

    NASA Astrophysics Data System (ADS)

    Hogue, H. H.; Mattson, R. B.; Stapelbroek, M. G.; Masterjohn, S. A.; Larsen, M. F.; Elwell, J. D.

    2007-09-01

    DRS Sensors & Targeting Systems, under contract to the Space Dynamics Laboratory of Utah State University, is providing the focal plane detector system for NASA's Wide-field Infrared Survey Explorer (WISE). The focal plane detector system consists of two mercury cadmium telluride (MCT) focal plane module assemblies (FPMAs), two arsenic doped silicon (Si:As) Blocked Impurity Band (BIB) FPMAs, electronics to drive the FPMAs and report digital data from them, and the cryogenic and ambient temperature cabling that connect the FPMAs and electronics. The MCT and Si:As BIB focal plane arrays (FPAs) utilized in the WISE FPMAs are both megapixel class indium-bump hybridized devices fabricated by Teledyne Imaging Systems and DRS Sensors & Targeting Systems, respectively. This paper reports performance of the WISE Si:As BIB FPAs that are used for the WISE 12- and 23-μm wavelength bands.

  2. Hemodynamics of focal choroidal excavations.

    PubMed

    Soma, Ryoko; Moriyama, Muka; Ohno-Matsui, Kyoko

    2015-04-01

    The purpose of this study was to investigate the hemodynamics of focal choroidal excavations (FCEs). Four eyes of four patients with a FCE were studied. Indocyanine green angiography (ICGA), laser speckle flowgraphy (LSFG), optical coherence tomography (OCT), and multi-focal electroretinography (mfERG) were performed to investigate the choroidal hemodynamics and the morphological and functional changes. The mean depth of the FCE determined by OCT was 222.5 ± 49.5 μm with a range of 164-272 μm. In one case, subretinal fluid was observed in the excavation, and in three cases, subretinal fluid was not observed. ICGA showed hypofluorescence, and laser flowgraphy (LSFG) showed decreased choroidal blood flow at the excavation in all cases. Three cases were symptomatic, and the amplitudes of the mfERGs were reduced. FCEs cause a decrease of choroidal blood flow. In three of four cases, the mfERGs were depressed over the FCEs leading to symptoms. PMID:25626897

  3. Rewritable photochromic focal plane masks

    NASA Astrophysics Data System (ADS)

    Molinari, Emilio; Bertarelli, Chiara; Bianco, Andrea; Bortoletto, Fabio; Conconi, Paolo; Crimi, Giuseppe; Gallazzi, Maria C.; Giro, Enrico; Lucotti, Andrea; Pernechele, Claudio; Zerbi, Filippo M.; Zerbi, Giuseppe

    2003-02-01

    The application of organic photochromic materials in astronomy is opening new possibilities which we are investigating in order to design innovative devices for future instrumentation. The photochromic property of transparent/opaque transition (although in a limited wavelength range) and the changes in intrinsic refractive index have led our studies to application in astronomic spectrographs, both as focal plane mask (for MOS application) and as dispersive elements (volume phase holographic gratings, VPHG), respectively. In both cases the possibility to write and erase devices with suitable irradiation has revealed a new perspective for non-disposable and fully customizable items for spectroscopy. Pursuing this goal we have synthesized a series of novel photochromic materials belonging to the diarylethenes. They fulfill the requirements of thermal stability and fatigue resistance necessary to build functional devices. Prototypes of high contrast focal plane mask working in the H-alpha spectral region have been manufactured and characterized both in laboratory and with the AFOSC camera at Asiago telescope (1.8 m). A custom writing robot (ARATRO) which, taking imaging frames and with the aid of interactive mask design software and ad hoc control electronics, is able to write MOS masks, has been constructed. The design of the MOS masks allow the fitting in the AFOSC slit wheel. The overall set-up is ready for the sky tests.

  4. Implementation of focal zooming on the Nike KrF laser

    SciTech Connect

    Kehne, D. M.; Karasik, M.; Weaver, J. L.; Chan, Y.; Obenschain, S. P.; Aglitsky, Y.; Smyth, Z.; Lehmberg, R. H.; Terrell, S.

    2013-01-15

    In direct drive inertial confinement laser fusion, a pellet containing D-T fuel is imploded by ablation arising from absorption of laser energy at its outer surface. For optimal coupling, the focal spot of the laser would continuously decrease to match the reduction in the pellet's diameter, thereby minimizing wasted energy. A krypton-fluoride laser ({lambda}= 248 nm) that incorporates beam smoothing by induced spatial incoherence has the ability to produce a high quality focal profile whose diameter varies with time, a property known as focal zooming. A two-stage focal zoom has been demonstrated on the Nike laser at the Naval Research Laboratory. In the experiment, a 4.4 ns laser pulse was created in which the on-target focal spot diameter was 1.3 mm (full width at half maximum) for the first 2.4 ns and 0.28 mm for the final 2 ns. These two diameters appear in time-integrated focal plane equivalent images taken at several locations in the amplification chain. Eight of the zoomed output beams were overlapped on a 60 {mu}m thick planar polystyrene target. Time resolved images of self-emission from the rear of the target show the separate shocks launched by the two corresponding laser focal diameters.

  5. Implementation of focal zooming on the Nike KrF laser.

    PubMed

    Kehne, D M; Karasik, M; Aglitsky, Y; Smyth, Z; Terrell, S; Weaver, J L; Chan, Y; Lehmberg, R H; Obenschain, S P

    2013-01-01

    In direct drive inertial confinement laser fusion, a pellet containing D-T fuel is imploded by ablation arising from absorption of laser energy at its outer surface. For optimal coupling, the focal spot of the laser would continuously decrease to match the reduction in the pellet's diameter, thereby minimizing wasted energy. A krypton-fluoride laser (λ = 248 nm) that incorporates beam smoothing by induced spatial incoherence has the ability to produce a high quality focal profile whose diameter varies with time, a property known as focal zooming. A two-stage focal zoom has been demonstrated on the Nike laser at the Naval Research Laboratory. In the experiment, a 4.4 ns laser pulse was created in which the on-target focal spot diameter was 1.3 mm (full width at half maximum) for the first 2.4 ns and 0.28 mm for the final 2 ns. These two diameters appear in time-integrated focal plane equivalent images taken at several locations in the amplification chain. Eight of the zoomed output beams were overlapped on a 60 μm thick planar polystyrene target. Time resolved images of self-emission from the rear of the target show the separate shocks launched by the two corresponding laser focal diameters. PMID:23387652

  6. Implementation of focal zooming on the Nike KrF laser

    NASA Astrophysics Data System (ADS)

    Kehne, D. M.; Karasik, M.; Aglitsky, Y.; Smyth, Z.; Terrell, S.; Weaver, J. L.; Chan, Y.; Lehmberg, R. H.; Obenschain, S. P.

    2013-01-01

    In direct drive inertial confinement laser fusion, a pellet containing D-T fuel is imploded by ablation arising from absorption of laser energy at its outer surface. For optimal coupling, the focal spot of the laser would continuously decrease to match the reduction in the pellet's diameter, thereby minimizing wasted energy. A krypton-fluoride laser (λ = 248 nm) that incorporates beam smoothing by induced spatial incoherence has the ability to produce a high quality focal profile whose diameter varies with time, a property known as focal zooming. A two-stage focal zoom has been demonstrated on the Nike laser at the Naval Research Laboratory. In the experiment, a 4.4 ns laser pulse was created in which the on-target focal spot diameter was 1.3 mm (full width at half maximum) for the first 2.4 ns and 0.28 mm for the final 2 ns. These two diameters appear in time-integrated focal plane equivalent images taken at several locations in the amplification chain. Eight of the zoomed output beams were overlapped on a 60 μm thick planar polystyrene target. Time resolved images of self-emission from the rear of the target show the separate shocks launched by the two corresponding laser focal diameters.

  7. EDA-containing cellular fibronectin induces fibroblast differentiation through binding to alpha4beta7 integrin receptor and MAPK/Erk 1/2-dependent signaling.

    PubMed

    Kohan, Martin; Muro, Andres F; White, Eric S; Berkman, Neville

    2010-11-01

    Fibroblast differentiation is an essential step during wound healing and fibrosis. Fibronectin (FN) is a major component of the extracellular matrix and occurs in two main forms: plasma and cellular FN. The latter includes the alternatively spliced domain A (EDA). Although EDA-containing cellular fibronectin (EDA-FN) is associated with fibroblast differentiation, how EDA-FN promotes differentiation is incompletely understood. In this study, we investigate the mechanism by which EDA-FN contributes to fibroblast differentiation with emphasis on the characterization of the EDA-FN receptor. We show that EDA-FN increases α-SMA expression (immunofluorescence), collagen deposition, cell contractility, and focal adhesion kinase (FAK) activation (immunoblotting); whereas plasma FN, a form lacking EDA, shows no effect. Primary lung fibroblasts constitutively express α(4)β(7) integrin receptor (FACS and RT-PCR). Blocking of α(4)β(7) reduces fibroblast adhesion to EDA-FN and inhibits α-SMA expression, collagen deposition, and FAK activation induced by EDA-FN. Using recombinant EDA-containing peptides, we demonstrate that the EDA segment is sufficient to induce fibroblast differentiation via binding to α(4)β(7). EDA-FN induces MAPK-Erk1/2 activation and inhibition of MEK1/2 attenuates EDA-FN-induced α-SMA expression. Our findings demonstrate that EDA-FN induces fibroblast differentiation by a mechanism that involves binding of EDA to α(4)β(7) integrin followed by activation of FAK and MAPK-associated signaling pathways. PMID:20643910

  8. Mechanotransduction at focal adhesions: integrating cytoskeletal mechanics in migrating cells

    PubMed Central

    Kuo, Jean-Cheng

    2013-01-01

    Focal adhesions (FAs) are complex plasma membrane-associated macromolecular assemblies that serve to physically connect the actin cytoskeleton to integrins that engage with the surrounding extracellular matrix (ECM). FAs undergo maturation wherein they grow and change composition differentially to provide traction and to transduce the signals that drive cell migration, which is crucial to various biological processes, including development, wound healing and cancer metastasis. FA-related signalling networks dynamically modulate the strength of the linkage between integrin and actin and control the organization of the actin cytoskeleton. In this review, we have summarized a number of recent investigations exploring how FA composition is affected by the mechanical forces that transduce signalling networks to modulate cellular function and drive cell migration. Understanding the fundamental mechanisms of how force governs adhesion signalling provides insights that will allow the manipulation of cell migration and help to control migration-related human diseases. PMID:23551528

  9. Focal axis resolver for offset reflector antennas

    NASA Technical Reports Server (NTRS)

    Schmidt, R. F. (Inventor)

    1983-01-01

    Method and apparatus for determining the focal axis of an asymmetrical antenna such as an offset paraboloid reflector whose physical rim is not coincident with the boundary of the electrical aperture but whose focal point is known is provided. A transmitting feed horn array consisting of at least two feed horn elements is positioned asymmetrically on either side of an estimated focal axis which is generally inclined with respect to the boresight axis of the antenna. The feed horn array is aligned with the estimated focal axis so that the phase centers (CP sub 1, CP sub 2) of the two feed horn elements are located on a common line running through the focal point (F) orthogonally with respect to the estimated focal axis.

  10. Signal processing of microbolometer infrared focal-plane arrays

    NASA Astrophysics Data System (ADS)

    Zhang, Junju; Qian, Yunsheng; Chang, Benkang; Xing, Suxia; Sun, Lianjun

    2005-01-01

    A 320×240-uncooled-microbolometer-based signal processing circuit for infrared focal-plane arrays is presented, and the software designs of this circuit system are also discussed in details. This signal processing circuit comprises such devices as FPGA, D/A, A/D, SRAM, Flash, DSP, etc., among which, FPGA is the crucial part, which realizing the generation of drive signals for infrared focal-plane, nonuniformity correction, image enhancement and video composition. The device of DSP, mainly offering auxiliary functions, carries out communication with PC and loads data when power-up. The phase locked loops (PLL) is used to generate high-quality clocks with low phase dithering and multiple clocks are to used satisfy the demands of focal-plane arrays, A/D, D/A and FPGA. The alternate structure is used to read or write SRAM in order to avoid the contradiction between different modules. FIFO embedded in FPGA not only makes full use of the resources of FPGA but acts as the channel between different modules which have different-speed clocks. What's more, working conditions, working process, physical design and management of the circuit are discussed. In software designing, all the function modules realized by FPGA and DSP devices, which are mentioned in the previous part, are discussed explicitly. Particularly to the nonuniformity correction module, the pipeline structure is designed to improve the working frequency and the ability to realize more complex algorithm.

  11. Multiplicative and subtractive focal volume engineering in coherent Raman microscopy

    PubMed Central

    Raghunathan, Varun; Potma, Eric Olaf

    2012-01-01

    Rigorous calculations are performed to study the effective reduction of the nonlinear excitation volumes when using phase-only masks to condition the pump and Stokes driving fields. Focal volume reduction was achieved using both a multiplicative operation of the excitation fields as well as a subtractive operation. Using a tunable optical bottle beam for the Stokes field, an effective reduction of the width of the excitation volume by a factor of 1.5 can be achieved in the focal plane. Further reduction of the focal volume introduces a rapid growth of sidelobes, which renders such volumes unsuitable for imaging applications. In addition, phase sensitive detection was found to provide information from selective sub-divisions of the engineered coherent anti-Stokes Raman scattering excitation volume. In the case of isolated nanoparticles, an apparent resolution improvement by a factor of 3 is demonstrated, and it is shown that the size of sub-diffraction-limited particles can be accurately determined using phase sensitive detection. PMID:21045900

  12. Functional Analysis of an S-Layer-Associated Fibronectin-Binding Protein in Lactobacillus acidophilus NCFM

    PubMed Central

    Hymes, Jeffrey P.; Johnson, Brant R.; Barrangou, Rodolphe

    2016-01-01

    Bacterial surface layers (S-layers) are crystalline arrays of self-assembling proteinaceous subunits called S-layer proteins (Slps) that comprise the outermost layer of the cell envelope. Many additional proteins that are associated with or embedded within the S-layer have been identified in Lactobacillus acidophilus NCFM, an S-layer-forming bacterium that is widely used in fermented dairy products and probiotic supplements. One putative S-layer-associated protein (SLAP), LBA0191, was predicted to mediate adhesion to fibronectin based on the in silico detection of a fibronectin-binding domain. Fibronectin is a major component of the extracellular matrix (ECM) of intestinal epithelial cells. Adhesion to intestinal epithelial cells is considered an important trait for probiotic microorganisms during transit and potential association with the intestinal mucosa. To investigate the functional role of LBA0191 (designated FbpB) in L. acidophilus NCFM, an fbpB-deficient strain was constructed. The L. acidophilus mutant with a deletion of fbpB lost the ability to adhere to mucin and fibronectin in vitro. Homologues of fbpB were identified in five additional putative S-layer-forming species, but no homologues were detected in species outside the L. acidophilus homology group. PMID:26921419

  13. Tenascin variants: differential binding to fibronectin and distinct distribution in cell cultures and tissues.

    PubMed Central

    Chiquet-Ehrismann, R; Matsuoka, Y; Hofer, U; Spring, J; Bernasconi, C; Chiquet, M

    1991-01-01

    In the chicken, three tenascin variants have been characterized that are generated by alternative splicing of 3 of its 11 fibronectin type III repeats. Using monoclonal antibodies that react with common regions versus extra repeats of tenascin, we could distinguish and separate tenascin variants and investigate their interaction with fibronectin using multiple experimental procedures. Interestingly, in all assays used the smallest tenascin variant bound more strongly to fibronectin than the larger ones. These biochemical data were paralleled by the observation that in chick embryo fibroblast cultures only the smallest form of tenascin could be detected in the fibronectin-rich extracellular matrix network laid down by the cells. Furthermore, each tissue present in adult chicken gizzard contained a distinct set of tenascin variants. Those tissues particularly rich in extracellular matrix, such as the tendon, contained the smallest tenascin only. Intermediate-sized tenascin was present in smooth muscle, whereas the largest form was exclusively detectable underneath the epithelial lining of the villi. Thus it appears that cell type-specific forms of tenascin exist that are appropriate for the functional requirements of the respective extracellular matrices. Images PMID:1725601

  14. Modification of shear modulus and creep compliance of fibrin clots by fibronectin.

    PubMed

    Kamykowski, G W; Mosher, D F; Lorand, L; Ferry, J D

    1981-02-01

    Shear moduli and creep compliances have been measured for four types of clots of human fibrin (about 7 mg/ml) clotted with and without human plasma fibronectin (usually 1.2 mg/ml). Fine clots (with little lateral aggregation of the fibrin protofibrils) were found at pH 8.5, ionic strength 0.45; coarse clots (with substantial lateral aggregation) were formed at pH 7.5, ionic strength 0.15; in both cases with and without ligation by fibrinoligase. In fine clots, the addition of fibronectin without ligation scarcely affected the shear modulus; with ligation, the modulus was decreased by a factor of 0.48. In coarse clots, the shear modulus was increased by addition of fibronectin. The increase was by a factor of 2.0 without ligation and by a factor of 2.4 with ligation. Creep and creep recovery in clots formed with and without fibronectin were similar except for the scale factor represented by the change in modulus. PMID:7260326

  15. Beta 8 integrins mediate interactions of chick sensory neurons with laminin-1, collagen IV, and fibronectin.

    PubMed Central

    Venstrom, K; Reichardt, L

    1995-01-01

    Integrins are major receptors used by cells to interact with extracellular matrices. In this paper, we identify the first ligands for the beta 8 family of integrins, presenting evidence that integrin heterodimers containing the beta 8 subunit mediate interactions of chick sensory neurons with laminin-1, collagen IV, and fibronectin. A polyclonal antibody, anti-beta 8-Ex, was prepared to a bacterial fusion protein expressing an extracellular portion of the chicken beta 8 subunit. In nonreducing conditions, this antibody immunoprecipitated from surface-labeled embryonic dorsal root ganglia neurons a M(r) 100 k protein, the expected M(r) of the beta 8 subunit, and putative alpha subunit(s) of M(r) 120 k. Affinity-purified anti-beta 8-Ex strongly inhibited sensory neurite outgrowth on laminin-1, collagen IV, and fibronectin-coated substrata. Binding sites were identified in a heat-resistant domain in laminin-1 and in the carboxyl terminal, 40-kDa fibronectin fragment. On substrates coated with the carboxyl terminal fragment of fibronectin, antibodies to beta 1 and beta 8 were only partially effective alone, but were completely effective in combination, at inhibiting neurite outgrowth. Results thus indicate that the integrin beta 8 subunit in association with one or more alpha subunits forms an important set of extracellular matrix receptors on sensory neurons. Images PMID:7542940

  16. Immunohistochemical demonstration of fibronectin in the most superficial layer of normal rabbit articular cartilage.

    PubMed Central

    Nishida, K; Inoue, H; Murakami, T

    1995-01-01

    OBJECTIVE--To locate fibronectin ultrastructurally in the most superficial layer of normal articular cartilage of rabbits, in order to clarify its role in joint physiology. METHODS--Articular cartilage was obtained from the femoral condyle of seven normal adult rabbits and prepared by a method that included tannic acid fixation. Polyclonal antibodies against rabbit fibronectin were used in an immunohistochemical electron microscopic study, without any enzymic digestion but with a pre-embedding method for the transmission electron microscopy. RESULTS--The cartilage surface was successfully preserved by tannic acid fixation. The most superficial layer in electron photomicrographs was approximately 200-300 nm thick, cell free, and appeared to have two parallel components: the more superficial lamina and the deeper lamina. Gold labelled fibronectin lined this layer in immunohistochemical electron photomicrographs. CONCLUSIONS--Fibronectin covering the surface of the articular cartilage may have a role in joint lubrication and protection of the cartilage by binding with the collagenous matrix and hyaluronic acid in synovial fluid. Chondroitin sulphates may act as a charge barrier in close relationship with the collagen fibrils in the deeper lamina. Significant alteration in these functions may be one of the first causal steps leading to destruction of the articular cartilage. Images PMID:8546534

  17. Fabrication of functional fibronectin patterns by nanosecond excimer laser direct write for tissue engineering applications.

    PubMed

    Grigorescu, S; Hindié, M; Axente, E; Carreiras, F; Anselme, K; Werckmann, J; Mihailescu, I N; Gallet, O

    2013-07-01

    Laser direct write techniques represent a prospective alternative for engineering a new generation of hybrid biomaterials via the creation of patterns consisting of biological proteins onto practically any type of substrate. In this paper we report on the characterization of fibronectin features obtained onto titanium substrates by UV nanosecond laser transfer. Fourier-transform infrared spectroscopy measurements evidenced no modification in the secondary structure of the post-transferred protein. The molecular weight of the transferred protein was identical to the initial fibronectin, no fragment bands being found in the transferred protein's Western blot migration profile. The presence of the cell-binding domain sequence and the mannose groups within the transferred molecules was revealed by anti-fibronectin monoclonal antibody immunolabelling and FITC-Concanavalin-A staining, respectively. The in vitro tests performed with MC3T3-E1 osteoblast-like cells and Swiss-3T3 fibroblasts showed that the cells' morphology and spreading were strongly influenced by the presence of the fibronectin spots. PMID:23615786

  18. Upregulation of fibronectin expression by COX-2 is mediated by interaction with ELMO1.

    PubMed

    Yang, Chen; Sorokin, Andrey

    2011-01-01

    Engulfment and cell motility 1 (ELMO1), a bipartite guanine nucleotide exchange factor (GEF) for the small GTPase Rac 1, was identified as a susceptibility gene for glomerular disease. Here, we reported that ELMO1 interacted with COX-2 in human mesangial cells. Furthermore, we identified ELMO1 as a posttranslational regulator of COX-2 activity. We demonstrated that COX-2 cyclooxygenase activity increased fibronectin promoter activity. The protein-protein interaction between ELMO1 and COX-2 increased the cyclooxygenase activity of COX-2 and, correspondingly, fibronectin expression. We also found that ET625, the dominant negative form of ELMO1 lacking Rac1 activity, interacted with COX-2, increased cyclooxygenase activity of COX-2 and enhanced COX-2-mediated fibronectin upregulation. To further rule out Rac1 as an ELMO1-mediated regulator of COX-2 activity, we employed the constitutive active Rac1, Rac1(Q63E), and demonstrated that Rac1 signaling has no effect on COX-2-mediated fibronectin promoter activity. These results suggest that ELMO1 contributes to the development of glomerular injury through serving as a regulator of COX-2 activity. The interaction of ELMO1 with COX-2 could play an important role in the development and progression of renal glomerular injury. PMID:20732417

  19. Digital scanner infrared focal plane technology

    NASA Astrophysics Data System (ADS)

    Ortiz, M. A.; Malone, N. R.; Harris, M.; Shin, J.; Byers, S.; Price, D.; Vampola, J.

    2011-09-01

    Advancements in finer geometry and technology advancements in circuit design now allow placement of digital architecture on cryogenic focal planes while using less power than heritage analog designs. These advances in technology reduce the size, weight, and power of modern focal planes. In addition, the interface to the focal plane is significantly simplified and is more immune to Electromagnetic Interference (EMI). The cost of the customer's instrument after integration with the digital scanning Focal Plane Array (FPA) has been significantly reduced by placing digital architecture such as Analog to digital convertors and Low Voltage Differential Signaling (LVDS) Inputs and Outputs (I/O) on the Read Out Integrated Circuit (ROIC).

  20. Focal axis resolver for offset reflector antennas

    NASA Technical Reports Server (NTRS)

    Schmidt, R. F.

    1980-01-01

    Described are electrical means for determining the focal axis of an offset reflector antenna whose physical rim is not coincident with the boundary of the electrical aperture. Even and odd sensing functions are employed in the focal region, leading to both amplitude and phase criteria for resolving a focal axis generally inclined with respect to the system axis. The analytical aspects of the problem are discussed, and an example related to a 4-meter Large-Antenna Multiple-Frequency Microwave Radiometer (LAMMR) is included. The technique is useful for focal axis determination in mathematical simulations and in the physical world.

  1. Optimal focal-plane restoration

    NASA Technical Reports Server (NTRS)

    Reichenbach, Stephen E.; Park, Stephen K.

    1989-01-01

    Image restoration can be implemented efficiently by calculating the convolution of the digital image and a small kernel during image acquisition. Processing the image in the focal-plane in this way requires less computation than traditional Fourier-transform-based techniques such as the Wiener filter and constrained least-squares filter. Here, the values of the convolution kernel that yield the restoration with minimum expected mean-square error are determined using a frequency analysis of the end-to-end imaging system. This development accounts for constraints on the size and shape of the spatial kernel and all the components of the imaging system. Simulation results indicate the technique is effective and efficient.

  2. Neurocysticercosis presenting as focal hydrocephalus

    PubMed Central

    Malik, Azharuddin Mohammed; Shamim, Md Dilawez; Ahmad, Mehtab; Abdali, Nasar

    2014-01-01

    A 40-year-old man presented with a 2-month history of headache, nausea and vomiting, with generalised seizures for the past 15 days. On examination he had bilateral papilloedema, visual acuity was 6/6 in both eyes but perimetry showed right homonymous inferior quadrantanopia. His MRI showed numerous small cystic lesions with eccentric nodules, diffusely distributed in bilateral cerebral and cerebellar hemispheres. There was also focal hydrocephalus involving occipital and temporal horns of the left lateral ventricle leading to its selective dilation. Stool examination showed ova of Taenia solium. He was treated with albendazole, prednisone and sustained release sodium valproate for 1 month. His headache resolved and he is free of seizures. Repeat perimetry at 1 month also showed resolution of visual field defect. PMID:24962486

  3. Regulation of the innate immune response by fibronectin: synergism between the III-1 and EDA domains.

    PubMed

    Kelsh, Rhiannon; You, Ran; Horzempa, Carol; Zheng, Mingzhe; McKeown-Longo, Paula J

    2014-01-01

    Fibronectin is a critical component of the extracellular matrix and alterations to its structure will influence cellular behavior. Matrix fibronectin is subjected to both mechanical and biochemical regulation. The Type III domains of fibronectin can be unfolded in response to increased cellular contractility, included or excluded from the molecule by alternative splicing mechanisms, or released from the matrix by proteolysis. Using Inflammatory Cytokine microarrays we found that the alternatively spliced fibronectin Type III domain, FnEDA, and the partially unfolded III-1 domain, FnIII-1c, induced the expression of a multitude of pro-inflammatory cytokines in human dermal fibroblasts, most notably CXCL1-3, IL-8 and TNF-α. FnIII-1c, a peptide representing an unfolded intermediate structure of the first Type III domain has been shown to initiate the toll-like receptor-4 (TLR4)-NFκB-dependent release of cytokines from human dermal fibroblasts (You, et al., J. Biol. Chem., 2010). Here we demonstrate that FnIII-1c and the alternatively spliced FnEDA domain induce a TLR4 dependent activation of p38 MAP kinase and its downstream effector, MAPKAP Kinase-2 (MK-2), to regulate cytokine expression in fibroblasts. RT-qPCR analysis indicated that the p38-MK-2 pathway regulates IL-8 mRNA stability. Interestingly, addition of FnIII-1c and FnEDA synergistically enhanced TLR4-dependent IL-8 release. These data indicate that Fn contains two Type III domains which can activate TLR signaling to induce an inflammatory response in fibroblasts. Furthermore, our data identifies the NF-κB and p38/MK2 signaling pathways as transducers of signals initiated in response to structural changes in fibronectin. PMID:25051083

  4. Regulation of the Innate Immune Response by Fibronectin: Synergism between the III-1 and EDA Domains

    PubMed Central

    Kelsh, Rhiannon; You, Ran; Horzempa, Carol; Zheng, Mingzhe; McKeown-Longo, Paula J.

    2014-01-01

    Fibronectin is a critical component of the extracellular matrix and alterations to its structure will influence cellular behavior. Matrix fibronectin is subjected to both mechanical and biochemical regulation. The Type III domains of fibronectin can be unfolded in response to increased cellular contractility, included or excluded from the molecule by alternative splicing mechanisms, or released from the matrix by proteolysis. Using Inflammatory Cytokine microarrays we found that the alternatively spliced fibronectin Type III domain, FnEDA, and the partially unfolded III-1 domain, FnIII-1c, induced the expression of a multitude of pro-inflammatory cytokines in human dermal fibroblasts, most notably CXCL1-3, IL-8 and TNF-α. FnIII-1c, a peptide representing an unfolded intermediate structure of the first Type III domain has been shown to initiate the toll-like receptor-4 (TLR4)-NFκB-dependent release of cytokines from human dermal fibroblasts (You, et al., J. Biol. Chem., 2010). Here we demonstrate that FnIII-1c and the alternatively spliced FnEDA domain induce a TLR4 dependent activation of p38 MAP kinase and its downstream effector, MAPKAP Kinase-2 (MK-2), to regulate cytokine expression in fibroblasts. RT-qPCR analysis indicated that the p38-MK-2 pathway regulates IL-8 mRNA stability. Interestingly, addition of FnIII-1c and FnEDA synergistically enhanced TLR4-dependent IL-8 release. These data indicate that Fn contains two Type III domains which can activate TLR signaling to induce an inflammatory response in fibroblasts. Furthermore, our data identifies the NF-κB and p38/MK2 signaling pathways as transducers of signals initiated in response to structural changes in fibronectin. PMID:25051083

  5. Structure and unfolding of the third type III domain from human fibronectin.

    PubMed

    Stine, Jessica M; Sun, Yizhi; Armstrong, Geoffrey; Bowler, Bruce E; Briknarová, Klára

    2015-11-10

    Fibronectin is a modular extracellular matrix protein that is essential for vertebrate development. The third type III domain (3FN3) in fibronectin interacts with other parts of fibronectin and with anastellin, a protein fragment that causes fibronectin aggregation. 3FN3 opens readily both as an isolated domain in solution and when part of fibronectin in stretched fibrils, and it was proposed that this opening is important for anastellin binding. We determined the structure of 3FN3 using nuclear magnetic resonance spectroscopy, and we investigated its stability, folding, and unfolding. Similar to most other FN3 domains, 3FN3 contains two antiparallel β-sheets that are composed of three (A, B, and E) and four (C, D, F, and G) β-strands, respectively, and are held together by a conserved hydrophobic interface. cis-trans isomerization of P847 at the end of β-strand C leads to observable conformational heterogeneity in 3FN3, with a cis peptide bond present in almost one-quarter of the molecules. The chemical stability of 3FN3 is relatively low, but the folding rate constant in the absence of denaturant is in the same range as those of other, more stable FN3 domains. Interestingly, the unfolding rate constant in the absence of denaturant is several orders of magnitude higher than the unfolding rate constants of other FN3 domains investigated to date. This unusually fast rate is comparable to the rate of binding of 3FN3 to anastellin at saturating anastellin concentrations, consistent with the model in which 3FN3 has to unfold to interact with anastellin. PMID:26517579

  6. The Fibronectin-Binding Protein Fnm Contributes to Adherence to Extracellular Matrix Components and Virulence of Enterococcus faecium

    PubMed Central

    Somarajan, Sudha R.; La Rosa, Sabina Leanti; Singh, Kavindra V.; Roh, Jung H.; Höök, Magnus

    2015-01-01

    The interaction between bacteria and fibronectin is believed to play an important role in the pathogenicity of clinically important Gram-positive cocci. In the present study, we identified a gene encoding a predicted fibronectin-binding protein of Enterococcus faecium (fnm), a homologue of Streptococcus pneumoniae pavA, in the genomes of E. faecium strain TX82 and all other sequenced E. faecium isolates. Full-length recombinant Fnm from strain TX82 bound to immobilized fibronectin in a concentration-dependent manner and also appeared to bind collagen type V and laminin, but not other proteins, such as transferrin, heparin, bovine serum albumin, mucin, or collagen IV. We demonstrated that the N-terminal fragment of Fnm is required for full fibronectin binding, since truncation of this region caused a 2.4-fold decrease (P < 0.05) in the adhesion of E. faecium TX82 to fibronectin. Deletion of fnm resulted in a significant reduction (P < 0.001) in the ability of the mutant, TX6128, to bind fibronectin relative to that of the wild-type strain; in situ reconstitution of fnm in the deletion mutant strain restored adherence. In addition, the Δfnm mutant was highly attenuated relative to TX82 (P ≤ 0.0001) in a mixed-inoculum rat endocarditis model. Taken together, these results demonstrate that Fnm affects the adherence of E. faecium to fibronectin and is important in the pathogenesis of experimental endocarditis. PMID:26371130

  7. Hormonal regulation of focal adhesions in bovine adrenocortical cells: induction of paxillin dephosphorylation by adrenocorticotropic hormone.

    PubMed Central

    Vilgrain, I; Chinn, A; Gaillard, I; Chambaz, E M; Feige, J J

    1998-01-01

    A study of bovine adrenocortical cell shape on adrenocorticotropic hormone (ACTH) challenge showed that the cells round up and develop arborized processes. This effect was found to be (1) specific for ACTH because angiotensin II and basic fibroblast growth factor have no effect; (2) mediated by a cAMP-dependent pathway because forskolin reproduces the effect of the hormone; (3) inhibited by sodium orthovanadate, a phosphotyrosine phosphatase inhibitor, but unchanged by okadaic acid, a serine/threonine phosphatase inhibitor; and (4) correlated with a complete loss of focal adhesions. Biochemical studies of the focal-adhesion-associated proteins showed that pp125fak, vinculin (110 kDa) and paxillin (70 kDa) were detected in the Triton X-100-insoluble fraction from adrenocortical cells. During cell adhesion on fibronectin as substratum, two major phosphotyrosine-containing proteins of molecular masses 125 and 68 kDa were immunodetected in the same fraction. A dramatic decrease in the extent of tyrosine phosphorylation of these proteins was observed within 60 min after treatment with ACTH. No change in pp125fak tyrosine phosphorylation nor in Src activity was detected. In contrast, paxillin was found to be tyrosine-dephosphorylated in a time-dependent manner in ACTH-treated cells. Sodium orthovanadate completely prevented the effect of ACTH. These observations suggest a possible role for phosphotyrosine phosphatases in hormone-dependent cellular regulatory processes. PMID:9601084

  8. Cleavage of extracellular matrix in periodontitis: gingipains differentially affect cell adhesion activities of fibronectin and tenascin-C.

    PubMed

    Ruggiero, Sabrina; Cosgarea, Raluca; Potempa, Jan; Potempa, Barbara; Eick, Sigrun; Chiquet, Matthias

    2013-04-01

    Gingipains are cysteine proteases that represent major virulence factors of the periodontopathogenic bacterium Porphyromonas gingivalis. Gingipains are reported to degrade extracellular matrix (ECM) of periodontal tissues, leading to tissue destruction and apoptosis. The exact mechanism is not known, however. Fibronectin and tenascin-C are pericellular ECM glycoproteins present in periodontal tissues. Whereas fibronectin mediates fibroblast adhesion, tenascin-C binds to fibronectin and inhibits its cell-spreading activity. Using purified proteins in vitro, we asked whether fibronectin and tenascin-C are cleaved by gingipains at clinically relevant concentrations, and how fragmentation by the bacterial proteases affects their biological activity in cell adhesion. Fibronectin was cleaved into distinct fragments by all three gingipains; however, only arginine-specific HRgpA and RgpB but not lysine-specific Kgp destroyed its cell-spreading activity. This result was confirmed with recombinant cell-binding domain of fibronectin. Of the two major tenascin-C splice variants, the large but not the small was a substrate for gingipains, indicating that cleavage occurred primarily in the alternatively spliced domain. Surprisingly, cleavage of large tenascin-C variant by all three gingipains generated fragments with increased anti-adhesive activity towards intact fibronectin. Fibronectin and tenascin-C fragments were detected in gingival crevicular fluid of a subset of periodontitis patients. We conclude that cleavage by gingipains directly affects the biological activity of both fibronectin and tenascin-C in a manner that might lead to increased cell detachment and loss during periodontal disease. PMID:23313574

  9. Cleavage of extracellular matrix in periodontitis: gingipains differentially affect cell adhesion activities of fibronectin and tenascin-C

    PubMed Central

    Ruggiero, Sabrina; Cosgarea, Raluca; Potempa, Jan; Potempa, Barbara; Eick, Sigrun; Chiquet, Matthias

    2014-01-01

    Gingipains are cysteine proteases that represent major virulence factors of the periodontopathogenic bacterium Porphyromonas gingivalis. Gingipains are reported to degrade extracellular matrix (ECM) of periodontal tissues, leading to tissue destruction and apoptosis. The exact mechanism is not known, however. Fibronectin and tenascin-C are pericellular ECM glycoproteins present in periodontal tissues. Whereas fibronectin mediates fibroblast adhesion, tenascin-C binds to fibronectin and inhibits its cell-spreading activity. Using purified proteins in vitro, we asked whether fibronectin and tenascin-C are cleaved by gingipains at clinically relevant concentrations, and how fragmentation by the bacterial proteases affects their biological activity in cell adhesion. Fibronectin was cleaved into distinct fragments by all three gingipains; however, only arginine-specific HRgpA and RgpB but not lysine-specific Kgp destroyed its cell-spreading activity. This result was confirmed with recombinant cell-binding domain of fibronectin. Of the two major tenascin-C splice variants, the large but not the small was a substrate for gingipains, indicating that cleavage occurred primarily in the alternatively spliced domain. Surprisingly, cleavage of large tenascin-C variant by all three gingipains generated fragments with increased anti-adhesive activity towards intact fibronectin. Fibronectin and tenascin-C fragments were detected in gingival crevicular fluid of a subset of periodontitis patients. We conclude that cleavage by gingipains directly affects the biological activity of both fibronectin and tenascin-C in a manner that might lead to increased cell detachment and loss during periodontal disease. PMID:23313574

  10. Production of Fibronectin by the Human Alveolar Macrophage: Mechanism for the Recruitment of Fibroblasts to Sites of Tissue Injury in Interstitial Lung Diseases

    NASA Astrophysics Data System (ADS)

    Rennard, Stephen I.; Hunninghake, Gary W.; Bitterman, Peter B.; Crystal, Ronald G.

    1981-11-01

    Because cells of the mononuclear phagocyte system are known to produce fibronectin and because alveolar macrophages are activated in many interstitial lung diseases, the present study was designed to evaluate a role for the alveolar macrophage as a source of the increased levels of fibronectin found in the lower respiratory tract in interstitial lung diseases and to determine if such fibronectin might contribute to the development of the fibrosis found in these disorders by being a chemoattractant for human lung fibroblasts. Production of fibronectin by human alveolar macrophages obtained by bronchoalveolar lavage and maintained in short-term culture in serum-free conditions was demonstrated; de novo synthesis was confirmed by the incorporation of [14C]proline. This fibronectin had a monomer molecular weight of 220,000 and was antigenically similar to plasma fibronectin. Macrophages from patients with idiopathic pulmonary fibrosis produced fibronectin at a rate 20 times higher than did normal macrophages; macrophages from patients with pulmonary sarcoidosis produced fibronectin at 10 times the normal rate. Macrophages from 6 of 10 patients with various other interstitial disorders produced fibronectin at rates greater than the rate of highest normal control. Human alveolar macrophage fibronectin was chemotactic for human lung fibroblasts, suggesting a functional role for this fibronectin in the derangement of the alveolar structures that is characteristic of these disorders.

  11. A liquid crystalline polymer microlens array with tunable focal intensity by the polarization control of a liquid crystal layer

    NASA Astrophysics Data System (ADS)

    Choi, Yoonseuk; Kim, Hak-Rin; Lee, Kwang-Ho; Lee, Yong-Min; Kim, Jae-Hoon

    2007-11-01

    We propose a focal intensity tunable microlens array by using a birefringent liquid crystalline polymer for lensing action. Due to the difference of effective refractive indices, it acts as a positive or negative microlens with respect to the polarization state. As we control the incident polarization by adding a liquid crystal layer, the focal intensity can be tuned by an applied voltage. Twisted nematic and bistable ferroelectric liquid crystal modes were applied to demonstrate the possibility of various driving features such as a continuously tunable focal intensity or fast switching with memory effect.

  12. Dementia and driving

    MedlinePlus

    ... not drive at times of the day when traffic is heaviest. Do not drive when the weather is bad. Do not drive long distances. Drive only on roads the person is used to. Caregivers should try to lessen ...

  13. GABAergic networks jump-start focal seizures.

    PubMed

    de Curtis, Marco; Avoli, Massimo

    2016-05-01

    Abnormally enhanced glutamatergic excitation is commonly believed to mark the onset of a focal seizure. This notion, however, is not supported by firm evidence, and it will be challenged here. A general reduction of unit firing has been indeed observed in association with low-voltage fast activity at the onset of seizures recorded during presurgical intracranial monitoring in patients with focal, drug-resistant epilepsies. Moreover, focal seizures in animal models start with increased γ-aminobutyric acid (GABA)ergic interneuronal activity that silences principal cells. In vitro studies have shown that synchronous activation of GABAA receptors occurs at seizure onset and causes sizeable elevations in extracellular potassium, thus facilitating neuronal recruitment and seizure progression. A paradoxical involvement of GABAergic networks is required for the initiation of focal seizures characterized by low-voltage fast activity, which represents the most common seizure-onset pattern in focal epilepsies. PMID:27061793

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

    PubMed Central

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

    2015-01-01

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

  15. Early vision and focal attention

    NASA Astrophysics Data System (ADS)

    Julesz, Bela

    1991-07-01

    At the thirty-year anniversary of the introduction of the technique of computer-generated random-dot stereograms and random-dot cinematograms into psychology, the impact of the technique on brain research and on the study of artificial intelligence is reviewed. The main finding-that stereoscopic depth perception (stereopsis), motion perception, and preattentive texture discrimination are basically bottom-up processes, which occur without the help of the top-down processes of cognition and semantic memory-greatly simplifies the study of these processes of early vision and permits the linking of human perception with monkey neurophysiology. Particularly interesting are the unexpected findings that stereopsis (assumed to be local) is a global process, while texture discrimination (assumed to be a global process, governed by statistics) is local, based on some conspicuous local features (textons). It is shown that the top-down process of "shape (depth) from shading" does not affect stereopsis, and some of the models of machine vision are evaluated. The asymmetry effect of human texture discrimination is discussed, together with recent nonlinear spatial filter models and a novel extension of the texton theory that can cope with the asymmetry problem. This didactic review attempts to introduce the physicist to the field of psychobiology and its problems-including metascientific problems of brain research, problems of scientific creativity, the state of artificial intelligence research (including connectionist neural networks) aimed at modeling brain activity, and the fundamental role of focal attention in mental events.

  16. Report on SEQUAL/FOCAL

    NASA Astrophysics Data System (ADS)

    Katz, E. J.; Philander, S. G. H.; Richardson, P. L.

    In Eos (April 6, 1982), United States plans for a program to study the dynamic response of the equatorial Atlantic to seasonally varying surface winds were described. Now, 6 years later, we report on progress toward our goal “to describe accurately, and to model correctly” the changes in the currents and density field of the upper equatorial Atlantic Ocean during a 2-year period. A major effort toward this goal was the field phase of SEQUAL (Seasonal Response of the Equatorial Atlantic) and the closely coordinated French program FOCAL (Français Océan et Climat dans l'Atlantique Equatorial).Between February 1983 and September 1984 changes in the surface winds and in oceanic conditions in the equatorial Atlantic were monitored continuously with a variety of instruments. Figure 1 shows key deployments and sections. The resulting data include six current meter moorings, 15 inverted echo sounders and island tide gauges, 57 near-surface drifters, and 18 French and five U.S. cruises that made 1200 hydrographic stations and 800 current profiles. All these data, as well as nearly 10,000 expendable bathythermographs (XBTs) (from the cruises, from air-dropped expendable bathythermograph (AXBT) programs, and from repeated ship of opportunity lines run between 1980 and 1985) were combined and documented by George Heimerdinger, of the National Oceanographic Data Center, and are available from NODC on request.

  17. Focal liver lesions found incidentally

    PubMed Central

    Algarni, Abdullah A; Alshuhri, Abdullah H; Alonazi, Majed M; Mourad, Moustafa Mabrouk; Bramhall, Simon R

    2016-01-01

    Incidentally found focal liver lesions are a common finding and a reason for referral to hepatobiliary service. They are often discovered in patients with history of liver cirrhosis, colorectal cancer, incidentally during work up for abdominal pain or in a trauma setting. Specific points should considered during history taking such as risk factors of liver cirrhosis; hepatitis, alcohol consumption, substance exposure or use of oral contraceptive pills and metabolic syndromes. Full blood count, liver function test and tumor markers can act as a guide to minimize the differential diagnosis and to categorize the degree of liver disease. Imaging should start with B-mode ultrasound. If available, contrast enhanced ultrasound is a feasible, safe, cost effective option and increases the ability to reach a diagnosis. Contrast enhanced computed tomography should be considered next. It is more accurate in diagnosis and better to study anatomy for possible operation. Contrast enhanced magnetic resonance is the gold standard with the highest sensitivity. If doubt still remains, the options are biopsy or surgical excision. PMID:27028805

  18. [The influence of immobilized fibronectin on karyotypic variability of two rat kangaroo kidney cell lines].

    PubMed

    Polianskaia, G G; Goriachaia, T S; Pinaev, G P

    2007-01-01

    The numerical and structural karyotypic variability has been investigated in "markerless" Rat kangaroo kidney cell lines NBL-3-17 and NBL-3-11 when cultivating on a fibronectin-coated surface. In cell line NBL-3-17, cultivated on the fibronectin-coated surface for 1, 2, 4 and 8 days, the character of cell distribution for the chromosome number has changed. These changes involve a significant decrease in frequency of cells with modal number of chromosomes, and an increase in frequency of cells with lower chromosomal number. Many new additional structural variants of the karyotype (SVK) appear. The observed alterations seem to be due preference adhesion of cells with lower chromosome number, disturbances of mitotic apparatus and selection of SVK, which are more adopted to changes in culture conditions. Detachment of cells from the fibronectin-coated surface, followed by 5 days cultivation on a hydrophilic surface restored control distribution. In cell line NBL-3-11, cultivated on the fibronectin-coated surface for 1, 2, 4 and 8 days, the character of numerical karyotypic variability did not change compared to control variants. In cell line NBL-3-17 the frequency of chromosomal aberrations under cultivation on the fibronectin-coated surface for 1, 2, 4 and 8 days did not change relative to control variants. In cell line NBL-3-11 the frequency of chromosomal aberrations under the same conditions significantly increases, mainly at the expence of chromosomal, chromatid breaks and dicentrics (telomeric association) relative to control variants. We discuss possible reasons of differences in the character of numerical and structural karyotypic variability between cell lines NBL-3-17 (hypotriploid) and NBL-3-11 (hypodiploid) under cultivation on fibronectin. The reasons of the observed interline karyotypic differences possibly consist in peculiarity of karyotypic structure of cell line NBL-3-11 and in the change of gene expression, namely in a dose of certain functioning

  19. Surface oxide net charge of a titanium alloy: modulation of fibronectin-activated attachment and spreading of osteogenic cells.

    PubMed

    Rapuano, Bruce E; MacDonald, Daniel E

    2011-01-01

    In the current study, we have altered the surface oxide properties of a Ti6Al4V alloy using heat treatment or radiofrequency glow discharge (RFGD) in order to evaluate the relationship between the physico-chemical and biological properties of the alloy's surface oxide. The effects of surface pretreatments on the attachment of cells from two osteogenic cell lines (MG63 and MC3T3) and a mesenchymal stem cell line (C3H10T1/2) to fibronectin adsorbed to the alloy were measured. Both heat and RFGD pretreatments produced a several-fold increase in the number of cells that attached to fibronectin adsorbed to the alloy at a range of coating concentrations (0.001-10nM FN) for each cell line tested. An antibody (HFN7.1) directed against the central integrin binding domain of fibronectin produced a 65-70% inhibition of cell attachment to fibronectin-coated disks, indicating that cell attachment to the metal discs was dependent on fibronectin binding to cell integrin receptors. Both treatments also accelerated the cell spreading response manifested by extensive flattening and an increase in mean cellular area. The treatment-induced increases in the cell attachment activity of adsorbed fibronectin were correlated with previously demonstrated increases in Ti6Al4V oxide negative net surface charge at physiological pH produced by both heat and RFGD pretreatments. Since neither treatment increased the adsorption mass of fibronectin, these findings suggest that negatively charged surface oxide functional groups in Ti6Al4V can modulate fibronectin's integrin receptor activity by altering the adsorbed protein's conformation. Our results further suggest that negatively charged functional groups in the surface oxide can play a prominent role in the osseointegration of metallic implant materials. PMID:20884181

  20. Fibronectin-binding protein of Streptococcus pyogenes: sequence of the binding domain involved in adherence of streptococci to epithelial cells.

    PubMed Central

    Talay, S R; Valentin-Weigand, P; Jerlström, P G; Timmis, K N; Chhatwal, G S

    1992-01-01

    The sequence of the fibronectin-binding domain of the fibronectin-binding protein of Streptococcus pyogenes (Sfb protein) was determined, and its role in streptococcal adherence was investigated by use of an Sfb fusion protein in adherence studies. A 1-kb DNA fragment coding for the binding domain of Sfb protein was cloned into the expression vector pEX31 to produce an Sfb fusion protein consisting of the N-terminal part of MS2 polymerase and a C-terminal fragment of the streptococcal protein. Induction of the vector promoter resulted in hyperexpression of fibronectin-binding fusion protein in the cytoplasm of the recombinant Escherichia coli cells. Sequence determination of the cloned 1-kb fragment revealed an in-frame reading frame for a 268-amino-acid peptide composed of a 37-amino-acid sequence which is completely repeated three times and incompletely repeated a fourth time. Cloning of one repeat into pEX31 resulted in expression of small fusion peptides that show fibronectin-binding activity, indicating that one repeat contains at least one binding domain. Each repeat exhibits two charged domains and shows high homology with the 38-amino-acid D3 repeat of the fibronectin-binding protein of Staphylococcus aureus. Sequence comparison with other streptococcal ligand-binding surface proteins, including M protein, failed to reveal significant homology, which suggests that Sfb protein represents a novel type of functional protein in S. pyogenes. The Sfb fusion protein isolated from the cytoplasm of recombinant cells was purified by fast protein liquid chromatography. It showed a strong competitive inhibition of fibronectin binding to S. pyogenes and of the adherence of bacteria to cultured epithelial cells. In contrast, purified streptococcal lipoteichoic acid showed only a weak inhibition of fibronectin binding and streptococcal adherence. These results demonstrate that Sfb protein is directly involved in the fibronectin-mediated adherence of S. pyogenes to

  1. EDA-containing fibronectin levels in the cerebrospinal fluid of children with meningitis.

    PubMed

    Pupek, Małgorzata; Jasonek, Jolanta; Kątnik-Prastowska, Iwona

    2013-01-01

    Fibronectin containing an alternatively spliced extra domain A (EDA-FN) participates in diverse biological cell functions, being also directly or indirectly engaged during an inflammatory response to brain injury and/or neuron regeneration. We analyzed FN and EDA-FN isoform levels by ELISA in 85 cerebrospinal fluid samples and 67 plasma samples obtained from children suffering from bacterial or viral meningitis and non-meningitis peripheral inflammation. We have found that the cerebrospinal level of EDA-FN was significantly lower in the bacterial meningitis group than in the viral- and non-meningitis groups. In the patients' plasma, EDA-FN was almost undetectable. The determination of fibronectin containing the EDA segment might be considered as an additional diagnostic marker of bacterial meningitis in children. PMID:23884219

  2. Simple application of fibronectin-mimetic coating enhances osseointegration of titanium implants

    PubMed Central

    Petrie, Timothy A.; Reyes, Catherine D.; Burns, Kellie L.; García, Andrés J.

    2009-01-01

    Integrin-mediated cell adhesion to biomolecules adsorbed onto biomedical devices regulates device integration and performance. Because of the central role of integrin-fibronectin (FN) interactions in osteoblastic function and bone formation, we evaluated the ability of fibronectin-inspired biomolecular coatings to promote osteoblastic differentiation and implant osseointegration. Notably, these biomolecular coatings relied on physical adsorption of FN-based ligands onto biomedical-grade titanium as a simple, clinically-translatable strategy to functionalize medical implants. Surfaces coated with a recombinant fragment of FN spanning the central cell binding domain enhanced osteoblastic differentiation and mineralization in bone marrow stromal cell cultures and increased implant osseointegration in a rat cortical bone model compared to passively adsorbed RGD peptides, serum proteins, and full-length FN. Differences in biological responses correlated with integrin binding specificity and signaling among surface coatings. This work validates a simple, clinically-translatable, surface biofunctionalization strategy to enhance biomedical device integration. PMID:18752639

  3. Identification of Surface Proteins from Lactobacillus casei BL23 Able to Bind Fibronectin and Collagen.

    PubMed

    Muñoz-Provencio, Diego; Pérez-Martínez, Gaspar; Monedero, Vicente

    2011-03-01

    Strains of lactobacilli show the capacity to attach to extracellular matrix proteins. Cell-wall fractions of Lactobacillus casei BL23 enriched in fibronectin, and collagen-binding proteins were isolated. Mass spectrometry analysis of their protein content revealed the presence of stress-related proteins (GroEL, ClpL), translational elongation factors (EF-Tu, EF-G), oligopeptide solute-binding proteins, and the glycolytic enzymes enolase and glyceraldehyde-3-phosphate dehydrogenase (GAPDH). The latter two enzymes were expressed in Escherichia coli and purified as glutathione-S-transferase (GST) fusion proteins, and their in vitro binding activity to fibronectin and collagen was confirmed. These results reinforce the idea that lactobacilli display on their surfaces a variety of moonlighting proteins that can be important in their adaptation to survive at intestinal mucosal sites and in the interaction with host cells. PMID:26781495

  4. Unilateral Isolated Proximal Femoral Focal Deficiency

    PubMed Central

    Doğer, Emek; Köpük, Şule Y.; Çakıroğlu, Yiğit; Çakır, Özgür; Yücesoy, Gülseren

    2013-01-01

    Objective. To discuss a patient with a prenatal diagnosis of unilateral isolated femoral focal deficiency. Case. Antenatal diagnosis of unilateral isolated femoral focal deficiency was made at 20 weeks of gestation. The length of left femur was shorter than the right, and fetal femur length was below the fifth percentile. Proximal femoral focal deficiency was diagnosed. After delivery, the diagnosis was confirmed with skeletal radiographs and magnetic resonance imaging. In prenatal ultrasonographic examination, the early recognition and exclusion of skeletal dysplasias is important; moreover, treatment plans should be initiated, and valuable information should be provided to the family. PMID:23984135

  5. Identification of a fibronectin interaction site in the extracellular matrix protein ameloblastin.

    PubMed

    Beyeler, Michael; Schild, Christof; Lutz, Roman; Chiquet, Matthias; Trueb, Beat

    2010-04-15

    Mammalian teeth are composed of hydroxyapatite crystals that are embedded in a rich extracellular matrix. This matrix is produced by only two cell types, the mesenchymal odontoblasts and the ectodermal ameloblasts. Ameloblasts secrete the enamel proteins amelogenin, ameloblastin, enamelin and amelotin. Odontoblasts secrete collagen type I and several calcium-binding phosphoproteins including dentin sialophosphoprotein, dentin matrix protein, bone sialoprotein and osteopontin. The latter four proteins have recently been grouped in the family of the SIBLINGs (small integrin-binding ligand, N-linked glycoproteins) because they display similar gene structures and because they contain an RGD tripeptide sequence that binds to integrin receptors and thus mediates cell adhesion. We have prepared all the other tooth-specific proteins in recombinant form and examined whether they might also promote cell adhesion similar to the SIBLINGs. We found that only ameloblastin consistently mediated adhesion of osteoblastic and fibroblastic cells to plastic or titanium surfaces. The activity was dependent on the intact three-dimensional structure of ameloblastin and required de novo protein synthesis of the adhering cells. By deletion analysis and in vitro mutagenesis, the active site could be narrowed down to a sequence of 13 amino acid residues (VPIMDFADPQFPT) derived from exon 7 of the rat ameloblastin gene or exons 7-9 of the human gene. Kinetic studies and RNA interference experiments further demonstrated that this sequence does not directly bind to a cell surface receptor but that it interacts with cellular fibronectin, which in turn binds to integrin receptors. The identification of a fibronectin-binding domain in ameloblastin might permit interesting applications for dental implantology. Implants could be coated with peptides containing the active sequence, which in turn would recruit fibronectin from the patient's blood. The recruited fibronectin should then promote cell

  6. Oxidative damage to fibronectin. 2. The effect of H2O2 and the hydroxyl radical

    SciTech Connect

    Vissers, M.C.; Winterbourn, C.C. )

    1991-03-01

    The effect of H2O2 and the hydroxyl radical (.OH) on fibronectin was investigated. .OH was generated in three ways: (1) by radiolysis with 60Co under N2O, or by the Fenton system using either (2) equimolar Fe(2+)-EDTA and H2O2 or (3) H2O2 and catalytic amounts of Fe(2+)-EDTA recycled with ascorbate. Each system had a different effect. H2O2 alone caused no changes, even at an 800-fold molar excess. Radiolytic .OH caused a rapid loss of tryptophan fluorescence, an increase in bityrosine fluorescence, and extensive crosslinking. The Fenton system using Fe-EDTA, H2O2, and ascorbate caused a loss in tryptophan fluorescence, a smaller increase in bityrosine than was seen with radiolytic .OH, and a threefold increase in carbonyl groups. On sodium dodecyl sulfate-polyacrylamide gel electrophoresis fragmentation of fibronectin was seen. In contrast, when .OH was generated with equimolar Fe-EDTA and H2O2, the only change was a small increase in bityrosine fluorescence at the highest dose of oxidant. None of the systems used affected cysteine. All the changes except the loss of tryptophan by radiolytic .OH were completely inhibited with mannitol. The differences seen with radiolytic .OH and the Fe-EDTA, H2O2, ascorbate system were not solely due to O2 in the latter system since similar results were obtained under N2. The differences between radiolytic .OH and the Fenton systems could be partly due to the components of the latter systems reacting with .OH and thus competing with fibronectin. The authors results demonstrate that the extent and type of fibronectin damage by .OH is dependent on the mode of radical generation.

  7. Fibronectin and vitronectin promote human fetal osteoblast cell attachment and proliferation on nanoporous titanium surfaces

    PubMed Central

    Rivera-Chacon, D. M.; Alvarado-Velez, M.; Acevedo-Morantes, C.Y.; Singh, S.P.; Gultepe, E.; Nagesha, D.; Sridhar, S.; Ramirez-Vick, J.E.

    2013-01-01

    Improvements in osteoconduction of implant biomaterials require focusing on the bone-implant interface, which is a complex multifactorial system. Surface topography of implants plays a crucial role at this interface. Nanostructured surfaces have been shown to promote serum protein adsorption and osteoblast adhesion when compared to microstructured surfaces for bone-implant materials. We studied the influence of the serum proteins fibronectin and vitronectin on the attachment and proliferation of osteoblasts onto nanostructured titania surfaces. Human fetal osteoblastic cells hFOB 1.19 were used as model osteoblasts and were grown on nanoporous TiO2 templates, using Ti6Al4V and commercially pure Ti substrates as controls. Results show a significant increase in cell proliferation on nanoporous TiO2 over flat substrates. Initial cell attachment data exhibited a significant effect by either fibronectin or vitronectin on cell adhesion at the surface of any of the tested materials. In addition, the extent of cell adhesion was significantly different between the nanoporous TiO2 and both Ti6Al4V and commercially pure Ti substrates, with the first showing the highest surface coverage. There was no significant difference on osteoblast attachment or proliferation between the presence of fibronectin or vitronectin using any of the material substrates. Taken together, these results suggest that the increase in osteoblast attachment and proliferation shown on the nanoporous TiO2 is due to an increase in the adsorption of fibronectin and vitronectin because of the higher surface area and to an enhanced protein unfolding, which allows access to osteoblast binding motifs within these proteins. PMID:23858975

  8. Fibronectin induction abrogates the BRAF inhibitor response of BRAF V600E/PTEN-null melanoma cells

    PubMed Central

    Fedorenko, Inna V.; Abel, Ethan V.; Koomen, John M.; Fang, Bin; Wood, Elizabeth R.; Chen, Y. Ann; Fisher, Kate J.; Iyengar, Sanjana; Dahlman, Kimberly B.; Wargo, Jennifer A.; Flaherty, Keith T.; Sosman, Jeffrey A.; Sondak, Vernon K.; Messina, Jane L.; Gibney, Geoffrey T.; Smalley, Keiran S.M.

    2015-01-01

    The mechanisms by which some melanoma cells adapt to BRAF inhibitor therapy are incompletely understood. In the present study, we used mass spectrometry-based phosphoproteomics to determine how BRAF inhibition remodeled the signaling network of melanoma cell lines that were BRAF-mutant and PTEN-null. Short-term BRAF inhibition was associated with marked changes in fibronectin-based adhesion signaling that were PTEN-dependent. These effects were recapitulated through BRAF siRNA knockdown and following treatment with chemotherapeutic drugs. Increased fibronectin expression was also observed in mouse xenograft models as well as specimens from melanoma patients undergoing BRAF inhibitor treatment. Analysis of a melanoma TMA showed loss of PTEN expression to predict for a lower overall survival, with a trend for even lower survival being seen when loss of fibronectin was included in the analysis. Mechanistically, the induction of fibronectin limited the responses of these PTEN-null melanoma cell lines to vemurafenib, with enhanced cytotoxicity observed following the knockdown of either fibronectin or its receptor α5β1 integrin. This in turn abrogated the cytotoxic response to BRAF inhibition via increased AKT signaling, which prevented the induction of cell death by maintaining the expression of the pro-survival protein Mcl-1. The protection conveyed by the induction of fibronectin expression could be overcome through combined treatment with a BRAF and PI3K inhibitor. PMID:26073081

  9. Characterization of a novel family of fibronectin-binding proteins with M23 peptidase domains from Treponema denticola

    PubMed Central

    Bamford, C.V.; Francescutti, T.; Cameron, C.E.; Jenkinson, H.F.; Dymock, D.

    2011-01-01

    SUMMARY Interactions with fibronectin are important in the virulence strategies of a range of disease-related bacteria. The periodontitis-associated oral spirochaete Treponema denticola expresses at least two fibronectin-binding proteins, designated Msp (major surface protein) and OppA (oligopeptide-binding protein homologue). To identify other T. denticola outer membrane fibronectin-binding proteins, the amino acid sequence of the Treponema pallidum fibronectin-binding protein Tp0155 was used to survey the T. denticola genome. Seven T. denticola genes encoding orthologous proteins were identified. All but two were expressed in Escherichia coli and purified recombinant proteins bound fibronectin. Using antibodies to the N-terminal region of Tp0155, it was demonstrated that T. denticola TDE2318, with highest homology to Tp0155, was cell surface localized. Like Tp0155, the seven T. denticola proteins contained an M23 peptidase domain and four (TDE2318, TDE2753, TDE1738, TDE1297) contained one or two LysM domains. M23 peptidases can degrade peptidoglycan whereas LysM domains recognize carbohydrate polymers. In addition, TDE1738 may act as a bacteriocin based on homology with other bacterial lysins and the presence of an adjacent gene encoding a putative immunity factor. Collectively, these results suggest that T. denticola expresses fibronectin-binding proteins associated with the cell surface that may also have cell wall modifying or lytic functions. PMID:21040511

  10. Fibronectin-Alginate microcapsules improve cell viability and protein secretion of encapsulated Factor IX-engineered human mesenchymal stromal cells.

    PubMed

    Sayyar, Bahareh; Dodd, Megan; Marquez-Curtis, Leah; Janowska-Wieczorek, Anna; Hortelano, Gonzalo

    2015-01-01

    Continuous delivery of proteins by engineered cells encapsu-lated in biocompatible polymeric microcapsules is of considerable therapeutic potential. However, this technology has not lived up to expectations due to inadequate cell--matrix interactions and subsequent cell death. In this study we hypoth-esize that the presence of fibronectin in an alginate matrix may enhance the viability and functionality of encapsulated human cord blood-derived mesenchymal stromal cells (MSCs) expressing the human Factor IX (FIX) gene. MSCs were encapsulated in alginate-PLL microcapsules containing 10, 100, or 500 μg/ml fibronectin to ameliorate cell survival. MSCs in microcapsules with 100 and 500 μg/ml fibronectin demonstrated improved cell viability and proliferation and higher FIX secretion compared to MSCs in non-supplemented microcapsules. In contrast, 10 μg/ml fibronectin did not significantly affect the viability and protein secretion from the encapsulated cells. Differentiation studies demonstrated osteogenic (but not chondrogenic or adipogenic) differentiation capability and efficient FIX secretion of the enclosed MSCs in the fibronectin-alginate suspension culture. Thus, the use of recombinant MSCs encapsulated in fibronectin-alginate microcapsules in basal or osteogenic cultures may be of practical use in the treatment of hemophilia B. PMID:24564349