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Sample records for actin cross-linking factor

  1. A Dictyostelium mutant lacking an F-actin cross-linking protein, the 120-kD gelation factor

    PubMed Central

    1990-01-01

    Actin-binding proteins are known to regulate in vitro the assembly of actin into supramolecular structures, but evidence for their activities in living nonmuscle cells is scarce. Amebae of Dictyostelium discoideum are nonmuscle cells in which mutants defective in several actin-binding proteins have been described. Here we characterize a mutant deficient in the 120-kD gelation factor, one of the most abundant F-actin cross- linking proteins of D. discoideum cells. No F-actin cross-linking activity attributable to the 120-kD protein was detected in mutant cell extracts, and antibodies recognizing different epitopes on the polypeptide showed the entire protein was lacking. Under the conditions used, elimination of the gelation factor did not substantially alter growth, shape, motility, or chemotactic orientation of the cells towards a cAMP source. Aggregates of the mutant developed into fruiting bodies consisting of normally differentiated spores and stalk cells. In cytoskeleton preparations a dense network of actin filaments as typical of the cell cortex, and bundles as they extend along the axis of filopods, were recognized. A significant alteration found was an enhanced accumulation of actin in cytoskeletons of the mutant when cells were stimulated with cyclic AMP. Our results indicate that control of cell shape and motility does not require the fine-tuned interactions of all proteins that have been identified as actin-binding proteins by in vitro assays. PMID:1698791

  2. Slow down of actin depolymerization by cross-linking molecules.

    PubMed

    Schmoller, Kurt M; Semmrich, Christine; Bausch, Andreas R

    2011-02-01

    The ability to control the assembly and disassembly dynamics of actin filaments is an essential property of the cellular cytoskeleton. While many different proteins are known which accelerate the polymerization of monomers into filaments or promote their disintegration, much less is known on mechanisms which guarantee the kinetic stability of the cytoskeletal filaments. Previous studies indicate that cross-linking molecules might fulfill these stabilizing tasks, which in addition facilitates their ability to regulate the organization of cytoskeletal structures in vivo. The effect of depolymerization factors on such structures or the mechanism which leads finally to their disintegration remain unknown. Here, we use multiple depolymerization methods in order to directly demonstrate that cross-linking and bundling proteins effectively suppress the actin depolymerization in a concentration dependent manner. Even the actin depolymerizing factor cofilin is not sufficient to facilitate a fast disintegration of highly cross-linked actin networks unless molecular motors are used simultaneously. The drastic modification of actin kinetics by cross-linking molecules can be expected to have wide-ranging implications for our understanding of the cytoskeleton, where cross-linking molecules are omnipresent and essential.

  3. Functional synergy of actin filament cross-linking proteins.

    PubMed

    Tseng, Yiider; Schafer, Benjamin W; Almo, Steven C; Wirtz, Denis

    2002-07-12

    The organization of filamentous actin (F-actin) in resilient networks is coordinated by various F-actin cross-linking proteins. The relative tolerance of cells to null mutations of genes that code for a single actin cross-linking protein suggests that the functions of those proteins are highly redundant. This apparent functional redundancy may, however, reflect the limited resolution of available assays in assessing the mechanical role of F-actin cross-linking/bundling proteins. Using reconstituted F-actin networks and rheological methods, we demonstrate how alpha-actinin and fascin, two F-actin cross-linking/bundling proteins that co-localize along stress fibers and in lamellipodia, could synergistically enhance the resilience of F-actin networks in vitro. These two proteins can generate microfilament arrays that "yield" at a strain amplitude that is much larger than each one of the proteins separately. F-actin/alpha-actinin/fascin networks display strain-induced hardening, whereby the network "stiffens" under shear deformations, a phenomenon that is non-existent in F-actin/fascin networks and much weaker in F-actin/alpha-actinin networks. Strain-hardening is further enhanced at high rates of deformation and high concentrations of actin cross-linking proteins. A simplified model suggests that the optimum results of the competition between the increased stiffness of bundles and their decreased density of cross-links. Our studies support a re-evaluation of the notion of functional redundancy among cytoskeletal regulatory proteins.

  4. Liquid behavior of cross-linked actin bundles.

    PubMed

    Weirich, Kimberly L; Banerjee, Shiladitya; Dasbiswas, Kinjal; Witten, Thomas A; Vaikuntanathan, Suriyanarayanan; Gardel, Margaret L

    2017-02-28

    The actin cytoskeleton is a critical regulator of cytoplasmic architecture and mechanics, essential in a myriad of physiological processes. Here we demonstrate a liquid phase of actin filaments in the presence of the physiological cross-linker, filamin. Filamin condenses short actin filaments into spindle-shaped droplets, or tactoids, with shape dynamics consistent with a continuum model of anisotropic liquids. We find that cross-linker density controls the droplet shape and deformation timescales, consistent with a variable interfacial tension and viscosity. Near the liquid-solid transition, cross-linked actin bundles show behaviors reminiscent of fluid threads, including capillary instabilities and contraction. These data reveal a liquid droplet phase of actin, demixed from the surrounding solution and dominated by interfacial tension. These results suggest a mechanism to control organization, morphology, and dynamics of the actin cytoskeleton.

  5. Cross-linking study on skeletal muscle actin: properties of suberimidate-treated actin.

    PubMed

    Ohara, O; Takahashi, S; Ooi, T; Fujiyoshi, Y

    1982-06-01

    Cross-linking experiments were performed on muscle skeletal actin, using imidoesters of various chain lengths. Chemical analyses on all products except one (derived from succinimidate) show evidence of the presence of intramolecular cross-links in the molecule. The detailed properties of suberimidate-treated actin (SA) are as follows: SA contains nearly 1 mol of intramolecular cross-link per mol of actin and less than 15% of intermolecularly cross-linked products. Even at a low salt concentration, SA is polymeric, exchanges slowly its bound nucleotide with free nucleotides in solution, and shows an F-actin-type CD spectrum. Electron micrographs of SA reveal that SA exists actually as fibrous polymers in solutions of low ionic strength, although the fibers seem to be less rigid than those at high salt concentration. The F-form of SA at a high salt concentration is indistinguishable from intact F-actin. SA can bind heavy meromyosin and activate the ATPase of heavy meromyosin as observed for intact F-actin. Tropomyosin binds SA only at a high salt concentration. These results show that SA possesses the properties of F-actin even in media of low salt concentration, which are favorable for depolymerization of F-actin. Thus, we may infer that the conformation of SA is frozen in the F-state of actin by the introduction of intramolecular cross-links in the protein.

  6. Actin polymerization is stimulated by actin cross-linking protein palladin.

    PubMed

    Gurung, Ritu; Yadav, Rahul; Brungardt, Joseph G; Orlova, Albina; Egelman, Edward H; Beck, Moriah R

    2016-02-15

    The actin scaffold protein palladin regulates both normal cell migration and invasive cell motility, processes that require the co-ordinated regulation of actin dynamics. However, the potential effect of palladin on actin dynamics has remained elusive. In the present study, we show that the actin-binding immunoglobulin-like domain of palladin, which is directly responsible for both actin binding and bundling, also stimulates actin polymerization in vitro. Palladin eliminated the lag phase that is characteristic of the slow nucleation step of actin polymerization. Furthermore, palladin dramatically reduced depolymerization, slightly enhanced the elongation rate, and did not alter the critical concentration. Microscopy and in vitro cross-linking assays reveal differences in actin bundle architecture when palladin is incubated with actin before or after polymerization. These results suggest a model whereby palladin stimulates a polymerization-competent form of globular or monomeric actin (G-actin), akin to metal ions, either through charge neutralization or through conformational changes.

  7. Tracer diffusion through F-actin: effect of filament length and cross-linking.

    PubMed Central

    Jones, J D; Luby-Phelps, K

    1996-01-01

    We have determined diffusion coefficients for small (50- to 70-nm diameter) fluorescein-thiocarbamoyl-labeled Ficoll tracers through F-actin as a function of filament length and cross-linking. fx45 was used to regulate filament length and avidin/biotinylated actin or ABP-280 was used to prepare cross-linked actin gels. We found that tracer diffusion was generally independent of filament length in agreement with theoretical predictions for diffusion through solutions of rods. However, in some experiments diffusion was slower through short (< or = 1.0 micron) filaments, although this result was not consistently reproducible. Measured diffusion coefficients through unregulated F-actin and filaments of lengths > 1.0 micron were more rapid than predicted by theory for tracer diffusion through rigid, random networks, which was consistent with some degree of actin bundling. Avidin-induced cross-linking of biotinylated F-actin did not affect diffusion through unregulated F-actin, but in cases where diffusion was slower through short filaments this cross-linking method resulted in enhanced tracer diffusion rates indistinguishable from unregulated F-actin. This finding, in conjunction with increased turbidity of 1.0-micron filaments upon avidin cross-linking, indicated that this cross-linking method induces F-actin bundling. By contrast, ABP-280 cross-linking retarded diffusion through unregulated F-actin and decreased turbidity. Tracer diffusion under these conditions was well approximated by the diffusion theory. Both cross-linking procedures resulted in gel formation as determined by falling ball viscometry. These results demonstrate that network microscopic geometry is dependent on the cross-linking method, although both methods markedly increase F-actin macroscopic viscosity. PMID:8913611

  8. Dexamethasone alters F-actin architecture and promotes cross-linked actin network formation in human trabecular meshwork tissue.

    PubMed

    Clark, Abbot F; Brotchie, Daniel; Read, A Thomas; Hellberg, Peggy; English-Wright, Sherry; Pang, Iok-Hou; Ethier, C Ross; Grierson, Ian

    2005-02-01

    Elevated intraocular pressure is an important risk factor for the development of glaucoma, a leading cause of irreversible blindness. This ocular hypertension is due to increased hydrodynamic resistance to the drainage of aqueous humor through specialized outflow tissues, including the trabecular meshwork (TM) and the endothelial lining of Schlemm's canal. We know that glucocorticoid therapy can cause increased outflow resistance and glaucoma in susceptible individuals, that the cytoskeleton helps regulate aqueous outflow resistance, and that glucocorticoid treatment alters the actin cytoskeleton of cultured TM cells. Our purpose was to characterize the actin cytoskeleton of cells in outflow pathway tissues in situ, to characterize changes in the cytoskeleton due to dexamethasone treatment in situ, and to compare these with changes observed in cell culture. Human ocular anterior segments were perfused with or without 10(-7) M dexamethasone, and F-actin architecture was investigated by confocal laser scanning microscopy. We found that outflow pathway cells contained stress fibers, peripheral actin staining, and occasional actin "tangles." Dexamethasone treatment caused elevated IOP in several eyes and increased overall actin staining, with more actin tangles and the formation of cross-linked actin networks (CLANs). The actin architecture in TM tissues was remarkably similar to that seen in cultured TM cells. Although CLANs have been reported previously in cultured cells, this is the first report of CLANs in tissue. These cytoskeletal changes may be associated with increased aqueous humor outflow resistance after ocular glucocorticoid treatment.

  9. Strain hardening, avalanches, and strain softening in dense cross-linked actin networks

    NASA Astrophysics Data System (ADS)

    Åström, Jan A.; Kumar, P. B. Sunil; Vattulainen, Ilpo; Karttunen, Mikko

    2008-05-01

    Actin filament networks enable the cytoskeleton to adjust to internal and external forcing. These dynamic networks can adapt to changes by dynamically adjusting their cross-links. Here, we model actin filaments as cross-linked elastic fibers of finite dimensions, with the cross-links being approximately 1μm apart, and employ a full three-dimensional model to study their elastic properties by computer simulations. The results show compelling evidence that dense actin networks are characterized by (a) strain hardening without entropic elasticity, (b) avalanches of cross-link slippage leading to strain softening in the case of breakable cross-links, and (c) spontaneous formation of stress fibers in the case of dynamic cross-link formation and destruction.

  10. Cross-Linking Molecules Modify Composite Actin Networks Independently

    NASA Astrophysics Data System (ADS)

    Schmoller, K. M.; Lieleg, O.; Bausch, A. R.

    2008-09-01

    While cells make use of many actin binding proteins (ABPs) simultaneously to tailor the mechanical properties of the cytoskeleton, the detailed interplay of different ABPs is not understood. By a combination of macrorheological measurements and confocal microscopy, we show that the ABPs fascin and filamin modify the structural and viscoelastic properties of composite in vitro actin networks independently. The outnumbering ABP dictates the local network structure and therefore also dominates the macromechanical network response.

  11. F actin bundles in Drosophila bristles. I. Two filament cross-links are involved in bundling

    PubMed Central

    1995-01-01

    Transverse sections though Drosophila bristles reveal 7-11 nearly round, plasma membrane-associated bundles of actin filaments. These filaments are hexagonally packed and in a longitudinal section they show a 12-nm periodicity in both the 1.1 and 1.0 views. From earlier studies this periodicity is attributable to cross-links and indicates that the filaments are maximally cross-linked, singed mutants also have 7-11 bundles, but the bundles are smaller, flattened, and the filaments within the bundles are randomly packed (not hexagonal); no periodicity can be detected in longitudinal sections. Another mutant, forked (f36a), also has 7-11 bundles but even though the bundles are very small, the filaments within them are hexagonally packed and display a 12-nm periodicity in longitudinal section. The singed-forked double mutant lacks filament bundles. Thus there are at least two species of cross-links between adjacent actin filaments. Hints of why two species of cross-links are necessary can be gleaned by studying bristle formation. Bristles sprout with only microtubules within them. A little later in development actin filaments appear. At early stages the filaments in the bundles are randomly packed. Later the filaments in the bundles become hexagonally packed and maximally cross-linked. We consider that the forked proteins may be necessary early in development to tie the filaments together in a bundle so that they can be subsequently zippered together by fascin (the singed gene product). PMID:7622563

  12. Espin cross-links cause the elongation of microvillus-type parallel actin bundles in vivo.

    PubMed

    Loomis, Patricia A; Zheng, Lili; Sekerková, Gabriella; Changyaleket, Benjarat; Mugnaini, Enrico; Bartles, James R

    2003-12-08

    The espin actin-bundling proteins, which are the target of the jerker deafness mutation, caused a dramatic, concentration-dependent lengthening of LLC-PK1-CL4 cell microvilli and their parallel actin bundles. Espin level was also positively correlated with stereocilium length in hair cells. Villin, but not fascin or fimbrin, also produced noticeable lengthening. The espin COOH-terminal peptide, which contains the actin-bundling module, was necessary and sufficient for lengthening. Lengthening was blocked by 100 nM cytochalasin D. Espin cross-links slowed actin depolymerization in vitro less than twofold. Elimination of an actin monomer-binding WASP homology 2 domain and a profilin-binding proline-rich domain from espin did not decrease lengthening, but made it possible to demonstrate that actin incorporation was restricted to the microvillar tip and that bundles continued to undergo actin treadmilling at approximately 1.5 s-1 during and after lengthening. Thus, through relatively subtle effects on actin polymerization/depolymerization reactions in a treadmilling parallel actin bundle, espin cross-links cause pronounced barbed-end elongation and, thereby, make a longer bundle without joining shorter modules.

  13. Myosin III-mediated cross-linking and stimulation of actin bundling activity of Espin.

    PubMed

    Liu, Haiyang; Li, Jianchao; Raval, Manmeet H; Yao, Ningning; Deng, Xiaoying; Lu, Qing; Nie, Si; Feng, Wei; Wan, Jun; Yengo, Christopher M; Liu, Wei; Zhang, Mingjie

    2016-01-19

    Class III myosins (Myo3) and actin-bundling protein Espin play critical roles in regulating the development and maintenance of stereocilia in vertebrate hair cells, and their defects cause hereditary hearing impairments. Myo3 interacts with Espin1 through its tail homology I motif (THDI), however it is not clear how Myo3 specifically acts through Espin1 to regulate the actin bundle assembly and stabilization. Here we discover that Myo3 THDI contains a pair of repeat sequences capable of independently and strongly binding to the ankyrin repeats of Espin1, revealing an unexpected Myo3-mediated cross-linking mechanism of Espin1. The structures of Myo3 in complex with Espin1 not only elucidate the mechanism of the binding, but also reveal a Myo3-induced release of Espin1 auto-inhibition mechanism. We also provide evidence that Myo3-mediated cross-linking can further promote actin fiber bundling activity of Espin1.

  14. Effect of nucleotides and actin on the intramolecular cross-linking of myosin subfragment-1.

    PubMed

    Blotnick, E; Muhlrad, A

    1994-06-07

    The heavy chain of myosin subfragment-1 (S1) is cleaved by limited trypsinolysis into three fragments, 27, 50, and 20 kDa--aligned in this order from the N-terminus. The tertiary structure of the molecule is essentially not affected by trypsinolysis. The spatial relations between the various regions of the molecule and the nucleotide- and actin-induced intramolecular movements were studied by cross-linking tryptic S1 with N-(ethoxycarbonyl)-2-ethoxy-1,2-dihydroquinoline (EEDQ), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (EDC), phenylenediglyoxal (PDG), and glutaraldehyde. The formation of cross-linked products was monitored by SDS-PAGE, using the fluorescent probes 9-anthronitrile and N-(iodoacetyl)-N'-(5-sulfo-1-naphthyl)ethylenediamine (IAEDANS), which specifically label the 27- and 20-kDa fragments, respectively. The reaction with the cross-linkers leads to the formation of 50-kDa/20-kDa, 27-kDa/20-kDa, 27-kDa/50-kDa, and 20-kDa/light chain cross-linked products. Of these, the most intensive was the formation of the 50-kDa/20-kDa products, which appeared as a doublet on the SDS-PAGE with all the cross-linkers. This indicates that the interface between the two fragments is rather extended. The presence of MgATP or MgADP promoted the formation of the 20-kDa/50-kDa cross-linked products, especially with the lower electrophoretic mobility band, when EEDQ was used as a cross-linker. With PDG as a cross-linker, MgATP also affected the cross-link formation between the 20-kDa fragment and the light chains whereas it had no influence on the formation of other products. On the other hand, the effect of actin on the cross-linking with the various cross-linkers was quite extensive, and it was manifested in the reduction of cross-link formation between the various S1 domains. It is concluded that both nucleotides and actin induce intramolecular movements in S1 and that the nucleotide-induced movements are more restricted than those induced by actin, which extend to larger

  15. Myosin III-mediated cross-linking and stimulation of actin bundling activity of Espin

    PubMed Central

    Liu, Haiyang; Li, Jianchao; Raval, Manmeet H; Yao, Ningning; Deng, Xiaoying; Lu, Qing; Nie, Si; Feng, Wei; Wan, Jun; Yengo, Christopher M; Liu, Wei; Zhang, Mingjie

    2016-01-01

    Class III myosins (Myo3) and actin-bundling protein Espin play critical roles in regulating the development and maintenance of stereocilia in vertebrate hair cells, and their defects cause hereditary hearing impairments. Myo3 interacts with Espin1 through its tail homology I motif (THDI), however it is not clear how Myo3 specifically acts through Espin1 to regulate the actin bundle assembly and stabilization. Here we discover that Myo3 THDI contains a pair of repeat sequences capable of independently and strongly binding to the ankyrin repeats of Espin1, revealing an unexpected Myo3-mediated cross-linking mechanism of Espin1. The structures of Myo3 in complex with Espin1 not only elucidate the mechanism of the binding, but also reveal a Myo3-induced release of Espin1 auto-inhibition mechanism. We also provide evidence that Myo3-mediated cross-linking can further promote actin fiber bundling activity of Espin1. DOI: http://dx.doi.org/10.7554/eLife.12856.001 PMID:26785147

  16. Prestressed F-actin networks cross-linked by hinged filamins replicate mechanical properties of cells

    NASA Astrophysics Data System (ADS)

    Gardel, M. L.; Nakamura, F.; Hartwig, J. H.; Crocker, J. C.; Stossel, T. P.; Weitz, D. A.

    2006-02-01

    We show that actin filaments, shortened to physiological lengths by gelsolin and cross-linked with recombinant human filamins (FLNs), exhibit dynamic elastic properties similar to those reported for live cells. To achieve elasticity values of comparable magnitude to those of cells, the in vitro network must be subjected to external prestress, which directly controls network elasticity. A molecular requirement for the strain-related behavior at physiological conditionsis a flexible hinge found in FLNa and some FLNb molecules. Basic physical properties of the in vitro filamin-F-actin network replicate the essential mechanical properties of living cells. This physical behavior could accommodate passive deformation and internal organelle trafficking at low strains yet resist externally or internally generated high shear forces. cytoskeleton | cell mechanics | nonlinear rheology

  17. Relating microstructure to rheology of a bundled and cross-linked F-actin network in vitro

    NASA Astrophysics Data System (ADS)

    Shin, J. H.; Gardel, M. L.; Mahadevan, L.; Matsudaira, P.; Weitz, D. A.

    2004-06-01

    The organization of individual actin filaments into higher-order structures is controlled by actin-binding proteins (ABPs). Although the biological significance of the ABPs is well documented, little is known about how bundling and cross-linking quantitatively affect the microstructure and mechanical properties of actin networks. Here we quantify the effect of the ABP scruin on actin networks by using imaging techniques, cosedimentation assays, multiparticle tracking, and bulk rheology. We show how the structure of the actin network is modified as the scruin concentration is varied, and we correlate these structural changes to variations in the resultant network elasticity.

  18. Gln-41 is intermolecularly cross-linked to Lys-113 in F-actin by N-(4-azidobenzoyl)-putrescine.

    PubMed Central

    Hegyi, G.; Michel, H.; Shabanowitz, J.; Hunt, D. F.; Chatterjie, N.; Healy-Louie, G.; Elzinga, M.

    1992-01-01

    The bifunctional reagent N-(4-azidobenzoyl)-putrescine was synthesized and covalently bound to rabbit skeletal muscle actin. The incorporation was mediated by guinea pig liver transglutaminase under conditions similar to those described by Takashi (1988, Biochemistry 27, 938-943); up to 0.5 M/M were incorporated into G-actin, whereas F-actin was refractory to incorporation. Peptide fractionation showed that at least 90% of the label was bound to Gln-41. The labeled G-actin was polymerized, and irradiation of the F-actin led to covalent intermolecular cross-linking. A cross-linked peptide complex was isolated from a tryptic digest of the cross-linked actin in which digestion was limited to arginine; sequence analysis as well as mass spectrometry indicated that the linked peptides contained residues 40-62 and residues 96-116, and that the actual cross-link was between Gln-41 and Lys-113. Thus the gamma-carboxyl group of Gln-41 must be within 10.7 A of the side chain (probably the amino group) of Lys-113 in an adjacent actin monomer. In the atomic model for F-actin proposed by Holmes et al. (1990, Nature 347, 44-49), the alpha-carbons of these residues in adjacent monomers along the two-start helices are sufficiently close to permit cross-linking of their side chains, and, pending atomic resolution of the side chains, the results presented here seem to support the proposed model. PMID:1363931

  19. Cytokines and growth factors cross-link heparan sulfate

    PubMed Central

    Migliorini, Elisa; Thakar, Dhruv; Kühnle, Jens; Sadir, Rabia; Dyer, Douglas P.; Li, Yong; Sun, Changye; Volkman, Brian F.; Handel, Tracy M.; Coche-Guerente, Liliane; Fernig, David G.; Lortat-Jacob, Hugues; Richter, Ralf P.

    2015-01-01

    The glycosaminoglycan heparan sulfate (HS), present at the surface of most cells and ubiquitous in extracellular matrix, binds many soluble extracellular signalling molecules such as chemokines and growth factors, and regulates their transport and effector functions. It is, however, unknown whether upon binding HS these proteins can affect the long-range structure of HS. To test this idea, we interrogated a supramolecular model system, in which HS chains grafted to streptavidin-functionalized oligoethylene glycol monolayers or supported lipid bilayers mimic the HS-rich pericellular or extracellular matrix, with the biophysical techniques quartz crystal microbalance (QCM-D) and fluorescence recovery after photobleaching (FRAP). We were able to control and characterize the supramolecular presentation of HS chains—their local density, orientation, conformation and lateral mobility—and their interaction with proteins. The chemokine CXCL12α (or SDF-1α) rigidified the HS film, and this effect was due to protein-mediated cross-linking of HS chains. Complementary measurements with CXCL12α mutants and the CXCL12γ isoform provided insight into the molecular mechanism underlying cross-linking. Fibroblast growth factor 2 (FGF-2), which has three HS binding sites, was also found to cross-link HS, but FGF-9, which has just one binding site, did not. Based on these data, we propose that the ability to cross-link HS is a generic feature of many cytokines and growth factors, which depends on the architecture of their HS binding sites. The ability to change matrix organization and physico-chemical properties (e.g. permeability and rigidification) implies that the functions of cytokines and growth factors may not simply be confined to the activation of cognate cellular receptors. PMID:26269427

  20. F-actin cross-linking enhances the stability of force generation in disordered actomyosin networks

    NASA Astrophysics Data System (ADS)

    Jung, Wonyeong; Murrell, Michael P.; Kim, Taeyoon

    2015-12-01

    Myosin molecular motors and actin cross-linking proteins (ACPs) are known to mediate the generation and transmission of mechanical forces within the cortical F-actin cytoskeleton that drive major cellular processes such as cell division and migration. However, how motors and ACPs interact collectively over diverse timescales to modulate the time-dependent mechanical properties of the cytoskeleton remains unclear. In this study, we present a three-dimensional agent-based computational model of the cortical actomyosin network to quantitatively determine the effects of motor activity and the density and kinetics of ACPs on the accumulation and maintenance of mechanical tension within a disordered actomyosin network. We found that motors accumulate large stress quickly by behaving as temporary cross-linkers although this stress is relaxed over time unless there are sufficient passive ACPs to stabilize the network. Stabilization by ACPs helps motors to generate forces up to their maximum potential, leading to significant enhancement of the efficiency and stability of stress generation. Thus, we demonstrated that the force-dependent kinetics of ACP dissociation plays a critical role for the accumulation and sustainment of stress and the structural remodeling of networks.

  1. F-actin structure destabilization and DNase I binding loop: fluctuations mutational cross-linking and electron microscopy analysis of loop states and effects on F-actin.

    PubMed

    Oztug Durer, Zeynep A; Diraviyam, Karthikeyan; Sept, David; Kudryashov, Dmitri S; Reisler, Emil

    2010-01-22

    The conformational dynamics of filamentous actin (F-actin) is essential for the regulation and functions of cellular actin networks. The main contribution to F-actin dynamics and its multiple conformational states arises from the mobility and flexibility of the DNase I binding loop (D-loop; residues 40-50) on subdomain 2. Therefore, we explored the structural constraints on D-loop plasticity at the F-actin interprotomer space by probing its dynamic interactions with the hydrophobic loop (H-loop), the C-terminus, and the W-loop via mutational disulfide cross-linking. To this end, residues of the D-loop were mutated to cysteines on yeast actin with a C374A background. These mutants showed no major changes in their polymerization and nucleotide exchange properties compared to wild-type actin. Copper-catalyzed disulfide cross-linking was investigated in equimolar copolymers of cysteine mutants from the D-loop with either wild-type (C374) actin or mutant S265C/C374A (on the H-loop) or mutant F169C/C374A (on the W-loop). Remarkably, all tested residues of the D-loop could be cross-linked to residues 374, 265, and 169 by disulfide bonds, demonstrating the plasticity of the interprotomer region. However, each cross-link resulted in different effects on the filament structure, as detected by electron microscopy and light-scattering measurements. Disulfide cross-linking in the longitudinal orientation produced mostly no visible changes in filament morphology, whereas the cross-linking of D-loop residues >45 to the H-loop, in the lateral direction, resulted in filament disruption and the presence of amorphous aggregates on electron microscopy images. A similar aggregation was also observed upon cross-linking the residues of the D-loop (>41) to residue 169. The effects of disulfide cross-links on F-actin stability were only partially accounted for by the simulations of current F-actin models. Thus, our results present evidence for the high level of conformational plasticity in

  2. Reversible mechano-memory in sheared cross-linked actin networks

    NASA Astrophysics Data System (ADS)

    Majumdar, Sayantan; Gardel, Margaret L.

    2015-03-01

    Is it possible to control the shear modulus of a material mechanically? We reconstitute a network of actin filaments cross-linked with Filamin A and show that the system has remarkable property to respond under shear in a deformation history dependent manner. When a large shear stress pulse is applied to the system, the system remembers the direction of deformation long after the stress pulse is removed. For the next loading cycle, shear response of the system becomes anisotropic; if the applied pulse direction is same as the previous one, the system behaves like a viscoelastic solid but a transient liquefaction is observed if the pulse direction is reversed. Imaging and normal force measurements under shear suggest that this anisotropic response comes from stretching and bending dominated deformation directions induced by the large shear deformation giving rise to a direction dependent mechano-memory. The long time scale over which the memory effect persists has relevance in various deformations in cellular and multicellular systems. S.M. acknowledges support from a Kadanoff-Rice Post Doctoral fellowship from MRSEC, University of Chicago.

  3. Avalanches, hardening and softening in dense cross-linked actin networks

    NASA Astrophysics Data System (ADS)

    Astrom, Jan; Kumar, Sunil; Vattulainen, Ilpo; Karttunen, Mikko

    2008-03-01

    Actin filament networks enable the cytoskeleton to adjust to internal and external forcing. These active networks can adapt to changes by dynamically adjusting their crosslinks. Here, we study actin filaments as elastic fibers having finite dimensions. We employ a full three-dimensional model to study the elastic properties of actin networks by computer simulations. We model a dense actin network with the crosslinks being approximately 1μm apart. The results show that dense actin networks, without any pre-straining, are characterized by (a) strain hardening without entropic elasticity, (b) 'viscotic' hysteresis in the case of strong crosslinks, (c) avalanches of crosslink slippage leading to strain softening in the case of breakable crosslinks, and (d) spontaneous formation of stress fibers in the case of active crosslink formation and destruction. We will discuss the relation to recent experimental observations.

  4. Passive and active microrheology for cross-linked F-actin networks in vitro.

    PubMed

    Lee, Hyungsuk; Ferrer, Jorge M; Nakamura, Fumihiko; Lang, Matthew J; Kamm, Roger D

    2010-04-01

    Actin filament (F-actin) is one of the dominant structural constituents in the cytoskeleton. Orchestrated by various actin-binding proteins (ABPs), F-actin is assembled into higher-order structures such as bundles and networks that provide mechanical support for the cell and play important roles in numerous cellular processes. Although mechanical properties of F-actin networks have been extensively studied, the underlying mechanisms for network elasticity are not fully understood, in part because different measurements probe different length and force scales. Here, we developed both passive and active microrheology techniques using optical tweezers to estimate the mechanical properties of F-actin networks at a length scale comparable to cells. For the passive approach we tracked the motion of a thermally fluctuating colloidal sphere to estimate the frequency-dependent complex shear modulus of the network. In the active approach, we used an optical trap to oscillate an embedded microsphere and monitored the response in order to obtain network viscoelasticity over a physiologically relevant force range. While both active and passive measurements exhibit similar results at low strain, the F-actin network subject to high strain exhibits non-linear behavior which is analogous to the strain-hardening observed in macroscale measurements. Using confocal and total internal reflection fluorescent microscopy, we also characterize the microstructure of reconstituted F-actin networks in terms of filament length, mesh size and degree of bundling. Finally, we propose a model of network connectivity by investigating the effect of filament length on the mechanical properties and structure.

  5. Phosphorylation of actin-binding protein (ABP-280; filamin) by tyrosine kinase p56lck modulates actin filament cross-linking.

    PubMed

    Pal Sharma, C; Goldmann, Wolfgang H

    2004-01-01

    Actin-binding protein (ABP-280; filamin) is a phosphoprotein present in the periphery of the cytoplasm where it can cross-link actin filaments, associate with lipid membranes, and bind to membrane surface receptors. Given its function and localization in the cell, we decided to investigate the possibility of whether it serves as substrate for p56lck, a lymphocyte-specific member of the src family of protein tyrosine kinases associated with cell surface glycoproteins. The interaction of p56lck with membrane glycoproteins is important for cell development and functional activation. Here, we show that purified p56lck interacts and catalyzes in vitro kinase reactions. Tyrosine phosphorylation by p56lck is restricted to a single peptide of labeled ABP-280 shown by protease digest. The addition of phorbol ester to cells results in the inhibition of phosphorylation of ABP-280 by p56lck. These results show a decrease in phosphorylation suggesting conformationally induced regulation. Dynamic light scattering confirmed increased actin filament cross-linking due to phosphorylation of ABP-280 by p56lck.

  6. A 27,000-D core of the Dictyostelium 34,000-D protein retains Ca(2+)- regulated actin cross-linking but lacks bundling activity

    PubMed Central

    1993-01-01

    Actin cross-linking proteins are important for formation of isotropic F- actin networks and anisotropic bundles of filaments in the cytoplasm of eucaryotic cells. A 34,000-D protein from the cellular slime mold Dictyostelium discoideum mediates formation of actin bundles in vitro, and is specifically incorporated into filopodia. The actin cross- linking activity of this protein is inhibited by the presence of micromolar calcium. A 27,000-D fragment obtained by digestion with alpha-chymotrypsin lacks the amino-terminal six amino acids and the carboxyl-terminal 7,000 D of the intact polypeptide. The 27,000-D fragment retains F-actin binding activity assessed by cosedimentation assays and by 125I-[F-actin] blot overlay technique, F-actin cross- linking activity as assessed by viscometry, and calcium binding activity. Ultrastructural analyses indicate that the 27,000-D fragment is deficient in the bundling activity characteristic of the intact 34,000-D protein. Actin filaments are aggregated into microdomains but not bundle in the presence of the 27,000-D fragment. A polarized light scattering assay was used to demonstrate that the 34,000-D protein increases the orientational correlation among F-actin filaments. The 27,000-D fragment does not increase the orientation of the actin filaments as assessed by this technique. A terminal segment(s) of the 34,000-D protein, lacking in the 27,000-D fragment, contributes significantly to the ability to cross-link actin filaments into bundles. PMID:8436589

  7. Crystal structure of the VgrG1 actin cross-linking domain of the Vibrio cholerae type VI secretion system.

    PubMed

    Durand, Eric; Derrez, Estelle; Audoly, Gilles; Spinelli, Silvia; Ortiz-Lombardia, Miguel; Raoult, Didier; Cascales, Eric; Cambillau, Christian

    2012-11-02

    Vibrio cholerae is the cause of the diarrheal disease cholera. V. cholerae produces RtxA, a large toxin of the MARTX family, which is targeted to the host cell cytosol, where its actin cross-linking domain (ACD) cross-links G-actin, leading to F-actin depolymerization, cytoskeleton rearrangements, and cell rounding. These effects on the cytoskeleton prevent phagocytosis and bacterial engulfment by macrophages, thus preventing V. cholerae clearance from the gut. The V. cholerae Type VI secretion-associated VgrG1 protein also contains a C-terminal ACD, which shares 61% identity with MARTX ACD and has been shown to covalently cross-link G-actin. Here, we purified the VgrG1 C-terminal domain and determined its crystal structure. The VgrG1 ACD exhibits a V-shaped three-dimensional structure, formed of 12 β-strands and nine α-helices. Its active site comprises five residues that are conserved in MARTX ACD toxin, within a conserved area of ∼10 Å radius. We showed that less than 100 ACD molecules are sufficient to depolymerize the actin filaments of a fibroblast cell in vivo. Mutagenesis studies confirmed that Glu-16 is critical for the F-actin depolymerization function. Co-crystals with divalent cations and ATP reveal the molecular mechanism of the MARTX/VgrG toxins and offer perspectives for their possible inhibition.

  8. Cross-linking myosin subfragment 1 Cys-697 and Cys-707 modifies ATP and actin binding site interactions.

    PubMed Central

    Kirshenbaum, K.; Papp, S.; Highsmith, S.

    1993-01-01

    Skeletal muscle myosin is an enzyme that interacts allosterically with MgATP and actin to transduce the chemical energy from ATP hydrolysis into work. By modifying myosin structure, one can change this allosteric interaction and gain insight into its mechanism. Chemical cross-linking with N,N'-p-phenylenedimaleimide (pPDM) of Cys-697 to Cys-707 of the myosin-ADP complex eliminates activity and produces a species that resembles myosin with ATP bound (Burke et al., 1976). Nucleotide-free pPDM-modified myosin subfragment 1 (S1) was prepared, and its structural and allosteric properties were investigated by comparing the nucleotide and actin interactions of S1 to those of pPDM-S1. The structural properties of the nucleotide-free pPDM-S1 are different from those of S1 in several respects. pPDM-S1 intrinsic tryptophan fluorescence intensity is reduced 28%, indicating a large increase of an internal quenching reaction (the fluorescence intensity of the related vanadate complex of S1, S1-MgADP-Vi, is reduced by a similar degree). Tryptophan fluorescence anisotropy increases from 0.168 for S1 to 0.192 for pPDM-S1, indicating that the unquenched tryptophan population in pPDM-S1 has reduced local freedom of motion. The actin affinity of pPDM-S1 is over 6,000-fold lower than that of S1, and the absolute value of the product of the net effective electric charges at the acto-S1 interface is reduced from 8.1 esu2 for S1 to 1.6 esu2 for pPDM-S1. In spite of these changes, the structural response of pPDM-S1 to nucleotide and the allosteric communication between its ATP and actin sites remain intact. Compared to pPDM-S1, the fluorescence intensity of pPDM-S1 *MgADP is increased 50%(compared to 8 and 31% increases, respectively, for MgADP and MgATP binding to S1). Compared to acto-pPDM-S1, the absolute value of the product of the net effective electric charge at the actin binding interface of acto-pPDM-S1 *MgADP increases 7.3 esu2 (compared to a 0.9 esu2 decrease and an 11.0 esu2

  9. TGFβ2 Induces the Formation of Cross-Linked Actin Networks (CLANs) in Human Trabecular Meshwork Cells Through the Smad and Non-Smad Dependent Pathways

    PubMed Central

    Montecchi-Palmer, Michela; Bermudez, Jaclyn Y.; Webber, Hannah C.; Patel, Gaurang C.; Clark, Abbot F.; Mao, Weiming

    2017-01-01

    Purpose Increased intraocular pressure results from increased aqueous humor (AH) outflow resistance at the trabecular meshwork (TM) due to pathologic changes including the formation of cross-linked actin networks (CLANs). Transforming growth factor β2 (TGFβ2) is elevated in the AH and TM of primary open angle glaucoma (POAG) patients and induces POAG-associated TM changes, including CLANs. We determined the role of individual TGFβ2 signaling pathways in CLAN formation. Methods Cultured nonglaucomatous human TM (NTM) cells were treated with control or TGFβ2, with or without the inhibitors of TGFβ receptor, Smad3, c-Jun N-terminal kinases (JNK), extracellular signal regulated kinase (ERK), P38, or Rho-associated protein kinase (ROCK). NTM cells were cotreated with TGFβ2 plus inhibitors for 10 days or pretreated with TGFβ2 for 10 days followed by 1-hour inhibitor treatment. NTM cells were immunostained with phalloidin-Alexa-488 and 4′,6-diamidino-2-phenylindole (DAPI). Data were analyzed using 1-way ANOVA and Dunnett's post hoc test. Results TGFβ2 significantly induced CLAN formation (n = 6 to 12, P < 0.05), which was completely inhibited by TGFβ receptor, Smad3, and ERK inhibitors, as well as completely or partially inhibited by JNK, P38, and ROCK inhibitors, depending on cell strains. One-hour exposure to ROCK inhibitor completely resolved formed CLANs (P < 0.05), whereas TGFβ receptor, Smad3 inhibitor, and ERK inhibitors resulted in partial or complete resolution. The JNK and P38 inhibitors showed partial or no resolution. Among these inhibitors, the ROCK inhibitor was the most disruptive to the actin stress fibers, whereas ERK inhibition showed the least disruption. Conclusions TGFβ2-induced CLANs in NTM cells were prevented and resolved using various pathway inhibitors. Apart from CLAN inhibition, some of these inhibitors also had different effects on actin stress fibers. PMID:28241317

  10. Why Are Two Different Cross-linkers Necessary for Actin Bundle Formation In Vivo and What Does Each Cross-link Contribute?

    PubMed Central

    Tilney, Lewis G.; Connelly, Patricia S.; Vranich, Kelly A.; Shaw, Michael K.; Guild, Gregory M.

    1998-01-01

    In developing Drosophila bristles two species of cross-linker, the forked proteins and fascin, connect adjacent actin filaments into bundles. Bundles form in three phases: (a) tiny bundles appear; (b) these bundles aggregate into larger bundles; and (c) the filaments become maximally cross-linked by fascin. In mutants that completely lack forked, aggregation of the bundles does not occur so that the mature bundles consist of <50 filaments versus ∼700 for wild type. If the forked concentration is genetically reduced to half the wild type, aggregation of the tiny bundles occurs but the filaments are poorly ordered albeit with small patches of fascin cross-linked filaments. In mutants containing an excess of forked, all the bundles tend to aggregate and the filaments are maximally crossbridged by fascin. Alternatively, if fascin is absent, phases 1 and 2 occur normally but the resultant bundles are twisted and the filaments within them are poorly ordered. By extracting fully elongated bristles with potassium iodide which removes fascin but leaves forked, the bundles change from being straight to twisted and the filaments within them become poorly ordered. From these observations we conclude that (a) forked is used early in development to aggregate the tiny bundles into larger bundles; and (b) forked facilitates fascin entry into the bundles to maximally cross-link the actin filaments into straight, compact, rigid bundles. Thus, forked aligns the filaments and then directs fascin binding so that inappropriate cross-linking does not occur. PMID:9763425

  11. Temperature-induced sol-gel transition and microgel formation in α-actinin cross-linked actin networks: A rheological study

    NASA Astrophysics Data System (ADS)

    Tempel, M.; Isenberg, G.; Sackmann, E.

    1996-08-01

    We have studied the sol-gel transition, the viscoelastic and the structural properties of networks constituted of semiflexible actin filaments cross-linked by α-actinin. Cross-linking was regulated in a reversible way by varying the temperature through the association-dissociation equilibrium of the actin-α-actinin system. Viscoelastic parameters [shear storage modulus G'(ω), phase shift tan(Φ)(ω), creep compliance J(t)] were measured as a function of temperature and actin-to-cross-linker ratio by a magnetically driven rotating disc rheometer. G'(ω) and tan(Φ)(ω) were studied at a frequency ω corresponding to the elastic plateau regime of the G'(ω) versus ω spectrum of the purely entangled solution. The microstructure of the networks was viewed by negative staining electron microscopy (EM). The phase shift tan(Φ) (or equivalently the viscosity η) diverges and reaches a maximum when approaching the apparent gel point from lower and higher temperatures, and the maximum defines the gel point (temperature Tg). The elastic plateau modulus G'N diverges at temperatures beyond this gel point TTg. The cross-linking transition (corresponding to a sol-gel transition at zero frequency) is interpreted in terms of a percolation model and the divergence of G'N at Tcross-linking transition (T>Tg), (2) that microscopic segregation takes place at T<=Tg leading to local formation of clusters (a state termed microgel), and (3) that at low actin-α-actinin ratios (rAα<=10) and low temperatures (T<=10 °C) macroscopic segregation into bundles of cross-linked actin filaments and a diluted solution of actin filaments is observed. The three regimes of network structure are represented by an

  12. Force generation and work production by covalently cross-linked actin-myosin cross-bridges in rabbit muscle fibers.

    PubMed Central

    Bershitsky, S Y; Tsaturyan, A K

    1995-01-01

    To separate a fraction of the myosin cross-bridges that are attached to the thin filaments and that participate in the mechanical responses, muscle fibers were cross-linked with 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide and then immersed in high-salt relaxing solution (HSRS) of 0.6 M ionic strength for detaching the unlinked myosin heads. The mechanical properties and force-generating ability of the cross-linked cross-bridges were tested with step length changes (L-steps) and temperature jumps (T-jumps) from 6-10 degrees C to 30-40 degrees C. After partial cross-linking, when instantaneous stiffness in HSRS was 25-40% of that in rigor, the mechanical behavior of the fibers was similar to that during active contraction. The kinetics of the T-jump-induced tension transients as well as the rate of the fast phase of tension recovery after length steps were close to those in unlinked fibers during activation. Under feedback force control, the T-jump initiated fiber shortening by up to 4 nm/half-sarcomere. Work produced by a cross-linked myosin head after the T-jump was up to 30 x 10(-21) J. When the extent of cross-linking was increased and fiber stiffness in HSRS approached that in rigor, the fibers lost their viscoelastic properties and ability to generate force with a rise in temperature. PMID:8519956

  13. Mammalian class I myosin, Myo1b, is monomeric and cross-links actin filaments as determined by hydrodynamic studies and electron microscopy.

    PubMed

    Stafford, Walter F; Walker, Matt L; Trinick, John A; Coluccio, Lynne M

    2005-01-01

    The class I myosin, Myo1b, is a calmodulin- and actin-associated molecular motor widely expressed in mammalian tissues. Analytical ultracentrifugation studies indicate that Myo1b purified from rat liver has a Stokes radius of 6.7 nm and a sedimentation coefficient, s(20,w), of 7.0 S with a predicted molar mass of 213 kg/mol. These results indicate that Myo1b is monomeric and consists primarily of a splice variant having five associated calmodulins. Molecular modeling based on the analytical ultracentrifugation studies are supported by electron microscopy studies that depict Myo1b as a single-headed, tadpole-shaped molecule with outer dimensions of 27.9 x 4.0 nm. Above a certain Myo1b/actin ratio, Myo1b bundles actin filaments presumably by virtue of a second actin-binding site. These studies provide new information regarding the oligomeric state and morphology of Myo1b and support a model in which Myo1b cross-links actin through a cryptic actin-binding site.

  14. Sequence and domain organization of scruin, an actin-cross-linking protein in the acrosomal process of Limulus sperm

    PubMed Central

    1995-01-01

    The acrosomal process of Limulus sperm is an 80-microns long finger of membrane supported by a crystalline bundle of actin filaments. The filaments in this bundle are crosslinked by a 102-kD protein, scruin present in a 1:1 molar ratio with actin. Recent image reconstruction of scruin decorated actin filaments at 13-A resolution shows that scruin is organized into two equally sized domains bound to separate actin subunits in the same filament. We have cloned and sequenced the gene for scruin from a Limulus testes cDNA library. The deduced amino acid sequence of scruin reflects the domain organization of scruin: it consists of a tandem pair of homologous domains joined by a linker region. The domain organization of scruin is confirmed by limited proteolysis of the purified acrosomal process. Three different proteases cleave the native protein in a 5-kD Protease-sensitive region in the middle of the molecule to generate an NH2-terminal 47-kD and a COOH-terminal 56-kD protease-resistant domains. Although the protein sequence of scruin has no homology to any known actin-binding protein, it has similarities to several proteins, including four open reading frames of unknown function in poxviruses, as well as kelch, a Drosophila protein localized to actin-rich ring canals. All proteins that show homologies to scruin are characterized by the presence of an approximately 50-amino acid residue motif that is repeated between two and seven times. Crystallographic studies reveal this motif represents a four beta-stranded fold that is characteristic of the "superbarrel" structural fold found in the sialidase family of proteins. These results suggest that the two domains of scruin seen in EM reconstructions are superbarrel folds, and they present the possibility that other members of this family may also bind actin. PMID:7822422

  15. Interactions among a Fimbrin, a Capping Protein, and an Actin-depolymerizing Factor in Organization of the Fission Yeast Actin Cytoskeleton

    PubMed Central

    Nakano, Kentaro; Satoh, Kazuomi; Morimatsu, Akeshi; Ohnuma, Masaaki; Mabuchi, Issei

    2001-01-01

    We report studies of the fission yeast fimbrin-like protein Fim1, which contains two EF-hand domains and two actin-binding domains (ABD1 and ABD2). Fim1 is a component of both F-actin patches and the F-actin ring, but not of F-actin cables. Fim1 cross-links F-actin in vitro, but a Fim1 protein lacking either EF-hand domains (Fim1A12) or both the EF-hand domains and ABD1 (Fim1A2) has no actin cross-linking activity. Overexpression of Fim1 induced the formation of F-actin patches throughout the cell cortex, whereas the F-actin patches disappear in cells overexpressing Fim1A12 or Fim1A2. Thus, the actin cross-linking activity of Fim1 is probably important for the formation of F-actin patches. The overexpression of Fim1 also excluded the actin-depolymerizing factor Adf1 from the F-actin patches and inhibited the turnover of actin in these structures. Thus, Fim1 may function in stabilizing the F-actin patches. We also isolated the gene encoding Acp1, a subunit of the heterodimeric F-actin capping protein. fim1 acp1 double null cells showed more severe defects in the organization of the actin cytoskeleton than those seen in each single mutant. Thus, Fim1 and Acp1 may function in a similar manner in the organization of the actin cytoskeleton. Finally, genetic studies suggested that Fim1 may function in cytokinesis in cooperation with Cdc15 (PSTPIP) and Rng2 (IQGAP), respectively. PMID:11694585

  16. Type VI secretion system translocates a phage tail spike-like protein into target cells where it cross-links actin.

    PubMed

    Pukatzki, Stefan; Ma, Amy T; Revel, Andrew T; Sturtevant, Derek; Mekalanos, John J

    2007-09-25

    Genes encoding type VI secretion systems (T6SS) are widely distributed in pathogenic Gram-negative bacterial species. In Vibrio cholerae, T6SS have been found to secrete three related proteins extracellularly, VgrG-1, VgrG-2, and VgrG-3. VgrG-1 can covalently cross-link actin in vitro, and this activity was used to demonstrate that V. cholerae can translocate VgrG-1 into macrophages by a T6SS-dependent mechanism. Protein structure search algorithms predict that VgrG-related proteins likely assemble into a trimeric complex that is analogous to that formed by the two trimeric proteins gp27 and gp5 that make up the baseplate "tail spike" of Escherichia coli bacteriophage T4. VgrG-1 was shown to interact with itself, VgrG-2, and VgrG-3, suggesting that such a complex does form. Because the phage tail spike protein complex acts as a membrane-penetrating structure as well as a conduit for the passage of DNA into phage-infected cells, we propose that the VgrG components of the T6SS apparatus may assemble a "cell-puncturing device" analogous to phage tail spikes to deliver effector protein domains through membranes of target host cells.

  17. Thiouracil cross-linking mass spectrometry: a cell-based method to identify host factors involved in viral amplification.

    PubMed

    Lenarcic, Erik M; Landry, Dori M; Greco, Todd M; Cristea, Ileana M; Thompson, Sunnie R

    2013-08-01

    Eukaryotic RNA viruses are known to utilize host factors; however, the identity of these factors and their role in the virus life cycle remain largely undefined. Here, we report a method to identify proteins bound to the viral RNA during amplification in cell culture: thiouracil cross-linking mass spectrometry (TUX-MS). TUX-MS relies on incorporation of a zero-distance cross-linker into the viral RNA during infection. Proteins bound to viral RNA are cross-linked prior to cell lysis, purified, and identified using mass spectrometry. Using the TUX-MS method, an unbiased screen for poliovirus (PV) host factors was conducted. All host and viral proteins that are known to interact with the poliovirus RNA were identified. In addition, TUX-MS identified an additional 66 host proteins that have not been previously described in poliovirus amplification. From these candidates, eight were selected and validated. Furthermore, we demonstrate that small interfering RNA (siRNA)-mediated knockdown of two of these uncharacterized host factors results in either a decrease in copy number of positive-stranded RNA or a decrease in PV translation. These data demonstrate that TUX-MS is a robust, unbiased method to identify previously unknown host cell factors that influence virus growth. This method is broadly applicable to a range of RNA viruses, such as flaviviruses, alphaviruses, picornaviruses, bunyaviruses, and coronaviruses.

  18. Effects of binding factors on structural elements in F-actin.

    PubMed

    Scoville, Damon; Stamm, John D; Altenbach, Christian; Shvetsov, Alexander; Kokabi, Kaveh; Rubenstein, Peter A; Hubbell, Wayne L; Reisler, Emil

    2009-01-20

    Understanding the dynamics of the actin filament is essential to a detailed description of their interactions and role in the cell. Previous studies have linked the dynamic properties of actin filaments (F-actin) to three structural elements contributing to a hydrophobic pocket, namely, the hydrophobic loop, the DNase I binding loop, and the C-terminus. Here, we examine how these structural elements are influenced by factors that stabilize or destabilize F-actin, using site-directed spin-labeled (SDSL) electron paramagnetic resonance (EPR), fluorescence, and cross-linking techniques. Specifically, we employ cofilin, an actin destabilizing protein that binds and severs filaments, and phalloidin, a fungal toxin that binds and stabilizes F-actin. We find that cofilin shifts both the DNase I binding loop and the hydrophobic loop away from the C-terminus in F-actin, as demonstrated by weakened spin-spin interactions, and alters the environment of spin probes on residues of these two loops. In contrast, although phalloidin strongly stabilizes F-actin, it causes little or no local change in the environment of the loop residues. This indicates that the stabilizing effect of phalloidin is achieved mainly through constraining structural fluctuations in F-actin and suggests that factors and interactions that control these fluctuations have an important role in the cytoskeleton dynamics.

  19. Molecular Basis of Actin Nucleation Factor Cooperativity

    PubMed Central

    Zeth, Kornelius; Pechlivanis, Markos; Samol, Annette; Pleiser, Sandra; Vonrhein, Clemens; Kerkhoff, Eugen

    2011-01-01

    The distinct actin nucleation factors of the Spir and formin subgroup families cooperate in actin nucleation. The Spir/formin cooperativity has been identified to direct two essential steps in mammalian oocyte maturation, the asymmetric spindle positioning and polar body extrusion during meiosis. Understanding the nature and regulation of the Spir/Fmn cooperation is an important requirement to comprehend mammalian reproduction. Recently we dissected the structural elements of the Spir and Fmn family proteins, which physically link the two actin nucleation factors. The trans-regulatory interaction is mediated by the Spir kinase non-catalytic C-lobe domain (KIND) and the C-terminal formin Spir interaction motif (FSI). The interaction inhibits formin nucleation activity and enhances the Spir activity. To get insights into the molecular mechanism of the Spir/Fmn interaction, we determined the crystal structure of the KIND domain alone and in complex with the C-terminal Fmn-2 FSI peptide. Together they confirm the proposed structural homology of the KIND domain to the protein kinase fold and reveal the basis of the Spir/formin interaction. The complex structure showed a large interface with conserved and positively charged residues of the Fmn FSI peptide mediating major contacts to an acidic groove on the surface of KIND. Protein interaction studies verified the electrostatic nature of the interaction. The data presented here provide the molecular basis of the Spir/formin interaction and give a first structural view into the mechanisms of actin nucleation factor cooperativity. PMID:21705804

  20. Factors essential for L,D-transpeptidase-mediated peptidoglycan cross-linking and β-lactam resistance in Escherichia coli

    PubMed Central

    Hugonnet, Jean-Emmanuel; Mengin-Lecreulx, Dominique; Monton, Alejandro; den Blaauwen, Tanneke; Carbonnelle, Etienne; Veckerlé, Carole; Brun, Yves, V.; van Nieuwenhze, Michael; Bouchier, Christiane; Tu, Kuyek; Rice, Louis B; Arthur, Michel

    2016-01-01

    The target of β-lactam antibiotics is the D,D-transpeptidase activity of penicillin-binding proteins (PBPs) for synthesis of 4→3 cross-links in the peptidoglycan of bacterial cell walls. Unusual 3→3 cross-links formed by L,D-transpeptidases were first detected in Escherichia coli more than four decades ago, however no phenotype has previously been associated with their synthesis. Here we show that production of the L,D-transpeptidase YcbB in combination with elevated synthesis of the (p)ppGpp alarmone by RelA lead to full bypass of the D,D-transpeptidase activity of PBPs and to broad-spectrum β-lactam resistance. Production of YcbB was therefore sufficient to switch the role of (p)ppGpp from antibiotic tolerance to high-level β-lactam resistance. This observation identifies a new mode of peptidoglycan polymerization in E. coli that relies on an unexpectedly small number of enzyme activities comprising the glycosyltransferase activity of class A PBP1b and the D,D-carboxypeptidase activity of DacA in addition to the L,D-transpeptidase activity of YcbB. DOI: http://dx.doi.org/10.7554/eLife.19469.001 PMID:27767957

  1. Alteration of mineral crystallinity and collagen cross-linking of bones in osteopetrotic toothless (tl/tl) rats and their improvement after treatment with colony stimulating factor-1

    NASA Technical Reports Server (NTRS)

    Wojtowicz, A.; Dziedzic-Goclawska, A.; Kaminski, A.; Stachowicz, W.; Wojtowicz, K.; Marks, S. C. Jr; Yamauchi, M.

    1997-01-01

    A common feature of various types of mammalian osteopetroses is a marked increase in bone mass accompanied by spontaneous bone fractures. The toothless (tl/tl) rat osteopetrotic mutation is characterized by drastically reduced bone resorption due to a profound deficiency of osteoclasts and their precursors. An altered bone morphology has also been observed. The mutants cannot be cured by bone marrow transplantation, but skeletal defects are greatly reduced after treatment with colony stimulating factor 1 (CSF-1). The objectives of this study were to characterize mineral and collagen matrices in cancellous and compact bone isolated from long bones of 6-week-old normal littermates, tl/tl osteopetrotic mutants and mutants (tl/tl) treated with CSF-1. There were no differences in bone mineral content, but a significant decrease in the crystallinity of mineral evaluated by the method based on electron paramagnetic resonance spectrometry was observed in all bones of tl/tl mutants as compared to that of controls. Within the collagen matrix, slight decreases in the labile cross-links, but significant increases in the content of the stable cross-links, pyridinoline, and deoxypyridinoline, were observed in both cancellous and compact bone of osteopetrotic mutants. In tl/tl mutants treated with human recombinant CSF-1, the normalization of the crystallinity of bone mineral as well as collagen cross-links was found. Our results indicate that remodeling of bone matrix in tl/tl mutants is highly suppressed, but that after treatment with CSF-1, this activity recovers significantly. Taken together, these data provide further support for the hypothesis that CSF-1 is an essential factor for normal osteoclast differentiation and bone remodelling.

  2. Study of several factors in RNA-protein cross-link formation induced by ionizing radiations within 70S ribosomes of E. coli MRE 600.

    PubMed

    Ekert, B; Giocanti, N; Sabattier, R

    1986-09-01

    The induction of RNA-protein crosslinks in E. coli 70S ribosomes by gamma-irradiation was studied by measuring the dependence of cross-link formation on ribosome concentration. The inverse dependence of cross-link percentage upon concentration up to at least 20 A260 nm units ml-1 indicate that indirect effects seem to play a more major part than direct effects for these ribosome concentrations. The effect of various gases and free radical scavengers was used to determine the roles of the radicals H., CO2-., OH. and e-aq and to estimate their relative efficiencies for cross-links. They were found to be: 7.2(H.), 6(CO2-.), 2(OH.) and 1(e-aq). The extent of RNA-protein cross-link production in 70S ribosomes induced by gamma-rays and neutrons in the presence and absence of oxygen was also investigated. Cross-link formation was estimated by separation of linked and unlinked material on nitrocellulose filters or after separation by SDS-sucrose gradient centrifugation under dissociating conditions. Oxygen inhibited cross-link formation by both neutrons and gamma-rays. However, very few cross-links were formed in de-aerated solutions by exposure to neutrons, compared to those produced by gamma-rays under the same conditions. This suggests that molecular oxygen generated along the secondary particle track can reduce the formation of RNA-protein cross-links.

  3. Cofilin-mediated actin dynamics promotes actin bundle formation during Drosophila bristle development

    PubMed Central

    Wu, Jing; Wang, Heng; Guo, Xuan; Chen, Jiong

    2016-01-01

    The actin bundle is an array of linear actin filaments cross-linked by actin-bundling proteins, but its assembly and dynamics are not as well understood as those of the branched actin network. Here we used the Drosophila bristle as a model system to study actin bundle formation. We found that cofilin, a major actin disassembly factor of the branched actin network, promotes the formation and positioning of actin bundles in the developing bristles. Loss of function of cofilin or AIP1, a cofactor of cofilin, each resulted in increased F-actin levels and severe defects in actin bundle organization, with the defects from cofilin deficiency being more severe. Further analyses revealed that cofilin likely regulates actin bundle formation and positioning by the following means. First, cofilin promotes a large G-actin pool both locally and globally, likely ensuring rapid actin polymerization for bundle initiation and growth. Second, cofilin limits the size of a nonbundled actin-myosin network to regulate the positioning of actin bundles. Third, cofilin prevents incorrect assembly of branched and myosin-associated actin filament into bundles. Together these results demonstrate that the interaction between the dynamic dendritic actin network and the assembling actin bundles is critical for actin bundle formation and needs to be closely regulated. PMID:27385345

  4. ACTIN DEPOLYMERIZING FACTOR4 regulates actin dynamics during innate immune signaling in Arabidopsis.

    PubMed

    Henty-Ridilla, Jessica L; Li, Jiejie; Day, Brad; Staiger, Christopher J

    2014-01-01

    Conserved microbe-associated molecular patterns (MAMPs) are sensed by pattern recognition receptors (PRRs) on cells of plants and animals. MAMP perception typically triggers rearrangements to actin cytoskeletal arrays during innate immune signaling. However, the signaling cascades linking PRR activation by MAMPs to cytoskeleton remodeling are not well characterized. Here, we developed a system to dissect, at high spatial and temporal resolution, the regulation of actin dynamics during innate immune signaling in plant cells. Within minutes of MAMP perception, we detected changes to single actin filament turnover in epidermal cells treated with bacterial and fungal MAMPs. These MAMP-induced alterations phenocopied an ACTIN DEPOLYMERIZING FACTOR4 (ADF4) knockout mutant. Moreover, actin arrays in the adf4 mutant were unresponsive to a bacterial MAMP, elf26, but responded normally to the fungal MAMP, chitin. Together, our data provide strong genetic and cytological evidence for the inhibition of ADF activity regulating actin remodeling during innate immune signaling. This work is the first to directly link an ADF/cofilin to the cytoskeletal rearrangements elicited directly after pathogen perception in plant or mammalian cells.

  5. Assessment of Actin FS and Actin FSL sensitivity to specific clotting factor deficiencies.

    PubMed

    Lawrie, A S; Kitchen, S; Purdy, G; Mackie, I J; Preston, F E; Machin, S J

    1998-06-01

    We present a two centre study designed to assess the sensitivity of Actin FS and Actin FSL to deficiencies of factor VIII, IX, XI or XII. The study was undertaken at two centres to avoid bias due to the investigations being undertaken on one analyser. Samples from patients with a factor VIII (n = 36, F VIII = < 1.0-50 iu/dl), factor IX (n = 22, F IX = 2-48 iu/dl), factor XI (n = 23, F XI = 5-50 u/dl) or a factor XII (n = 18, F XII = 1-50 u/dl) deficient state were studied. Activated partial thromboplastin times (APTT) were determined using two batches of Actin FS and of Actin FSL; comparison of APTT results between centres was facilitated by the conversion of clotting times to ratios (test divided by geometric mean normal clotting time). APTT ratios were considered to be elevated if greater than two standard deviations above the mean normal. The factor deficient status of each sample was verified by assaying all samples for factors VIII, IX, XI and XII. Clotting factor assays were performed on a Sysmex CA-1000 fitted with research software, which permitted the auto-dilution and testing of three serial dilution of both a reference preparation and each patient's sample. Assay results were calculated using parallel-line Bioassay principles. This procedure allowed for variation in clotting times due to the effect of temporal drift of any of the reagents within the assay system. Actin FS and Actin FSL demonstrate acceptable sensitivity to factor VIII deficiency, however, both reagents failed to detect a large proportion of factor XI (17.4% and 30.4% of samples, respectively) and factor XII (66.7% and 72.2%, respectively) deficiencies. The detection rate with Actin FSL for factor IX deficiency was also poor (36.4% not detected). As factor IX and XI deficiencies are both associated with haemorrhagic disorders, the inability of these reagents to detect such abnormalities gave cause for concern.

  6. A membrane cytoskeleton from Dictyostelium discoideum. I. Identification and partial characterization of an actin-binding activity

    PubMed Central

    1981-01-01

    Dictyostelium discoideum plasma membranes isolated by each of three procedures bind F-actin. The interactions between these membranes and actin are examined by a novel application of falling ball viscometry. Treating the membranes as multivalent actin-binding particles analogous to divalent actin-gelation factors, we observe large increases in viscosity (actin cross-linking) when membranes of depleted actin and myosin are incubated with rabbit skeletal muscle F-actin. Pre- extraction of peripheral membrane proteins with chaotropes or the inclusion of Triton X-100 during the assay does not appreciably diminish this actin cross-linking activity. Lipid vesicles, heat- denatured membranes, proteolyzed membranes, or membranes containing endogenous actin show minimal actin cross-linking activity. Heat- denatured, but not proteolyzed, membranes regain activity when assayed in the presence of Triton X-100. Thus, integral membrane proteins appear to be responsible for some or all of the actin cross-linking activity of D. discoideum membranes. In the absence of MgATP, Triton X- 100 extraction of isolated D. discoideum membranes results in a Triton- insoluble residue composed of actin, myosin, and associated membrane proteins. The inclusion of MgATP before and during Triton extraction greatly diminishes the amount of protein in the Triton-insoluble residue without appreciably altering its composition. Our results suggest the existence of a protein complex stabilized by actin and/or myosin (membrane cytoskeleton) associated with the D. discoideum plasma membrane. PMID:6894148

  7. Arabidopsis ACTIN-DEPOLYMERIZING FACTOR7 Severs Actin Filaments and Regulates Actin Cable Turnover to Promote Normal Pollen Tube Growth[W

    PubMed Central

    Zheng, Yiyan; Xie, Yurong; Jiang, Yuxiang; Qu, Xiaolu; Huang, Shanjin

    2013-01-01

    Actin filaments are often arranged into higher-order structures, such as the longitudinal actin cables that generate the reverse fountain cytoplasmic streaming pattern present in pollen tubes. While several actin binding proteins have been implicated in the generation of these cables, the mechanisms that regulate their dynamic turnover remain largely unknown. Here, we show that Arabidopsis thaliana ACTIN-DEPOLYMERIZING FACTOR7 (ADF7) is required for turnover of longitudinal actin cables. In vitro biochemical analyses revealed that ADF7 is a typical ADF that prefers ADP-G-actin over ATP-G-actin. ADF7 inhibits nucleotide exchange on actin and severs filaments, but its filament severing and depolymerizing activities are less potent than those of the vegetative ADF1. ADF7 primarily decorates longitudinal actin cables in the shanks of pollen tubes. Consistent with this localization pattern, the severing frequency and depolymerization rate of filaments significantly decreased, while their maximum lifetime significantly increased, in adf7 pollen tube shanks. Furthermore, an ADF7–enhanced green fluorescent protein fusion with defective severing activity but normal G-actin binding activity could not complement adf7, providing compelling evidence that the severing activity of ADF7 is vital for its in vivo functions. These observations suggest that ADF7 evolved to promote turnover of longitudinal actin cables by severing actin filaments in pollen tubes. PMID:24058157

  8. Structure of a Longitudinal Actin Dimer Assembled by Tandem W Domains: Implications for Actin Filament Nucleation

    SciTech Connect

    Rebowski, Grzegorz; Namgoong, Suk; Boczkowska, Malgorzata; Leavis, Paul C.; Navaza, Jorge; Dominguez, Roberto

    2013-11-20

    Actin filament nucleators initiate polymerization in cells in a regulated manner. A common architecture among these molecules consists of tandem WASP homology 2 domains (W domains) that recruit three to four actin subunits to form a polymerization nucleus. We describe a low-resolution crystal structure of an actin dimer assembled by tandem W domains, where the first W domain is cross-linked to Cys374 of the actin subunit bound to it, whereas the last W domain is followed by the C-terminal pointed end-capping helix of thymosin {beta}4. While the arrangement of actin subunits in the dimer resembles that of a long-pitch helix of the actin filament, important differences are observed. These differences result from steric hindrance of the W domain with intersubunit contacts in the actin filament. We also determined the structure of the first W domain of Vibrio parahaemolyticus VopL cross-linked to actin Cys374 and show it to be nearly identical with non-cross-linked W-Actin structures. This result validates the use of cross-linking as a tool for the study of actin nucleation complexes, whose natural tendency to polymerize interferes with most structural methods. Combined with a biochemical analysis of nucleation, the structures may explain why nucleators based on tandem W domains with short inter-W linkers have relatively weak activity, cannot stay bound to filaments after nucleation, and are unlikely to influence filament elongation. The findings may also explain why nucleation-promoting factors of the Arp2/3 complex, which are related to tandem-W-domain nucleators, are ejected from branch junctions after nucleation. We finally show that the simple addition of the C-terminal pointed end-capping helix of thymosin {beta}4 to tandem W domains can change their activity from actin filament nucleation to monomer sequestration.

  9. Structure of a longitudinal actin dimer assembled by tandem w domains: implications for actin filament nucleation.

    PubMed

    Rebowski, Grzegorz; Namgoong, Suk; Boczkowska, Malgorzata; Leavis, Paul C; Navaza, Jorge; Dominguez, Roberto

    2010-10-15

    Actin filament nucleators initiate polymerization in cells in a regulated manner. A common architecture among these molecules consists of tandem WASP homology 2 domains (W domains) that recruit three to four actin subunits to form a polymerization nucleus. We describe a low-resolution crystal structure of an actin dimer assembled by tandem W domains, where the first W domain is cross-linked to Cys374 of the actin subunit bound to it, whereas the last W domain is followed by the C-terminal pointed end-capping helix of thymosin β4. While the arrangement of actin subunits in the dimer resembles that of a long-pitch helix of the actin filament, important differences are observed. These differences result from steric hindrance of the W domain with intersubunit contacts in the actin filament. We also determined the structure of the first W domain of Vibrio parahaemolyticus VopL cross-linked to actin Cys374 and show it to be nearly identical with non-cross-linked W-Actin structures. This result validates the use of cross-linking as a tool for the study of actin nucleation complexes, whose natural tendency to polymerize interferes with most structural methods. Combined with a biochemical analysis of nucleation, the structures may explain why nucleators based on tandem W domains with short inter-W linkers have relatively weak activity, cannot stay bound to filaments after nucleation, and are unlikely to influence filament elongation. The findings may also explain why nucleation-promoting factors of the Arp2/3 complex, which are related to tandem-W-domain nucleators, are ejected from branch junctions after nucleation. We finally show that the simple addition of the C-terminal pointed end-capping helix of thymosin β4 to tandem W domains can change their activity from actin filament nucleation to monomer sequestration.

  10. Induction of Fc epsilon receptors on normal murine T cells and IgE binding factor(s) by cross-linked IgE or IgE-pulsed adherent cells.

    PubMed Central

    Firer, M A; Eshhar, Z

    1986-01-01

    This study aimed to compare the efficiency and extent of induction by monomeric versus cross-linked IgE of specific receptors for IgE on normal murine splenic T cells (Fc epsilon R-T), and to study the ability of IgE-pulsed splenic adherent cells to induce receptors for IgE on T cells. Chemically cross-linked IgE was found to be both more effective and more efficient than monomeric IgE in inducing Fc epsilon R-T as measured by the ability of IgE-pulsed T cells to form specific rosettes with IgE-sensitized trinitrophenylated sheep red blood cells (TNP-SRBC). This phenomenon was dependent on both DNA and protein synthesis, suggesting that induction caused the production of new IgE receptors. It was also found that cross-linked but not monomeric IgE-pulsed normal murine adherent cells as well as their cell-free products could actively induce significant levels of specific Fc epsilon R-T. Both cross-linked IgE-pulsed T cells and adherent cells released IgE binding factor(s). These materials were capable of specifically inhibiting the binding of IgE to rat basophilic leukaemic cells (RBL) in vitro and to rat tissue mast cells in vivo. Collectively, these data provide further evidence to suggest that polymerized forms of IgE and adherent cells play important roles in the regulation of IgE responses. Images Figure 3 PMID:2937716

  11. Single-molecule studies of actin assembly and disassembly factors.

    PubMed

    Smith, Benjamin A; Gelles, Jeff; Goode, Bruce L

    2014-01-01

    The actin cytoskeleton is very dynamic and highly regulated by multiple associated proteins in vivo. Understanding how this system of proteins functions in the processes of actin network assembly and disassembly requires methods to dissect the mechanisms of activity of individual factors and of multiple factors acting in concert. The advent of single-filament and single-molecule fluorescence imaging methods has provided a powerful new approach to discovering actin-regulatory activities and obtaining direct, quantitative insights into the pathways of molecular interactions that regulate actin network architecture and dynamics. Here we describe techniques for acquisition and analysis of single-molecule data, applied to the novel challenges of studying the filament assembly and disassembly activities of actin-associated proteins in vitro. We discuss the advantages of single-molecule analysis in directly visualizing the order of molecular events, measuring the kinetic rates of filament binding and dissociation, and studying the coordination among multiple factors. The methods described here complement traditional biochemical approaches in elucidating actin-regulatory mechanisms in reconstituted filamentous networks.

  12. NMR solution structures of actin depolymerizing factor homology domains

    PubMed Central

    Goroncy, Alexander K; Koshiba, Seizo; Tochio, Naoya; Tomizawa, Tadashi; Sato, Manami; Inoue, Makato; Watanabe, Satoru; Hayashizaki, Yoshihide; Tanaka, Akiko; Kigawa, Takanori; Yokoyama, Shigeyuki

    2009-01-01

    Actin is one of the most conserved proteins in nature. Its assembly and disassembly are regulated by many proteins, including the family of actin-depolymerizing factor homology (ADF-H) domains. ADF-H domains can be divided into five classes: ADF/cofilin, glia maturation factor (GMF), coactosin, twinfilin, and Abp1/drebrin. The best-characterized class is ADF/cofilin. The other four classes have drawn much less attention and very few structures have been reported. This study presents the solution NMR structure of the ADF-H domain of human HIP-55-drebrin-like protein, the first published structure of a drebrin-like domain (mammalian), and the first published structure of GMF β (mouse). We also determined the structures of mouse GMF γ, the mouse coactosin-like domain and the C-terminal ADF-H domain of mouse twinfilin 1. Although the overall fold of the five domains is similar, some significant differences provide valuable insights into filamentous actin (F-actin) and globular actin (G-actin) binding, including the identification of binding residues on the long central helix. This long helix is stabilized by three or four residues. Notably, the F-actin binding sites of mouse GMF β and GMF γ contain two additional β-strands not seen in other ADF-H structures. The G-actin binding site of the ADF-H domain of human HIP-55-drebrin-like protein is absent and distorted in mouse GMF β and GMF γ. PMID:19768801

  13. Low temperature cross linking polyimides

    NASA Technical Reports Server (NTRS)

    Serafini, T. T.; Delvigs, P. (Inventor)

    1982-01-01

    A polyimide is formed by cross linking a prepolymer formed by reacting a polyfunctional ester, a polyfunctional amine, and an end-capping unit. By providing an end-capping unit, the prepolymer is curable at a relatively low temperature of about 175 to 245 C.

  14. Actin-interacting Protein 1 Promotes Disassembly of Actin-depolymerizing Factor/Cofilin-bound Actin Filaments in a pH-dependent Manner*

    PubMed Central

    Nomura, Kazumi; Hayakawa, Kimihide; Tatsumi, Hitoshi; Ono, Shoichiro

    2016-01-01

    Actin-interacting protein 1 (AIP1) is a conserved WD repeat protein that promotes disassembly of actin filaments when actin-depolymerizing factor (ADF)/cofilin is present. Although AIP1 is known to be essential for a number of cellular events involving dynamic rearrangement of the actin cytoskeleton, the regulatory mechanism of the function of AIP1 is unknown. In this study, we report that two AIP1 isoforms from the nematode Caenorhabditis elegans, known as UNC-78 and AIPL-1, are pH-sensitive in enhancement of actin filament disassembly. Both AIP1 isoforms only weakly enhance disassembly of ADF/cofilin-bound actin filaments at an acidic pH but show stronger disassembly activity at neutral and basic pH values. However, a severing-defective mutant of UNC-78 shows pH-insensitive binding to ADF/cofilin-decorated actin filaments, suggesting that the process of filament severing or disassembly, but not filament binding, is pH-dependent. His-60 of AIP1 is located near the predicted binding surface for the ADF/cofilin-actin complex, and an H60K mutation of AIP1 partially impairs its pH sensitivity, suggesting that His-60 is involved in the pH sensor for AIP1. These biochemical results suggest that pH-dependent changes in AIP1 activity might be a novel regulatory mechanism of actin filament dynamics. PMID:26747606

  15. Human CAP1 is a key factor in the recycling of cofilin and actin for rapid actin turnover.

    PubMed

    Moriyama, Kenji; Yahara, Ichiro

    2002-04-15

    Cofilin-ADF (actin-depolymerizing factor) is an essential driver of actin-based motility. We discovered two proteins, p65 and p55, that are components of the actin-cofilin complex in a human HEK293 cell extract and identified p55 as CAP1/ASP56, a human homologue of yeast CAP/SRV2 (cyclase-associated protein). CAP is a bifunctional protein with an N-terminal domain that binds to Ras-responsive adenylyl cyclase and a C-terminal domain that inhibits actin polymerization. Surprisingly, we found that the N-terminal domain of CAP1, but not the C-terminal domain, is responsible for the interaction with the actin-cofilin complex. The N-terminal domain of CAP1 was also found to accelerate the depolymerization of F-actin at the pointed end, which was further enhanced in the presence of cofilin and/or the C-terminal domain of CAP1. Moreover, CAP1 and its C-terminal domain were observed to facilitate filament elongation at the barbed end and to stimulate ADP-ATP exchange on G-actin, a process that regenerates easily polymerizable G-actin. Although cofilin inhibited the nucleotide exchange on G-actin even in the presence of the C-terminal domain of CAP1, its N-terminal domain relieved this inhibition. Thus, CAP1 plays a key role in speeding up the turnover of actin filaments by effectively recycling cofilin and actin and through its effect on both ends of actin filament.

  16. Viral Replication Protein Inhibits Cellular Cofilin Actin Depolymerization Factor to Regulate the Actin Network and Promote Viral Replicase Assembly

    PubMed Central

    Kovalev, Nikolay; de Castro Martín, Isabel Fernández; Barajas, Daniel; Risco, Cristina; Nagy, Peter D.

    2016-01-01

    RNA viruses exploit host cells by co-opting host factors and lipids and escaping host antiviral responses. Previous genome-wide screens with Tomato bushy stunt virus (TBSV) in the model host yeast have identified 18 cellular genes that are part of the actin network. In this paper, we show that the p33 viral replication factor interacts with the cellular cofilin (Cof1p), which is an actin depolymerization factor. Using temperature-sensitive (ts) Cof1p or actin (Act1p) mutants at a semi-permissive temperature, we find an increased level of TBSV RNA accumulation in yeast cells and elevated in vitro activity of the tombusvirus replicase. We show that the large p33 containing replication organelle-like structures are located in the close vicinity of actin patches in yeast cells or around actin cable hubs in infected plant cells. Therefore, the actin filaments could be involved in VRC assembly and the formation of large viral replication compartments containing many individual VRCs. Moreover, we show that the actin network affects the recruitment of viral and cellular components, including oxysterol binding proteins and VAP proteins to form membrane contact sites for efficient transfer of sterols to the sites of replication. Altogether, the emerging picture is that TBSV, via direct interaction between the p33 replication protein and Cof1p, controls cofilin activities to obstruct the dynamic actin network that leads to efficient subversion of cellular factors for pro-viral functions. In summary, the discovery that TBSV interacts with cellular cofilin and blocks the severing of existing filaments and the formation of new actin filaments in infected cells opens a new window to unravel the way by which viruses could subvert/co-opt cellular proteins and lipids. By regulating the functions of cofilin and the actin network, which are central nodes in cellular pathways, viruses could gain supremacy in subversion of cellular factors for pro-viral functions. PMID:26863541

  17. Elasticity of cross-linked semiflexible biopolymers under tension.

    PubMed

    von der Heydt, Alice; Wilkin, Daniel; Benetatos, Panayotis; Zippelius, Annette

    2013-09-01

    Aiming at the mechanical properties of cross-linked biopolymers, we set up and analyze a model of two weakly bending wormlike chains subjected to a tensile force, with regularly spaced inter-chain bonds (cross-links) represented by harmonic springs. Within this model, we compute the force-extension curve and the differential stiffness exactly and discuss several limiting cases. Cross-links effectively stiffen the chain pair by reducing thermal fluctuations transverse to the force and alignment direction. The extra alignment due to cross-links increases both with growing number and with growing strength of the cross-links, and is most prominent for small force f. For large f, the additional, cross-link-induced extension is subdominant except for the case of linking the chains rigidly and continuously along their contour. In this combined limit, we recover asymptotically the elasticity of a weakly bending wormlike chain without constraints, stiffened by a factor of 4. The increase in differential stiffness can be as large as 100% for small f or large numbers of cross-links.

  18. Electrospinning formaldehyde cross-linked zein solutions

    Technology Transfer Automated Retrieval System (TEKTRAN)

    In order to develop zein fibers with improved physical properties and solvent resistance, formaldehyde was used as the cross-linking reagent before spinning. The cross-linking reaction was carried out in either acetic acid or ethanolic-HCl where the amount of cross-linking reagent was between 1 and...

  19. Electrospun cross linked rosin fibers

    NASA Astrophysics Data System (ADS)

    Baek, Woo-il; Nirmala, R.; Barakat, Nasser A. M.; El-Newehy, Mohamed H.; Al-Deyab, Salem S.; Kim, Hak Yong

    2011-12-01

    In this study, we describe the first reported preparation of rosin in fiber form through use of an electrospinning technique utilizing various solvent systems. The polymer concentration of the formed fiber was studied by using various solvents such as chloroform, ethanol, N-N dimethylformamide (DMF), tetrahydrofuran (THF), acetone, and methylene chloride (MC). An electrospray of the solution resulted in the beaded form of the rosin. By varying the polymer concentration with MC, we were then able to obtain uniform fibers. However, the fibers exhibited large diameter. We believe that it is possible to reduce the diameter of the rosin fibers through appropriate selection of electrospinning parameters. In addition, the morphological transitions from beads, to beaded fiber, to fiber were studied at different polymer concentrations. We propose a possible physical cross linking mechanism for the formation of rosin fibers during the electrospinning process. Our results demonstrate the feasibility of producing fiber nanostructures of rosin by using an electrospinning technique.

  20. Bundling of actin filaments by elongation factor 1 alpha inhibits polymerization at filament ends

    PubMed Central

    1996-01-01

    Elongation factor 1 alpha (EF1 alpha) is an abundant protein that binds aminoacyl-tRNA and ribosomes in a GTP-dependent manner. EF1 alpha also interacts with the cytoskeleton by binding and bundling actin filaments and microtubules. In this report, the effect of purified EF1 alpha on actin polymerization and depolymerization is examined. At molar ratios present in the cytosol, EF1 alpha significantly blocks both polymerization and depolymerization of actin filaments and increases the final extent of actin polymer, while at high molar ratios to actin, EF1 alpha nucleates actin polymerization. Although EF1 alpha binds actin monomer, this monomer-binding activity does not explain the effects of EF1 alpha on actin polymerization at physiological molar ratios. The mechanism for the inhibition of polymerization is related to the actin-bundling activity of EF1 alpha. Both ends of the actin filament are inhibited for polymerization and both bundling and the inhibition of actin polymerization are affected by pH within the same physiological range; at high pH both bundling and the inhibition of actin polymerization are reduced. Additionally, it is seen that the binding of aminoacyl-tRNA to EF1 alpha releases EF1 alpha's inhibiting effect on actin polymerization. These data demonstrate that EF1 alpha can alter the assembly of F-actin, a filamentous scaffold on which non- membrane-associated protein translation may be occurring in vivo. PMID:8947553

  1. Corneal Collagen Cross-Linking

    PubMed Central

    Jankov II, Mirko R.; Jovanovic, Vesna; Nikolic, Ljubisa; Lake, Jonathan C.; Kymionis, Georgos; Coskunseven, Efekan

    2010-01-01

    Corneal collagen cross-linking (CXL) with riboflavin and ultraviolet-A (UVA) is a new technique of corneal tissue strengthening by using riboflavin as a photosensitizer and UVA to increase the formation of intra and interfibrillar covalent bonds by photosensitized oxidation. Keratocyte apoptosis in the anterior segment of the corneal stroma all the way down to a depth of about 300 microns has been described and a demarcation line between the treated and untreated cornea has been clearly shown. It is important to ensure that the cytotoxic threshold for the endothelium has not been exceeded by strictly respecting the minimal corneal thickness. Confocal microscopy studies show that repopulation of keratocytes is already visible 1 month after the treatment, reaching its pre-operative quantity and quality in terms of functional morphology within 6 months after the treatment. The major indication for the use of CXL is to inhibit the progression of corneal ectasias, such as keratoconus and pellucid marginal degeneration. CXL may also be effective in the treatment and prophylaxis of iatrogenic keratectasia, resulting from excessively aggressive photoablation. This treatment has also been used to treat infectious corneal ulcers with apparent favorable results. Combination with other treatments, such as intracorneal ring segment implantation, limited topography-guided photoablation and conductive keratoplasty have been used with different levels of success. PMID:20543933

  2. Porous Cross-Linked Polyimide Networks

    NASA Technical Reports Server (NTRS)

    Meador, Mary Ann B. (Inventor); Guo, Haiquan (Inventor)

    2015-01-01

    Porous cross-linked polyimide networks are provided. The networks comprise an anhydride end-capped polyamic acid oligomer. The oligomer (i) comprises a repeating unit of a dianhydride and a diamine and terminal anhydride groups, (ii) has an average degree of polymerization of 10 to 50, (iii) has been cross-linked via a cross-linking agent, comprising three or more amine groups, at a balanced stoichiometry of the amine groups to the terminal anhydride groups, and (iv) has been chemically imidized to yield the porous cross-linked polyimide network. Also provided are porous cross-linked polyimide aerogels comprising a cross-linked and imidized anhydride end-capped polyamic acid oligomer, wherein the oligomer comprises a repeating unit of a dianhydride and a diamine, and the aerogel has a density of 0.10 to 0.333 g/cm.sup.3 and a Young's modulus of 1.7 to 102 MPa. Also provided are thin films comprising aerogels, and methods of making porous cross-linked polyimide networks.

  3. The effect of cross-link distributions in axially-ordered, cross-linked networks

    NASA Astrophysics Data System (ADS)

    Bennett, C. Brad; Kruczek, James; Rabson, D. A.; Matthews, W. Garrett; Pandit, Sagar A.

    2013-07-01

    Cross-linking between the constituent chains of biopolymers has a marked effect on their materials’ properties. In certain of these materials, such as fibrillar collagen, increases in cross-linking lead to an increase in the melting temperature. Fibrillar collagen is an axially-ordered network of cross-linked polymer chains exhibiting a broadened denaturation transition, which has been explained in terms of the successive denaturation with temperature of multiple species. We model axially-ordered, cross-linked materials as stiff chains with distinct arrangements of cross-link-forming sites. Simulations suggest that systems composed of chains with identical arrangements of cross-link-forming sites exhibit critical behavior. In contrast, systems composed of non-identical chains undergo a crossover. This model suggests that the arrangement of cross-link-forming sites may contribute to the broadening of the denaturation transition in fibrillar collagen.

  4. Highly cross-linked nanoporous polymers

    DOEpatents

    Steckle, Jr., Warren P.; Apen, Paul G.; Mitchell, Michael A.

    1998-01-01

    Condensation polymerization followed by a supercritical extraction step can be used to obtain highly cross-linked nanoporous polymers with high surface area, controlled pore sizes and rigid structural integrity. The invention polymers are useful for applications requiring separation membranes.

  5. Highly cross-linked nanoporous polymers

    DOEpatents

    Steckle, Jr., Warren P.; Apen, Paul G.; Mitchell, Michael A.

    1997-01-01

    Condensation polymerization followed by a supercritical extraction step can be used to obtain highly cross-linked nanoporous polymers with high surface area, controlled pore sizes and rigid structural integrity. The invention polymers are useful for applications requiring separation membranes.

  6. Cross-linked biopolymer bundles: Cross-link reversibility leads to cooperative binding/unbinding phenomena

    NASA Astrophysics Data System (ADS)

    Vink, Richard L. C.; Heussinger, Claus

    2012-01-01

    We consider a biopolymer bundle consisting of filaments that are cross-linked together. The cross-links are reversible: they can dynamically bind and unbind adjacent filament pairs as controlled by a binding enthalpy. The bundle is subjected to a bending deformation and the corresponding distribution of cross-links is measured. For a bundle consisting of two filaments, upon increasing the bending amplitude, a first-order transition is observed. The transition is from a state where the filaments are tightly coupled by many bound cross-links, to a state of nearly independent filaments with only a few bound cross-links. For a bundle consisting of more than two filaments, a series of first-order transitions is observed. The transitions are connected with the formation of an interface between regions of low and high cross-link densities. Combining umbrella sampling Monte Carlo simulations with analytical calculations, we present a detailed picture of how the competition between cross-link shearing and filament stretching drives the transitions. We also find that, when the cross-links become soft, collective behavior is not observed: the cross-links then unbind one after the other leading to a smooth decrease of the average cross-link density.

  7. DNA interstrand cross-linking by epichlorohydrin.

    PubMed

    Romano, Keith P; Newman, Adam G; Zahran, Rami W; Millard, Julie T

    2007-05-01

    Epichlorohydrin (ECH), an important industrial chemical, is a bifunctional alkylating agent with the potential to form DNA cross-links. Occupational exposure to this suspect carcinogen leads to chromosomal aberrations, and ECH has been shown previously to undergo reaction with DNA in vivo and in vitro. We used denaturing polyacrylamide gel electrophoresis to monitor the possible formation of interstrand cross-links within DNA oligomers by ECH and the related compound, epibromohydrin (EBH). Although both compounds did indeed form cross-links between deoxyguanosine residues, EBH was a more efficient cross-linker than ECH. The optimal pH for cross-linking also varied, with ECH more efficient at pH 5.0 and EBH more efficient at pH 7.0. Both agents were relatively flexible in the sequences targeted, with comparable efficiencies for 5'-GGC and 5'GC sites. Furthermore, interstrand cross-linking by the two optical isomers of ECH correlated with their relative cytotoxicities, with R-ECH about twice as potent as S-ECH.

  8. Cross-linking chemistry of squid beak.

    PubMed

    Miserez, Ali; Rubin, Daniel; Waite, J Herbert

    2010-12-03

    In stark contrast to most aggressive predators, Dosidicus gigas (jumbo squids) do not use minerals in their powerful mouthparts known as beaks. Their beaks instead consist of a highly sclerotized chitinous composite with incremental hydration from the tip to the base. We previously reported l-3,4-dihydroxyphenylalanine (dopa)-histidine (dopa-His) as an important covalent cross-link providing mechanical strengthening to the beak material. Here, we present a more complete characterization of the sclerotization chemistry and describe additional cross-links from D. gigas beak. All cross-links presented in this report share common building blocks, a family of di-, tri-, and tetra-histidine-catecholic adducts, that were separated by affinity chromatography and high performance liquid chromatography (HPLC) and identified by tandem mass spectroscopy and proton nuclear magnetic resonance ((1)H NMR). The data provide additional insights into the unusually high cross-link density found in mature beaks. Furthermore, we propose both a low molecular weight catechol, and peptidyl-dopa, to be sclerotization agents of squid beak. This appears to represent a new strategy for forming hard tissue in animals. The interplay between covalent cross-linking and dehydration on the graded properties of the beaks is discussed.

  9. Nuclear actin activates human transcription factor genes including the OCT4 gene.

    PubMed

    Yamazaki, Shota; Yamamoto, Koji; Tokunaga, Makio; Sakata-Sogawa, Kumiko; Harata, Masahiko

    2015-01-01

    RNA microarray analyses revealed that nuclear actin activated many human transcription factor genes including OCT4, which is required for gene reprogramming. Oct4 is known to be activated by nuclear actin in Xenopus oocytes. Our findings imply that this process of OCT4 activation is conserved in vertebrates and among cell types and could be used for gene reprogramming of human cells.

  10. In vivo protein cross-linking.

    PubMed

    Agou, Fabrice; Ye, Fei; Véron, Michel

    2004-01-01

    In the cell, homo- and heteroassociations of polypeptide chains evolve and take place within subcellular compartments that are crowded with many other cellular macromolecules. In vivo chemical cross-linking of proteins is a powerful method to examine changes in protein oligomerization and protein-protein interactions upon cellular events such as signal transduction. This chapter is intended to provide a guide to the selection of the cell-membrane-permeable cross-linkers, the optimization of in vivo cross-linking conditions, and the identification of specific cross-links in a cellular context where the frequency of random collisions is high. By combining the chemoselectivity of the homo-bifunctional cross-linker and the length of its spacer arm with knowledge on the protein structure, we show that selective cross-links can be introduced specifically on either the dimer or the hexamer form of the same polypeptide in vitro as well as in vivo, using the human type B nucleoside diphosphate kinase as a protein model.

  11. Cross-linked structure of network evolution

    SciTech Connect

    Bassett, Danielle S.; Wymbs, Nicholas F.; Grafton, Scott T.; Porter, Mason A.; Mucha, Peter J.

    2014-03-15

    We study the temporal co-variation of network co-evolution via the cross-link structure of networks, for which we take advantage of the formalism of hypergraphs to map cross-link structures back to network nodes. We investigate two sets of temporal network data in detail. In a network of coupled nonlinear oscillators, hyperedges that consist of network edges with temporally co-varying weights uncover the driving co-evolution patterns of edge weight dynamics both within and between oscillator communities. In the human brain, networks that represent temporal changes in brain activity during learning exhibit early co-evolution that then settles down with practice. Subsequent decreases in hyperedge size are consistent with emergence of an autonomous subgraph whose dynamics no longer depends on other parts of the network. Our results on real and synthetic networks give a poignant demonstration of the ability of cross-link structure to uncover unexpected co-evolution attributes in both real and synthetic dynamical systems. This, in turn, illustrates the utility of analyzing cross-links for investigating the structure of temporal networks.

  12. Highly cross-linked nanoporous polymers

    DOEpatents

    Steckle, W.P. Jr.; Apen, P.G.; Mitchell, M.A.

    1998-01-20

    Condensation polymerization followed by a supercritical extraction step can be used to obtain highly cross-linked nanoporous polymers with high surface area, controlled pore sizes and rigid structural integrity. The invention polymers are useful for applications requiring separation membranes. 1 fig.

  13. Actin dynamics tune the integrated stress response by regulating eukaryotic initiation factor 2α dephosphorylation

    PubMed Central

    Chambers, Joseph E; Dalton, Lucy E; Clarke, Hanna J; Malzer, Elke; Dominicus, Caia S; Patel, Vruti; Moorhead, Greg; Ron, David; Marciniak, Stefan J

    2015-01-01

    Four stress-sensing kinases phosphorylate the alpha subunit of eukaryotic translation initiation factor 2 (eIF2α) to activate the integrated stress response (ISR). In animals, the ISR is antagonised by selective eIF2α phosphatases comprising a catalytic protein phosphatase 1 (PP1) subunit in complex with a PPP1R15-type regulatory subunit. An unbiased search for additional conserved components of the PPP1R15-PP1 phosphatase identified monomeric G-actin. Like PP1, G-actin associated with the functional core of PPP1R15 family members and G-actin depletion, by the marine toxin jasplakinolide, destabilised the endogenous PPP1R15A-PP1 complex. The abundance of the ternary PPP1R15-PP1-G-actin complex was responsive to global changes in the polymeric status of actin, as was its eIF2α-directed phosphatase activity, while localised G-actin depletion at sites enriched for PPP1R15 enhanced eIF2α phosphorylation and the downstream ISR. G-actin's role as a stabilizer of the PPP1R15-containing holophosphatase provides a mechanism for integrating signals regulating actin dynamics with stresses that trigger the ISR. DOI: http://dx.doi.org/10.7554/eLife.04872.001 PMID:25774599

  14. G-actin sequestering protein thymosin-β4 regulates the activity of myocardin-related transcription factor.

    PubMed

    Morita, Tsuyoshi; Hayashi, Ken'ichiro

    2013-08-02

    Myocardin-related transcription factors (MRTFs) are robust coactivators of serum response factor (SRF). MRTFs contain three copies of the RPEL motif at their N-terminus, and they bind to monomeric globular actin (G-actin). Previous studies illustrate that G-actin binding inhibits MRTF activity by preventing the MRTFs nuclear accumulation. In the living cells, the majority of G-actin is sequestered by G-actin binding proteins that prevent spontaneous actin polymerization. Here, we demonstrate that the most abundant G-actin sequestering protein thymosin-β4 (Tβ4) was involved in the regulation of subcellular localization and activity of MRTF-A. Tβ4 competed with MRTF-A for G-actin binding; thus, interfering with G-actin-MRTF-A complex formation. Tβ4 overexpression induced the MRTF-A nuclear accumulation and activation of MRTF-SRF signaling. The activation rate of MRTF-A by the Tβ4 mutant L17A, whose affinity for G-actin is very low, was lower than that by wild-type Tβ4. In contrast, the β-actin mutant 3DA, which has a lower affinity for Tβ4, more effectively suppressed MRTF-A activity than wild-type β-actin. Furthermore, ectopic Tβ4 increased the endogenous expression of SRF-dependent actin cytoskeletal genes. Thus, Tβ4 is an important MRTF regulator that controls the G-actin-MRTFs interaction.

  15. Cross-Linking Studies of Lysozyme Nucleation

    NASA Technical Reports Server (NTRS)

    Forsythe, Elizabeth; Pusey, Marc

    2000-01-01

    Tetragonal chicken egg white crystals consist of 4(sub 3) helices running in alternating directions, the helix rows having a two fold symmetry with each other. The unit cell consists of one complete tetrameric turn from each of two adjacent helices (an octamer). PBC analysis indicates that the helix intermolecular bonds are the strongest in the crystal, therefore likely formed first. AFM analysis of the (110) surface shows only complete helices, no half steps or bisected helices being found, while AFM line scans to measure the growth step increments show that they are multiples of the 4(sub 3) helix tetramer dimensions. This supports our thesis that the growth units are in fact multiples of the four molecule 4(sub 3) helix unit, the "average" growth unit size for the (110) face being an octamer (two turns about the helix) and the (101) growth unit averaging about the size of a hexamer. In an effort to better understand the species involved in the crystal nucleation and growth process, we have initiated an experimental program to study the species formed in solution compared to what is found in the crystal through covalent cross-linking studies. These experiments use the heterobifunctional cross-linking agent aminoethyl-4-azidonitroanaline (AEANA). An aliphatic amine at one end is covalently attached to the protein by a carbodiimide-mediated reaction, and a photo reactive group at the other can be used to initiate crosslinking. Modifications to the parent structure can be used to alter the distance between the two reactive groups and thus the cross-linking agents "reach". In practice, the cross-linking agent is first coupled to the asp101 side chain through the amine group. Asp101 lies within the active site cleft, and previous work with fluorescent probes had shown that derivatives at this site still crystallize in the tetragonal space group. This was also found to be the case with the AEANA derivative, which gave red tetragonal crystals. The protein now has a

  16. Ion exchange selectivity for cross-linked polyacrylic acid

    NASA Technical Reports Server (NTRS)

    May, C. E.; Philipp, W. H.

    1983-01-01

    The ion separation factors for 21 common metal ions with cross-linked polyacrylic acid were determined as a function of pH and the percent of the cross-linked polyacrylic acid neutralized. The calcium ion was used as a reference. At a pH of 5 the decreasing order of affinity of the ions for the cross-linked polyacrylic acid was found to be: Hg++, Fe+++, Pb++, Cr+++, Cu++, Cd++, Al+++, Ag+, Zn++, Ni++, Mn++, Co++, Ca++, Sr++, Ba++, Mg++, K+, Rb+, Cs+, Na+, and Li+. Members of a chemical family exhibited similar selectivities. The Hg++ ion appeared to be about a million times more strongly bound than the alkali metal ions. The relative binding of most of the metal ions varied with pH; the very tightly and very weakly bound ions showed the largest variations with pH. The calcium ion-hydrogen ion equilibrium was perturbed very little by the presence of the other ions. The separation factors and selectivity coefficients are discussed in terms of equilibrium and thermodynamic significance.

  17. DNA Gel with dynamic cross-links

    NASA Astrophysics Data System (ADS)

    Park, Chang-Young; Fygenson, Deborah; Saleh, Omar

    2014-03-01

    The mechanical properties of a living cell are strongly related to the cytoskeletal network, which is comprised of diverse protein filaments connected by cross-linking proteins, some of which are dynamic. Gels comprised of dynamic cross-linkers exhibit unique mechanical properties not seen in those using permanent cross-linkers. To investigate the effect of a dynamic cross-linker on mechanical properties of a material, we have synthesized biopolymer gels with a well-known semi-flexible biopolymer, DNA, and probed the mechanics of the system using microrheological techniques. We discuss these results in comparison to cytoskeletal systems, and seek to establish universal principles of dynamic cross-link based gels. This work was supported by the NSF-funded UCSB MRSEC program, Award No. DMR-0520415.

  18. Positive tone cross-linked resists based on photoacid inhibition of cross linking

    NASA Astrophysics Data System (ADS)

    Lawson, Richard A.; Chun, Jun Sung; Neisser, Mark; Tolbert, Laren M.; Henderson, Clifford L.

    2014-03-01

    A resist imaging design that utilizes photoacid inhibition of cationic polymerization and cross-linking during a postexposure bake step has been studied. The key to the design approach is the use of two different polymerization catalysts/initiators: (1) a photoacid produced from a photoacid generator (PAG) upon exposure of the resist that can result in polymerization and cross-linking of the resist matrix and (2) a thermal cross-linking catalyst (TCC) designed to thermally catalyze epoxide-phenol cross-linking. The TCC can be chosen from a variety of compounds such as triphenylphosphine (TPP) or imidazole. When only one of these catalysts (e.g TPP or photoacid) is present in an epoxide and phenol containing resist matrix, it will individually catalyze cross-linking. When they are present together, they effectively quench one another and little to no cross-linking occurs. This approach can be used to switch the tone of a resist from negative (photoacid catalyzed) to positive (TCC catalyzed and photoacid inhibited). The effect of the ratio of TCC:PAG was examined and the optimal ratio for positive tone behavior was determined. Resist contrast can be modified by optimization of epoxide:phenol ratio in the formulation. Dual tone behavior with positive tone at low dose and negative tone at higher doses can be observed in certain formulation conditions. Initial EUV patterning shows poor results, but the source of the poor imaging is not yet understood.

  19. Magnetic macromolecular cross linked enzyme aggregates (CLEAs) of glucoamylase.

    PubMed

    Nadar, Shamraja S; Rathod, Virendra K

    2016-02-01

    This work illustrates the preparation of magnetic macromolecular glucoamylase CLEAs using dialdehydic pectin, as a cross linker instead of traditional glutaraldehyde. The effect of precipitators type and amount, cross linker concentration, cross linking time and amount of amino functionalized magnetic nanoparticles (AFMNs) on glucoamylase activity was studied. Glucoamylase magnetic macromolecular CLEAs prepared by precipitation in presence of AFMNs by ammonium sulfate were subsequently cross linked by dialdehydic pectin. After cross-linked by pectin, 95.4% activity recovery was achieved in magnetic macromolecular CLEAs, whereas in case of glutaraldehyde cross linker, 85.3% activity recovery was achieved. Magnetic macromolecular CLEAs showed 2.91 and 1.27 folds higher thermal stability as compared to free and magnetic glutaraldehyde CLEAs. In kinetics study, magnetic macromolecular CLEAs retained same Km values, whereas magnetic glutaraldehyde CLEAs showed higher Km value than free enzyme. The porous structure of magnetic macromolecular CLEAs was not only enhanced mass transfer toward macromolecular substrates, but also showed compression resistance for 5 consecutive cycles which was checked in terms of effectiveness factor. At the end, in reusability study; magnetic macromolecular CLEAs were retained 84% activity after 10(th) cycle without leaching of enzyme which is 22% higher than traditional magnetic CLEAs.

  20. Cellular Levels of Signaling Factors Are Sensed by β-actin Alleles to Modulate Transcriptional Pulse Intensity.

    PubMed

    Kalo, Alon; Kanter, Itamar; Shraga, Amit; Sheinberger, Jonathan; Tzemach, Hadar; Kinor, Noa; Singer, Robert H; Lionnet, Timothée; Shav-Tal, Yaron

    2015-04-21

    The transcriptional response of β-actin to extra-cellular stimuli is a paradigm for transcription factor complex assembly and regulation. Serum induction leads to a precisely timed pulse of β-actin transcription in the cell population. Actin protein is proposed to be involved in this response, but it is not known whether cellular actin levels affect nuclear β-actin transcription. We perturbed the levels of key signaling factors and examined the effect on the induced transcriptional pulse by following endogenous β-actin alleles in single living cells. Lowering serum response factor (SRF) protein levels leads to loss of pulse integrity, whereas reducing actin protein levels reveals positive feedback regulation, resulting in elevated gene activation and a prolonged transcriptional response. Thus, transcriptional pulse fidelity requires regulated amounts of signaling proteins, and perturbations in factor levels eliminate the physiological response, resulting in either tuning down or exaggeration of the transcriptional pulse.

  1. The actin depolymerizing factor (ADF)/cofilin signaling pathway and DNA damage responses in cancer.

    PubMed

    Chang, Chun-Yuan; Leu, Jyh-Der; Lee, Yi-Jang

    2015-02-13

    The actin depolymerizing factor (ADF)/cofilin protein family is essential for actin dynamics, cell division, chemotaxis and tumor metastasis. Cofilin-1 (CFL-1) is a primary non-muscle isoform of the ADF/cofilin protein family accelerating the actin filamental turnover in vitro and in vivo. In response to environmental stimulation, CFL-1 enters the nucleus to regulate the actin dynamics. Although the purpose of this cytoplasm-nucleus transition remains unclear, it is speculated that the interaction between CFL-1 and DNA may influence various biological responses, including DNA damage repair. In this review, we will discuss the possible involvement of CFL-1 in DNA damage responses (DDR) induced by ionizing radiation (IR), and the implications for cancer radiotherapy.

  2. The effects of collapsing factors on F-actin content and microtubule distribution of Helisoma growth cones.

    PubMed

    Torreano, Paul J; Waterman-Storer, Clare M; Cohan, Christopher S

    2005-03-01

    Growth cone collapsing factors induce growth cone collapse or repulsive growth cone turning by interacting with membrane receptors that induce alterations in the growth cone cytoskeleton. A common change induced by collapsing factors in the cytoskeleton of the peripheral domain, the thin lamellopodial area of growth cones, is a decline in the number of radially aligned F-actin bundles that form the core of filopodia. The present study examined whether ML-7, a myosin light chain kinase inhibitor, serotonin, a neurotransmitter and TPA, an activator of protein kinase C, which induce growth cone collapse of Helisoma growth cones, depolymerized or debundled F-actin. We report that these collapsing factors had different effects. ML-7 induced F-actin reorganization consistent with debundling whereas serotonin and TPA predominately depolymerized and possibly debundled F-actin. Additionally, these collapsing factors induced the formation of a dense actin-ring around the central domain, the thicker proximal area of growth cones [Zhou and Cohan, 2001: J. Cell Biol. 153:1071-1083]. The formation of the actin-ring occurred subsequent to the loss of actin bundles. The ML-7-induced actin-ring was found to inhibit microtubule extension into the P-domain. Thus, ML-7, serotonin, and TPA induce growth cone collapse associated with a decline in radially aligned F-actin bundles through at least two mechanisms involving debundling of actin filaments and/or actin depolymerization.

  3. Cross-linked carbon nanotube heat spreader

    NASA Astrophysics Data System (ADS)

    Konesky, Gregory

    2014-09-01

    Isolated individual carbon nanotubes (CNTs) have shown exceptional thermal conductivity along their axis, but have poor thermal transfer between adjacent CNTs. Thick bundles of aligned CNTs have been used as heat pipes, but the thermal input and output areas are the same, providing no heat spreading effect. Energetic argon ion beams are used to join, or cross-link overlapping CNTs in a thick film to form an interpenetrating network with an isotropic thermal conductivity of 2150 W/m-K. Such thick films may be used as heat spreaders to enlarge the thermal footprint of various electronic and semiconductor devices, laser diodes and CPU chips, for example, to enhance cooling.

  4. DNA cross-linking by intermediates in the mitomycin activation cascade

    SciTech Connect

    Cera, C.; Egbertson, M.; Teng, S.P.; Crothers, D.M.; Danishefsky, S.J. )

    1989-06-27

    The authors have assayed the cross-linking of oligonucleotides containing repeated mitomycin-reactive CpG sites in order to assess the factors that enhance activation of the carbamoyl function at C{sub 10}, yielding efficient mitomycin cross-linking. Drugs studied include mitomycin C (MC), N-methylmitomycin A (NMA), and the aziridinomitosene of NMA (MS). Drugs were reduced both by catalytic hydrogenation and by dithionite. They find that cross-linking by fully reduced NMA can be increased severalfold by addition of either excess dithionite reductant or the oxidant FeCl{sub 3}. Enhancement by FeCl{sub 3} is not seen with MC or MS, but excess dithionite increases cross-linking by all three compounds. They explain the action of Fe{sup 3+} by postulating production of the semiquinone of the monoadduct of mitomycin reacted at the C{sub 1}-position; according to this mechanism, departure of the carbamate from C{sub 10} is more efficient for the semiquinone than for the hydroquinone. However, the results imply that the hydroquinone can also function as a cross-linking agent. Excess dithionite beyond that required for stoichiometric reduction, activates the carbamate 2-3-fold for cross-linking. They find that the fully reduced leucoaziridinomitosene is highly unstable in solution, yet it produces efficient cross-linking. Hence, this compound is highly reactive in DNA alkylation and a good candidate for the role of primary alkylating agent.

  5. Understanding chemical reactivity for homo- and heterobifunctional protein cross-linking agents.

    PubMed

    Chen, Fan; Nielsen, Simone; Zenobi, Renato

    2013-07-01

    Chemical cross-linking, combined with mass spectrometry, has been applied to map three-dimensional protein structures and protein-protein interactions. Proper choice of the cross-linking agent, including its reactive groups and spacer arm length, is of great importance. However, studies to understand the details of reactivity of the chemical cross-linkers with proteins are quite sparse. In this study, we investigated chemical cross-linking from the aspects of the protein structures and the cross-linking reagents involved, by using two structurally well-known proteins, glyceraldehyde 3-phosohate dehydrogenase and ribonuclease S. Chemical cross-linking reactivity was compared using a series of homo- and hetero-bifunctional cross-linkers, including bis(sulfosuccinimidyl) suberate, dissuccinimidyl suberate, bis(succinimidyl) penta (ethylene glycol), bis(succinimidyl) nona (ethylene glycol), m-maleimidobenzoyl-N-hydroxysulfosuccinimide ester, 2-pyridyldithiol-tetraoxaoctatriacontane-N-hydrosuccinimide and succinimidyl-[(N-maleimidopropionamido)-tetracosaethyleneglycol]ester. The protein structure itself, especially the distances between target amino acid residues, was found to be a determining factor for the cross-linking efficiency. Moreover, the reactive groups of the chemical cross-linker also play an important role; a higher cross-linking reaction efficiency was found for maleimides compared to 2-pyrimidyldithiols. The reaction between maleimides and sulfhydryl groups is more favorable than that between N-hydroxysuccinimide esters and amine groups, although cysteine residues are less abundant in proteins compared to lysine residues.

  6. A structural study of F-actin - filamin networks

    NASA Astrophysics Data System (ADS)

    Ahrens-Braunstein, Ashley; Nguyen, Lam; Hirst, Linda

    2010-03-01

    The cell's ability to move and contract is attributed to the semi-flexible filamentous protein, F -actin, one of the three filaments in the cytoskeleton. Actin bundling can be formed by a cross-linking actin binding protein (ABP) filamin. By examining filamin's cross-linking abilities at different concentrations and molar ratios, we can study the flexibility, structure and multiple network formations created when cross-linking F-actin with this protein. We have studied the phase diagram of this protein system using fluorescence microscopy, analyzing the network structures observed in the context of a coarse grained molecular dynamics simulation carried out by our group.

  7. Corneal collagen cross-linking: a review.

    PubMed

    O'Brart, David P S

    2014-01-01

    The aim was to review the published literature on corneal collagen cross-linking. The emphasis was on the seminal publications, systemic reviews, meta-analyses and randomized controlled trials. Where such an evidence did not exist, selective large series cohort studies, case controlled studies and case series with follow-up preferably greater than 12 months were included. Riboflavin/Ultraviolet A (UVA) corneal collagen cross-linking appears to be the first treatment modality to halt the progression of keratoconus and other corneal ectatic disorders with improvement in visual, keratometric and topographic parameters documented by most investigators. Its precise mechanism of action at a molecular level is as yet not fully determined. Follow-up is limited to 4-6 years at present but suggests continued stability and improvement in corneal shape with time. Most published data are with epithelium-off techniques. Epithelium-on studies suggest some efficacy but less than with the epithelium-off procedures and long-term data are not currently available. The use of Riboflavin/UVA CXL for the management of infectious and non-infectious keratitis appears very promising. Its use in the management of bullous keratopathy is equivocal. Investigation of other methodologies for CXL are under investigation.

  8. Corneal Collagen Cross-Linking Outcomes: Review

    PubMed Central

    Jankov II, Mirko R; Jovanovic, Vesna; Delevic, Sladjana; Coskunseven, Efekan

    2011-01-01

    Keratoconus is a condition characterized by biomechanical instability of the cornea, presenting in a progressive, asymmetric and bilateral way. Corneal collagen cross-linking with riboflavin and UVA (CXL) is a new technique of corneal tissue strengthening that combines the use of riboflavin as a photo sensitizer and UVA irradiation. The studies showed that CXL was effective in halting the progression of keratoconus over a period of up to four years. The published studies also revealed a reduction of max K readings by more than 2 D, while the postoperative SEQ was reduced by an average of more than 1 D, and refractive cylinder decreased by about 1 D. No eyes lost any line of BCDVA. Moreover, there was no significant decrease in endothelial cell density. It was also found that CXL treatment was effective with reducing corneal and total wavefront aberrations. Corneal cross-linking has also led to an arrest and/or even a partial reversal of keratectasia in the treatment of iatrogenic ectasia after excimer laser ablation. A primary intervention such as CXL should be considered to potentially increase the biomechanical stability of the corneal tissue and postpone the need of lamellar or penetrating keratoplasty. PMID:21448301

  9. Corneal collagen cross-linking: A review

    PubMed Central

    O’Brart, David P.S.

    2014-01-01

    The aim was to review the published literature on corneal collagen cross-linking. The emphasis was on the seminal publications, systemic reviews, meta-analyses and randomized controlled trials. Where such an evidence did not exist, selective large series cohort studies, case controlled studies and case series with follow-up preferably greater than 12 months were included. Riboflavin/Ultraviolet A (UVA) corneal collagen cross-linking appears to be the first treatment modality to halt the progression of keratoconus and other corneal ectatic disorders with improvement in visual, keratometric and topographic parameters documented by most investigators. Its precise mechanism of action at a molecular level is as yet not fully determined. Follow-up is limited to 4–6 years at present but suggests continued stability and improvement in corneal shape with time. Most published data are with epithelium-off techniques. Epithelium-on studies suggest some efficacy but less than with the epithelium-off procedures and long-term data are not currently available. The use of Riboflavin/UVA CXL for the management of infectious and non-infectious keratitis appears very promising. Its use in the management of bullous keratopathy is equivocal. Investigation of other methodologies for CXL are under investigation. PMID:25000866

  10. To Cross-Link or Not to Cross-Link? Cross-Linking Associated Foreign Body Response of Collagen-Based Devices

    PubMed Central

    Delgado, Luis M.; Bayon, Yves; Pandit, Abhay

    2015-01-01

    Collagen-based devices, in various physical conformations, are extensively used for tissue engineering and regenerative medicine applications. Given that the natural cross-linking pathway of collagen does not occur in vitro, chemical, physical, and biological cross-linking methods have been assessed over the years to control mechanical stability, degradation rate, and immunogenicity of the device upon implantation. Although in vitro data demonstrate that mechanical properties and degradation rate can be accurately controlled as a function of the cross-linking method utilized, preclinical and clinical data indicate that cross-linking methods employed may have adverse effects on host response, especially when potent cross-linking methods are employed. Experimental data suggest that more suitable cross-linking methods should be developed to achieve a balance between stability and functional remodeling. PMID:25517923

  11. New insights into the pros and cons of cross-linking decellularized bioartificial organs.

    PubMed

    Hussein, Kamal H; Park, Kyung-Mee; Lee, Yun-Suk; Woo, Jae-Seok; Kang, Byung-Jae; Choi, Ki-Young; Kang, Kyung-Sun; Woo, Heung-Myong

    2017-01-25

    ABSTRACTDecellularization is an attractive method for scaffold designing in regenerative medicine. The resulting extracellular matrix (ECM) consists of structural proteins such as collagen and elastin, growth factors, and glycosaminoglycans, which can direct site-appropriate remodeling after in vivo implantation. Mainly, collagen and elastin of ECM are exposed to the enzymatic biodegradation in the host. To control the biodegradation process, treatment of decellularized tissue by a cross-linking agent is required. Cross-linking also reduces antigenicity and increases the storage properties. Cross-linkers should be nontoxic, with the ability to preserve the ECM components, especially glycosaminoglycans and associated growth factors for retention of scaffold bioactivity. In this review, we describe the different cross-linking agents and methods of evaluation of cross-linking efficiency.

  12. Combinatorial genetic analysis of a network of actin disassembly‐promoting factors

    PubMed Central

    Ydenberg, Casey A.; Johnston, Adam; Weinstein, Jaclyn; Bellavance, Danielle; Jansen, Silvia

    2015-01-01

    The patterning of actin cytoskeleton structures in vivo is a product of spatially and temporally regulated polymer assembly balanced by polymer disassembly. While in recent years our understanding of actin assembly mechanisms has grown immensely, our knowledge of actin disassembly machinery and mechanisms has remained comparatively sparse. Saccharomyces cerevisiae is an ideal system to tackle this problem, both because of its amenabilities to genetic manipulation and live‐cell imaging and because only a single gene encodes each of the core disassembly factors: cofilin (COF1), Srv2/CAP (SRV2), Aip1 (AIP1), GMF (GMF1/AIM7), coronin (CRN1), and twinfilin (TWF1). Among these six factors, only the functions of cofilin are essential and have been well defined. Here, we investigated the functions of the nonessential actin disassembly factors by performing genetic and live‐cell imaging analyses on a combinatorial set of isogenic single, double, triple, and quadruple mutants in S. cerevisiae. Our results show that each disassembly factor makes an important contribution to cell viability, actin organization, and endocytosis. Further, our data reveal new relationships among these factors, providing insights into how they work together to orchestrate actin turnover. Finally, we observe specific combinations of mutations that are lethal, e.g., srv2Δ aip1Δ and srv2Δ crn1Δ twf1Δ, demonstrating that while cofilin is essential, it is not sufficient in vivo, and that combinations of the other disassembly factors perform vital functions. © 2015 The Authors. Cytoskeleton Published by Wiley Periodicals, Inc. PMID:26147656

  13. Sorption of substituted indoles on highly cross-linked polystyrene from water-acetonitrile solutions

    NASA Astrophysics Data System (ADS)

    Shafigulin, R. V.; Myakishev, A. A.; Il'Ina, E. A.; Il'in, M. M.; Davankov, V. A.; Bulanova, A. V.

    2011-07-01

    The sorption of first synthesized indole derivatives by highly cross-linked polystyrenes from water-acetonitrile solutions was studied by high-performance liquid chromatography. The retention factors and differences in the Gibbs energy of adsorption from infinite diluted solutions were calculated, and the applicability of the Snyder-Soczewinski and Scott-Kucera models for describing the chromatographic retention of sorbates on a polymer network of highly cross-linked polystyrene was shown.

  14. Identification of Arabidopsis cyclase-associated protein 1 as the first nucleotide exchange factor for plant actin.

    PubMed

    Chaudhry, Faisal; Guérin, Christophe; von Witsch, Matthias; Blanchoin, Laurent; Staiger, Christopher J

    2007-08-01

    The actin cytoskeleton powers organelle movements, orchestrates responses to abiotic stresses, and generates an amazing array of cell shapes. Underpinning these diverse functions of the actin cytoskeleton are several dozen accessory proteins that coordinate actin filament dynamics and construct higher-order assemblies. Many actin-binding proteins from the plant kingdom have been characterized and their function is often surprisingly distinct from mammalian and fungal counterparts. The adenylyl cyclase-associated protein (CAP) has recently been shown to be an important regulator of actin dynamics in vivo and in vitro. The disruption of actin organization in cap mutant plants indicates defects in actin dynamics or the regulated assembly and disassembly of actin subunits into filaments. Current models for actin dynamics maintain that actin-depolymerizing factor (ADF)/cofilin removes ADP-actin subunits from filament ends and that profilin recharges these monomers with ATP by enhancing nucleotide exchange and delivery of subunits onto filament barbed ends. Plant profilins, however, lack the essential ability to stimulate nucleotide exchange on actin, suggesting that there might be a missing link yet to be discovered from plants. Here, we show that Arabidopsis thaliana CAP1 (AtCAP1) is an abundant cytoplasmic protein; it is present at a 1:3 M ratio with total actin in suspension cells. AtCAP1 has equivalent affinities for ADP- and ATP-monomeric actin (Kd approximately 1.3 microM). Binding of AtCAP1 to ATP-actin monomers inhibits polymerization, consistent with AtCAP1 being an actin sequestering protein. However, we demonstrate that AtCAP1 is the first plant protein to increase the rate of nucleotide exchange on actin. Even in the presence of ADF/cofilin, AtCAP1 can recharge actin monomers and presumably provide a polymerizable pool of subunits to profilin for addition onto filament ends. In turnover assays, plant profilin, ADF, and CAP act cooperatively to promote flux

  15. Bacterial nucleators: actin' on actin

    PubMed Central

    Bugalhão, Joana N.; Mota, Luís Jaime; Franco, Irina S.

    2015-01-01

    The actin cytoskeleton is a key target of numerous microbial pathogens, including protozoa, fungi, bacteria and viruses. In particular, bacterial pathogens produce and deliver virulence effector proteins that hijack actin dynamics to enable bacterial invasion of host cells, allow movement within the host cytosol, facilitate intercellular spread or block phagocytosis. Many of these effector proteins directly or indirectly target the major eukaryotic actin nucleator, the Arp2/3 complex, by either mimicking nucleation promoting factors or activating upstream small GTPases. In contrast, this review is focused on a recently identified class of effector proteins from Gram-negative bacteria that function as direct actin nucleators. These effector proteins mimic functional activities of formins, WH2-nucleators and Ena/VASP assembly promoting factors demonstrating that bacteria have coopted the complete set of eukaryotic actin assembly pathways. Structural and functional analyses of these nucleators have revealed several motifs and/or mechanistic activities that are shared with eukaryotic actin nucleators. However, functional effects of these proteins during infection extend beyond plain actin polymerization leading to interference with other host cell functions such as vesicle trafficking, cell cycle progression and cell death. Therefore, their use as model systems could not only help in the understanding of the mechanistic details of actin polymerization but also provide novel insights into the connection between actin dynamics and other cellular pathways. PMID:26416078

  16. Kojak: Efficient analysis of chemically cross-linked protein complexes

    PubMed Central

    Hoopmann, Michael R.; Zelter, Alex; Johnson, Richard S.; Riffle, Michael; MacCoss, Michael J.; Davis, Trisha N.; Moritz, Robert L.

    2015-01-01

    Protein chemical cross-linking and mass spectrometry enable the analysis of protein-protein interactions and protein topologies, however complicated cross-linked peptide spectra require specialized algorithms to identify interacting sites. The Kojak cross-linking software application is a new, efficient approach to identify cross-linked peptides, enabling large-scale analysis of protein-protein interactions by chemical cross-linking techniques. The algorithm integrates spectral processing and scoring schemes adopted from traditional database search algorithms, and can identify cross-linked peptides using many different chemical cross-linkers, with or without heavy isotope labels. Kojak was used to analyze both novel and existing datasets, and was compared with existing cross-linking algorithms. The algorithm provided increased cross-link identifications over existing algorithms, and equally importantly, the results in a fraction of computational time. The Kojak algorithm is open-source, cross-platform, and freely available. This software provides both existing and new cross-linking researchers alike an effective way to derive additional cross-link identifications from new or existing datasets. For new users, it provides a simple analytical resource resulting in more cross-link identifications than other methods. PMID:25812159

  17. Corneal cross-linking treatment of keratoconus

    PubMed Central

    Farjadnia, Mahgol; Naderan, Mohammad

    2015-01-01

    Keratoconus as the most common cause of ectasia is one of the leading cause of corneal transplants worldwide. The current available therapies do not modify the underlying pathogenesis of the disease, and none of the available approaches but corneal transplant hinder the ongoing ectasia. Several studies document Crosslink defect between collagen fibrils in the pathogenesis of keratoconus. Collagen cross link is a relatively new approach that with the application of the riboflavin and ultraviolet A, new covalent bands reform. Subjective and objective results following this method seem to be promising. Endothelial damage besides other deep structural injury, which is the major concern of this technique have not yet been reported, when applying the standard method. PMID:26622134

  18. Chemistry of the collagen cross-links. Origin and partial characterization of a putative mature cross-link of collagen.

    PubMed Central

    Barnard, K; Light, N D; Sims, T J; Bailey, A J

    1987-01-01

    The conversion of the reducible divalent cross-links in collagen to non-reducible multivalent cross-links in mature collagen has resulted in the identification of several new amino acids as the putative mature cross-link. None of these compounds has completely satisfied the necessary criteria. We have now isolated an amino acid of high Mr, derived from lysine, that is only present in high-Mr peptides derived from mature collagen. Its increase with age of the tissue correlates with the decrease in the reducible cross-links, and it is present both in mature skin and bone, which are initially cross-linked through the aldimine and oxo-imine divalent cross-link respectively. We propose that this amino acid, as yet incompletely characterized and designated compound M, is a major cross-link of mature collagen. PMID:3117039

  19. Photocontrolled Cargo Release from Dual Cross-Linked Polymer Particles.

    PubMed

    Tan, Shereen; Cui, Jiwei; Fu, Qiang; Nam, Eunhyung; Ladewig, Katharina; Ren, Jing M; Wong, Edgar H H; Caruso, Frank; Blencowe, Anton; Qiao, Greg G

    2016-03-09

    Burst release of a payload from polymeric particles upon photoirradiation was engineered by altering the cross-linking density. This was achieved via a dual cross-linking concept whereby noncovalent cross-linking was provided by cyclodextrin host-guest interactions, and irreversible covalent cross-linking was mediated by continuous assembly of polymers (CAP). The dual cross-linked particles (DCPs) were efficiently infiltrated (∼80-93%) by the biomacromolecule dextran (molecular weight up to 500 kDa) to provide high loadings (70-75%). Upon short exposure (5 s) to UV light, the noncovalent cross-links were disrupted resulting in increased permeability and burst release of the cargo (50 mol % within 1 s) as visualized by time-lapse fluorescence microscopy. As sunlight contains UV light at low intensities, the particles can potentially be incorporated into systems used in agriculture, environmental control, and food packaging, whereby sunlight could control the release of nutrients and antimicrobial agents.

  20. Protein Interactions Captured by Chemical Cross-linking: Simple Cross-linking Screen Using Sulfo-MBS.

    PubMed

    Nadeau, Owen W; Carlson, Gerald M

    2007-04-01

    INTRODUCTIONThis protocol describes a method for chemical cross-linking of proteins using sulfo-MBS (m-maleimidobenzoyl-N-hydroxysulfo-succinimide ester). Optimal conditions for cross-linking can be determined rapidly for a fixed concentration of a protein complex by varying the time of cross-linking, pH of the reaction, and concentration of sulfo-MBS. Typically, these screens require only small amounts of target proteins and can be carried out in less than a day.

  1. The wear of cross-linked polyethylene against itself.

    PubMed

    Joyce, T J; Ash, H E; Unsworth, A

    1996-01-01

    Cross-linked polyethylene (XLPE) may have an application as a material for an all-plastic surface replacement finger joint. It is inexpensive, biocompatible and can be injection-moulded into the complex shapes that are found on the ends of the finger bones. Further, the cross-linking of polyethylene has significantly improved its mechanical properties. Therefore, the opportunity exists for an all-XLPE joint, and so the wear characteristics of XLPE sliding against itself have been investigated. Wear tests were carried out on both reciprocating pin-on-plate machines and a finger function simulator. The reciprocating pin-on-plate machines had pins loaded at 10 N and 40 N. All pin-on-plate tests show wear factors from the plates very much greater than those of the pins. After 349 km of sliding, a mean wear factor of 0.46 x 10(-6) mm3/N m was found for the plates compared with 0.021 x 10(-6) mm3/N m for the pins. A fatigue mechanism may be causing this phenomenon of greater plate wear. Tests using the finger function simulator give an average wear rate of 0.22 x 10(-6) mm3/N m after 368 km. This sliding distance is equivalent to 12.5 years of use in vivo. The wear factors found were comparable with those of ultra-high molecular weight polyethylene (UHMWPE) against a metallic counterface and, therefore, as the loads across the finger joint are much less than those across the knee or the hip, it is probable that an all-XLPE finger joint will be viable from a wear point of view.

  2. Cytoplasmic Actin Is an Extracellular Insect Immune Factor which Is Secreted upon Immune Challenge and Mediates Phagocytosis and Direct Killing of Bacteria, and Is a Plasmodium Antagonist

    PubMed Central

    Sandiford, Simone L.; Dong, Yuemei; Pike, Andrew; Blumberg, Benjamin J.; Bahia, Ana C.; Dimopoulos, George

    2015-01-01

    Actin is a highly versatile, abundant, and conserved protein, with functions in a variety of intracellular processes. Here, we describe a novel role for insect cytoplasmic actin as an extracellular pathogen recognition factor that mediates antibacterial defense. Insect actins are secreted from cells upon immune challenge through an exosome-independent pathway. Anopheles gambiae actin interacts with the extracellular MD2-like immune factor AgMDL1, and binds to the surfaces of bacteria, mediating their phagocytosis and direct killing. Globular and filamentous actins display distinct functions as extracellular immune factors, and mosquito actin is a Plasmodium infection antagonist. PMID:25658622

  3. Actin depolymerizing factors ADF7 and ADF10 play distinct roles during pollen development and pollen tube growth.

    PubMed

    Daher, Firas Bou; Geitmann, Anja

    2012-07-01

    An important player in actin remodeling is the actin depolymerizing factor (ADF) which increases actin filament treadmilling rates. Previously, we had prepared fluorescent protein fusions of two Arabidopsis pollen specific ADFs, ADF7 and ADF10. These had enabled us to determine the temporal expression patterns and subcellular localization of these proteins during male gametophyte development. Here we generated stable transformants containing both chimeric genes allowing for simultaneous imaging and direct comparison. One of the striking differences between the two proteins was the localization profile in the growing pollen tube apex. Whereas ADF10 was associated with the filamentous actin array forming the subapical actin fringe, ADF7 was present in the same cytoplasmic region, but in diffuse form. This suggests that ADF7 is involved in the high actin turnover that is likely to occur in the fringe by continuously and efficiently depolymerizing filamentous actin and supplying monomeric actin to the advancing end of the fringe. The possibility to visualize both of these pollen-specific ADFs simultaneously opens avenues for future research into the regulatory function of actin binding proteins in pollen.

  4. Multi-Scale Modeling of Cross-Linked Nanotube Materials

    NASA Technical Reports Server (NTRS)

    Frankland, S. J. V.; Odegard, G. M.; Herzog, M. N.; Gates, T. S.; Fay, C. C.

    2005-01-01

    The effect of cross-linking single-walled carbon nanotubes on the Young's modulus of a nanotube-reinforced composite is modeled with a multi-scale method. The Young's modulus is predicted as a function of nanotube volume fraction and cross-link density. In this method, the constitutive properties of molecular representative volume elements are determined using molecular dynamics simulation and equivalent-continuum modeling. The Young's modulus is subsequently calculated for cross-linked nanotubes in a matrix which consists of the unreacted cross-linking agent. Two different cross-linking agents are used in this study, one that is short and rigid (Molecule A), and one that is long and flexible (Molecule B). Direct comparisons between the predicted elastic constants are made for the models in which the nanotubes are either covalently bonded or not chemically bonded to the cross-linking agent. At a nanotube volume fraction of 10%, the Young's modulus of Material A is not affected by nanotube crosslinking, while the Young's modulus of Material B is reduced by 64% when the nanotubes are cross-linked relative to the non-cross-linked material with the same matrix.

  5. TaADF3, an Actin-Depolymerizing Factor, Negatively Modulates Wheat Resistance Against Puccinia striiformis

    PubMed Central

    Tang, Chunlei; Deng, Lin; Chang, Dan; Chen, Shuntao; Wang, Xiaojie; Kang, Zhensheng

    2016-01-01

    The actin cytoskeleton has been implicated in plant defense against pathogenic fungi, oomycetes, and bacteria. Actin depolymerizing factors (ADFs) are stimulus responsive actin cytoskeleton modulators. However, there is limited evidence linking ADFs with plant defense against pathogens. In this study, we have isolated and functionally characterized a stress-responsive ADF gene (TaADF3) from wheat, which was detectable in all examined wheat tissues. TaADF3 is a three-copy gene located on chromosomes 5AL, 5BL, and 5DL. A particle bombardment assay in onion epidermal cells revealed the cytoplasmic and nuclear localization of TaADF3. The expression of TaADF3 was inducible by abscisic acid (ABA), as well as various abiotic stresses (drought and cold) and virulent Puccinia striiformis f. sp. tritici (Pst) but was down regulated in response to avirulent Pst. Virus-induced silencing of TaADF3 copies enhanced wheat resistance to avirulent Pst, with decreased reactive oxygen species (ROS) accumulation and hypersensitive response (HR). Upon treatment with virulent Pst, TaADF3-knockdown plants exhibited reduced susceptibility, which was accompanied by increased ROS production and HR. Interestingly, the silencing of TaADF3 resulted in hindered pathogen penetration and haustoria formation for both avirulent and virulent Pst. Moreover, the array and distribution of actin filaments was transformed in TaADF3-knockdown epidermal cells, which possibly facilitated attenuating the fungus penetration. Thus, our findings suggest that TaADF3 positively regulates wheat tolerance to abiotic stresses and negatively regulates wheat resistance to Pst in an ROS-dependent manner, possibly underlying the mechanism of impeding fungal penetration dependent on the actin architecture dynamics. PMID:26834758

  6. Cross-linked polyvinyl alcohol and method of making same

    NASA Technical Reports Server (NTRS)

    Hsu, L. C.; Sheibley, D. W.; Philipp, W. H. (Inventor)

    1981-01-01

    A film-forming polyvinyl alcohol polymer is mixed with a polyaldehyde-polysaccharide cross-linking agent having at least two monosaccharide units and a plurality of aldehyde groups per molecule, perferably an average of at least one aldehyde group per monosaccharide units. The cross-linking agent, such as a polydialdehyde starch, is used in an amount of about 2.5 to 20% of the theoretical amount required to cross-link all of the available hydroxyl groups of the polyvinyl alcohol polymer. Reaction between the polymer and cross-linking agent is effected in aqueous acidic solution to produce the cross-linked polymer. The polymer product has low electrical resistivity and other properties rendering it suitable for making separators for alkaline batteries.

  7. Cross-linked polyvinyl alcohol films as alkaline battery separators

    NASA Technical Reports Server (NTRS)

    Sheibley, D. W.; Manzo, M. A.; Gonzalez-Sanabria, O. D.

    1983-01-01

    Cross-linking methods have been investigated to determine their effect on the performance of polyvinyl alcohol (PVA) films as alkaline battery separators. The following types of cross-linked PVA films are discussed: (1) PVA-dialdehyde blends post-treated with an acid or acid periodate solution (two-step method) and (2) PVA-dialdehyde blends cross-linked during film formation (drying) by using a reagent with both aldehyde and acid functionality (one-step method). Laboratory samples of each cross-linked type of film were prepared and evaluated in standard separator screening tests. Then pilot-plant batches of films were prepared and compared to measure differences due to the cross-linking method. The pilot-plant materials were then tested in nickel oxide-zinc cells to compare the two methods with respect to performance characteristics and cycle life. Cell test results are compared with those from tests with Celgard.

  8. Cross-linked polyvinyl alcohol films as alkaline battery separators

    NASA Technical Reports Server (NTRS)

    Sheibley, D. W.; Manzo, M. A.; Gonzalez-Sanabria, O. D.

    1982-01-01

    Cross-linking methods were investigated to determine their effect on the performance of polyvinyl alcohol (PVA) films as alkaline battery separators. The following types of cross-linked PVA films are discussed: (1) PVA-dialdehyde blends post-treated with an acid or acid periodate solution (two-step method) and (2) PVA-dialdehyde blends cross-linked during film formation (drying) by using a reagent with both aldehyde and acid functionality (one-step method). Laboratory samples of each cross-linked type of film were prepared and evaluated in standard separator screening tests. The pilot-plant batches of films were prepared and compared to measure differences due to the cross-linking method. The pilot-plant materials were then tested in nickel oxide - zinc cells to compare the two methods with respect to performance characteristics and cycle life. Cell test results are compared with those from tests with Celgard.

  9. Polycation induced actin bundles.

    PubMed

    Muhlrad, Andras; Grintsevich, Elena E; Reisler, Emil

    2011-04-01

    Three polycations, polylysine, the polyamine spermine and the polycationic protein lysozyme were used to study the formation, structure, ionic strength sensitivity and dissociation of polycation-induced actin bundles. Bundles form fast, simultaneously with the polymerization of MgATP-G-actins, upon the addition of polycations to solutions of actins at low ionic strength conditions. This indicates that nuclei and/or nascent filaments bundle due to attractive, electrostatic effect of polycations and the neutralization of repulsive interactions of negative charges on actin. The attractive forces between the filaments are strong, as shown by the low (in nanomolar range) critical concentration of their bundling at low ionic strength. These bundles are sensitive to ionic strength and disassemble partially in 100 mM NaCl, but both the dissociation and ionic strength sensitivity can be countered by higher polycation concentrations. Cys374 residues of actin monomers residing on neighboring filaments in the bundles can be cross-linked by the short span (5.4Å) MTS-1 (1,1-methanedyl bismethanethiosulfonate) cross-linker, which indicates a tight packing of filaments in the bundles. The interfilament cross-links, which connect monomers located on oppositely oriented filaments, prevent disassembly of bundles at high ionic strength. Cofilin and the polysaccharide polyanion heparin disassemble lysozyme induced actin bundles more effectively than the polylysine-induced bundles. The actin-lysozyme bundles are pathologically significant as both proteins are found in the pulmonary airways of cystic fibrosis patients. Their bundles contribute to the formation of viscous mucus, which is the main cause of breathing difficulties and eventual death in this disorder.

  10. Structural dynamics of the actomyosin complex probed by a bifunctional spin label that cross-links SH1 and SH2.

    PubMed

    Thompson, Andrew R; Naber, Nariman; Wilson, Clyde; Cooke, Roger; Thomas, David D

    2008-12-01

    We have used a bifunctional spin label (BSL) to cross-link Cys(707) (SH1) and Cys(697) (SH2) in the catalytic domain of myosin subfragment 1 (S1). BSL induces the same weakened ATPase activity and actin-binding affinity that is observed when SH1 and SH2 are cross-linked with pPDM, which traps an analog of the post-hydrolysis state A.M.ADP.P. Electron paramagnetic resonance showed that BSL reports the global orientation and dynamics of S1. When bound to actin in oriented muscle fibers in the absence of ATP, BSL-S1 showed almost complete orientational disorder, as reported previously for the weakly bound A.M.ADP. In contrast, helical order is observed for the strongly bound state A.M. Saturation transfer electron paramagnetic resonance showed that the disorder of cross-linked S1 on actin is nearly static on the microsecond timescale, at least 30 times slower than that of A.M.ADP. We conclude that cross-linked S1 exhibits rotational disorder comparable to that of A.M.ADP, slow rotational mobility comparable to that of A.M, and intermediate actin affinity. These results support the hypothesis that the catalytic domain of myosin is orientationally disordered on actin in a post-hydrolysis state in the early stages of force generation.

  11. Grass Cell Walls: A Story of Cross-Linking

    PubMed Central

    Hatfield, Ronald D.; Rancour, David M.; Marita, Jane M.

    2017-01-01

    Cell wall matrices are complex composites mainly of polysaccharides, phenolics (monomers and polymers), and protein. We are beginning to understand the synthesis of these major wall components individually, but still have a poor understanding of how cell walls are assembled into complex matrices. Valuable insight has been gained by examining intact components to understand the individual elements that make up plant cell walls. Grasses are a prominent group within the plant kingdom, not only for their important roles in global agriculture, but also for the complexity of their cell walls. Ferulate incorporation into grass cell wall matrices (C3 and C4 types) leads to a cross-linked matrix that plays a prominent role in the structure and utilization of grass biomass compared to dicot species. Incorporation of p-coumarates as part of the lignin structure also adds to the complexity of grass cell walls. Feruoylation results in a wall with individual hemicellulosic polysaccharides (arabinoxylans) covalently linked to each other and to lignin. Evidence strongly suggests that ferulates not only cross-link arabinoxylans, but may be important factors in lignification of the cell wall. Therefore, the distribution of ferulates on arabinoxylans could provide a means of structuring regions of the matrix with the incorporation of lignin and have a significant impact upon localized cell wall organization. The role of other phenolics in cell wall formation such as p-coumarates (which can have concentrations higher than ferulates) remains unknown. It is possible that p-coumarates assist in the formation of lignin, especially syringyl rich lignin. The uniqueness of the grass cell wall compared to dicot sepcies may not be so much in the gross composition of the wall, but how the distinctive individual components are organized into a functional wall matrix. These features are discussed and working models are provided to illustrate how changing the organization of feruoylation and p

  12. Myocardin-Related Transcription Factor A Activation by Competition with WH2 Domain Proteins for Actin Binding

    PubMed Central

    Weissbach, Julia; Schikora, Franziska; Weber, Anja; Kessels, Michael

    2016-01-01

    The myocardin-related transcription factors (MRTFs) are coactivators of serum response factor (SRF)-mediated gene expression. Activation of MRTF-A occurs in response to alterations in actin dynamics and critically requires the dissociation of repressive G-actin–MRTF-A complexes. However, the mechanism leading to the release of MRTF-A remains unclear. Here we show that WH2 domains compete directly with MRTF-A for actin binding. Actin nucleation-promoting factors, such as N-WASP and WAVE2, as well as isolated WH2 domains, including those of Spire2 and Cobl, activate MRTF-A independently of changes in actin dynamics. Simultaneous inhibition of Arp2-Arp3 or mutation of the CA region only partially reduces MRTF-A activation by N-WASP and WAVE2. Recombinant WH2 domains and the RPEL domain of MRTF-A bind mutually exclusively to cellular and purified G-actin in vitro. The competition by different WH2 domains correlates with MRTF-SRF activation. Following serum stimulation, nonpolymerizable actin dissociates from MRTF-A, and de novo formation of the G-actin–RPEL complex is impaired by a transferable factor. Our work demonstrates that WH2 domains activate MRTF-A and contribute to target gene regulation by a competitive mechanism, independently of their role in actin filament formation. PMID:26976641

  13. Effects of alginate hydrogel cross-linking density on mechanical and biological behaviors for tissue engineering.

    PubMed

    Jang, Jinah; Seol, Young-Joon; Kim, Hyeon Ji; Kundu, Joydip; Kim, Sung Won; Cho, Dong-Woo

    2014-09-01

    An effective cross-linking of alginate gel was made through reaction with calcium carbonate (CaCO3). We used human chondrocytes as a model cell to study the effects of cross-linking density. Three different pore size ranges of cross-linked alginate hydrogels were fabricated. The morphological, mechanical, and rheological properties of various alginate hydrogels were characterized and responses of biosynthesis of cells encapsulated in each gel to the variation in cross-linking density were investigated. Desired outer shape of structure was maintained when the alginate solution was cross-linked with the applied method. The properties of alginate hydrogel could be tailored through applying various concentrations of CaCO3. The rate of synthesized GAGs and collagens was significantly higher in human chondrocytes encapsulated in the smaller pore structure than that in the larger pore structure. The expression of chondrogenic markers, including collagen type II and aggrecan, was enhanced in the smaller pore structure. It was found that proper structural morphology is a critical factor to enhance the performance and tissue regeneration.

  14. Synthesis of surface protein-imprinted nanoparticles endowed with reversible physical cross-links.

    PubMed

    Yang, Chongchong; Yan, Xianming; Guo, Hao; Fu, Guoqi

    2016-01-15

    Researches on protein molecularly imprinted polymers have been challenged by the difficulties in facilitating biomacromolecular transfer, in particular upon the template removal step, and enhancing their recognition performance. Addressing these issues, herein we report synthesis of core–shell structured surface protein-imprinted nanoparticles with reversible physical cross-links formed in the imprinted nanoshells. The imprinted layers over nanoparticle supports are fabricated via aqueous precipitation polymerization (PP) of di(ethylene glycol) methyl ether methacrylate (MEO2MA), a thermo-responsive monomer bearing no strong H-bond donor, and other functional and cross-linking monomers. During polymerization, physical cross-links together with chemical cross-links are in site produced within the imprinted shells based on hydrophobic association among the PMEO2MA, favoring formation of high-quality imprints. While cooled appropriately below the polymerization temperature, these physical cross-links can be dissociated rapidly, thus facilitating removal of the embedded template. For proof of this concept, lysozyme-imprinted nanoparticles were synthesized at 37 °C over the nanoparticles functionalized with carboxylic and vinyl groups. The template removal from the imprinted nanoparticles was readily achieved by washing with a dilute acidic detergent solution at 4 °C. As-prepared imprinted nanoparticles showed greatly higher imprinting factor and specific rebinding than obtained with the same recipe but by solution polymerization (SP). Moreover, such imprinted nanomaterials exhibited satisfactory rebinding selectivity, kinetics and reusability.

  15. Identification of two nuclear factor-binding domains on the chicken cardiac actin promoter: implications for regulation of the gene.

    PubMed Central

    Quitschke, W W; DePonti-Zilli, L; Lin, Z Y; Paterson, B M

    1989-01-01

    The cis-acting regions that appear to be involved in negative regulation of the chicken alpha-cardiac actin promoter both in vivo and in vitro have been identified. A nuclear factor(s) binding to the proximal region mapped over the TATA element between nucleotides -50 and -25. In the distal region, binding spanned nucleotides -136 to -112, a region that included a second CArG box (CArG2) 5' to the more familiar CCAAT-box (CArG1) consensus sequence. Nuclear factors binding to these different domains were found in both muscle and nonmuscle preparations but were detectable at considerably lower levels in tissues expressing the alpha-cardiac actin gene. In contrast, concentrations of the beta-actin CCAAT-box binding activity were similar in all extracts tested. The role of these factor-binding domains on the activity of the cardiac actin promoter in vivo and in vitro and the prevalence of the binding factors in nonmuscle extracts are consistent with the idea that these binding domains and their associated factors are involved in the tissue-restricted expression of cardiac actin through both positive and negative regulatory mechanisms. In the absence of negative regulatory factors, these same binding domains act synergistically, via other factors, to activate the cardiac actin promoter during myogenesis. Images PMID:2552286

  16. Effective-medium approach for stiff polymer networks with flexible cross-links

    NASA Astrophysics Data System (ADS)

    Broedersz, C. P.; Storm, C.; Mackintosh, F. C.

    2009-06-01

    Recent experiments have demonstrated that the nonlinear elasticity of in vitro networks of the biopolymer actin is dramatically altered in the presence of a flexible cross-linker such as the abundant cytoskeletal protein filamin. The basic principles of such networks remain poorly understood. Here we describe an effective-medium theory of flexibly cross-linked stiff polymer networks. We argue that the response of the cross-links can be fully attributed to entropic stiffening, while softening due to domain unfolding can be ignored. The network is modeled as a collection of randomly oriented rods connected by flexible cross-links to an elastic continuum. This effective medium is treated in a linear elastic limit as well as in a more general framework, in which the medium self-consistently represents the nonlinear network behavior. This model predicts that the nonlinear elastic response sets in at strains proportional to cross-linker length and inversely proportional to filament length. Furthermore, we find that the differential modulus scales linearly with the stress in the stiffening regime. These results are in excellent agreement with bulk rheology data.

  17. Molecular Structures of Isolevuglandin-Protein Cross-Links.

    PubMed

    Bi, Wenzhao; Jang, Geeng-Fu; Zhang, Lei; Crabb, John W; Laird, James; Linetsky, Mikhail; Salomon, Robert G

    2016-10-17

    Isolevuglandins (isoLGs) are stereo and structurally isomeric γ-ketoaldehydes produced through free radical-induced oxidation of arachidonates. Some isoLG isomers are also generated through enzymatic cyclooxygenation. Post-translational modification of proteins by isoLGs is associated with loss-of-function, cross-linking and aggregation. We now report that a low level of modification by one or two molecules of isoLG has a profound effect on the activity of a multi subunit protease, calpain-1. Modification of one or two key lysyl residues apparently suffices to abolish catalytic activity. Covalent modification of calpain-1 led to intersubunit cross-linking. Hetero- and homo-oligomers of the catalytic and regulatory subunits of calpain-1 were detected by SDS-PAGE with Western blotting. N-Acetyl-glycyl-lysine methyl ester and β-amyloid(11-17) peptide EVHHQKL were used as models for characterizing the cross-linking of protein lysyl residues resulting from adduction of iso[4]LGE2. Aminal, bispyrrole, and trispyrrole cross-links of these two peptides were identified and fully characterized by mass spectrometry. Aminal and bispyrrole dimers were both detected. Furthermore, a complex mixture of derivatives of the bispyrrole cross-link containing one or more additional atoms of oxygen was found. Interesting differences are evident in the predominant cross-link type generated in the reaction of iso[4]LGE2 with these peptides. More aminal cross-links versus bispyrrole are formed during the reaction of the dipeptide with iso[4]LGE2. In contrast, more bispyrrole versus aminal cross-links are formed during the reaction of EVHHQKL with iso[4]LGE2. It is tempting to speculate that the EVHHQKL peptide-pyrrole modification forms noncovalent aggregates that favor the production of covalent bispyrrole cross-links because β-amyloid(11-17) tends to spontaneously oligomerize.

  18. Zyxin regulates endothelial von Willebrand factor secretion by reorganizing actin filaments around exocytic granules

    PubMed Central

    Han, Xiaofan; Li, Pin; Yang, Zhenghao; Huang, Xiaoshuai; Wei, Guoqin; Sun, Yujie; Kang, Xuya; Hu, Xueting; Deng, Qiuping; Chen, Liangyi; He, Aibin; Huo, Yingqing; Li, Dong; Betzig, Eric; Luo, Jincai

    2017-01-01

    Endothelial exocytosis of Weibel–Palade body (WPB) is one of the first lines of defence against vascular injury. However, the mechanisms that control WPB exocytosis in the final stages (including the docking, priming and fusion of granules) are poorly understood. Here we show that the focal adhesion protein zyxin is crucial in this process. Zyxin downregulation inhibits the secretion of von Willebrand factor (VWF), the most abundant cargo in WPBs, from human primary endothelial cells (ECs) induced by cAMP agonists. Zyxin-deficient mice exhibit impaired epinephrine-stimulated VWF release, prolonged bleeding time and thrombosis, largely due to defective endothelial secretion of VWF. Using live-cell super-resolution microscopy, we visualize previously unappreciated reorganization of pre-existing actin filaments around WPBs before fusion, dependent on zyxin and an interaction with the actin crosslinker α-actinin. Our findings identify zyxin as a physiological regulator of endothelial exocytosis through reorganizing local actin network in the final stage of exocytosis. PMID:28256511

  19. Gelation threshold of cross-linked polymer brushes.

    PubMed

    Hoffmann, Max; Lang, Michael; Sommer, Jens-Uwe

    2011-02-01

    The cross-linking of polymer brushes is studied using the bond-fluctuation model. By mapping the cross-linking process into a two-dimensional (2D) percolation problem within the lattice of grafting points, we investigate the gelation transition in detail. We show that the particular properties of cross-linked polymer brushes can be reduced to the distribution of bonds which are formed between the grafted chains, and we propose scaling arguments to relate the gelation threshold to the chain length and the grafting density. The gelation threshold is lower than the percolation threshold for 2D bond percolation because of the longer range and broad distribution of bonds formed by the cross-linking process. We term this type of percolation problem star percolation. We observe a broad crossover from mean-field to critical percolation behavior by analyzing the cluster size distribution near the gelation threshold.

  20. Photoreactivities and thermal properties of psoralen cross-links

    SciTech Connect

    Yeung, A.T.; Jones, B.K.; Chu, C.T.

    1988-05-03

    The authors have studied the photoreaction of 8-methoxypsoralen (8-MOP), 4,5',8-trimethylpsoralen (TMP), and 4'-(hydroxymethyl)-4,5',8-trimethylpsoralen (HMT) with a pair of 18-base-long oligonucleotides in which a 14-base region is complementary. Only one 5'TpA site, favored for both monoadduct and cross-link formation with psoralen, is present in this oligonucleotide pair. They have used this model system to demonstrate, for the first time, strand specificity in the photoreaction of psoralen with DNA. They found that the two types of cross-links which form at this site have large differences in thermal stabilities. In addition, the denaturation of each cross-links isomer duplex occurred in at least three stages, which can be visualized as three bands in thermal equilibrium under the conditions of a denaturing polyacrylamide gel. This novel observation suggests that there are several domains differing in thermal stability in a psoralen cross-link.

  1. Cross-linking and rheological changes of whey proteins treated with microbial transglutaminase.

    PubMed

    Truong, Van-Den; Clare, Debra A; Catignani, George L; Swaisgood, Harold E

    2004-03-10

    Modification of the functionality of whey proteins using microbial transglutaminase (TGase) has been the subject of recent studies. However, changes in rheological properties of whey proteins as affected by extensive cross-linking with TGase are not well studied. The factors affecting cross-linking of whey protein isolate (WPI) using both soluble and immobilized TGase were examined, and the rheological properties of the modified proteins were characterized. The enzyme was immobilized on aminopropyl glass beads (CPG-3000) by selective adsorption of the biotinylated enzyme on avidin that had been previously immobilized. WPI (4 and 8% w/w) in deionized water, pH 7.5, containing 10 mM dithiothreitol was cross-linked using enzyme/substrate ratios of 0.12-10 units of activity/g WPI. The reaction was carried out in a jacketed bioreactor for 8 h at 40 degrees C with continuous circulation. The gel point temperature of WPI solutions treated with 0.12 unit of immobilized TGase/g was slightly decreased, but the gel strength was unaffected. However, increasing the enzyme/substrate ratio resulted in extensive cross-linking of WPI that was manifested by increases in apparent viscosity and changes in the gelation properties. For example, using 10 units of soluble TGase/g resulted in extensive cross-linking of alpha-lactalbumin and beta-lactoglobulin in WPI, as evidenced by SDS-PAGE and Western blotting results. Interestingly, the gelling point of WPI solutions increased from 68 to 94 degrees C after a 4-h reaction, and the gel strength was drastically decreased (lower storage modulus, G'). Thus, extensive intra- and interchain cross-linking probably caused formation of polymers that were too large for effective network development. These results suggest that a process could be developed to produce heat-stable whey proteins for various food applications.

  2. Diepoxybutane cross-links DNA at 5'-GNC sequences.

    PubMed

    Millard, J T; White, M M

    1993-03-02

    Epoxides are cancer-causing agents chemically analogous to the nitrogen mustards, a family of powerful antitumor drugs. We found that the DNA interstrand cross-linking sequence preference of diepoxybutane is the same as that of the mustard mechlorethamine: 5'-GNC. Therefore, the genomic site of cross-linking alone cannot explain why some interstrand cross-linkers act as antitumor agents whereas others are deadly toxins.

  3. Large Scale Chemical Cross-linking Mass Spectrometry Perspectives

    PubMed Central

    Zybailov, Boris L.; Glazko, Galina V.; Jaiswal, Mihir; Raney, Kevin D.

    2014-01-01

    The spectacular heterogeneity of a complex protein mixture from biological samples becomes even more difficult to tackle when one’s attention is shifted towards different protein complex topologies, transient interactions, or localization of PPIs. Meticulous protein-by-protein affinity pull-downs and yeast-two-hybrid screens are the two approaches currently used to decipher proteome-wide interaction networks. Another method is to employ chemical cross-linking, which gives not only identities of interactors, but could also provide information on the sites of interactions and interaction interfaces. Despite significant advances in mass spectrometry instrumentation over the last decade, mapping Protein-Protein Interactions (PPIs) using chemical cross-linking remains time consuming and requires substantial expertise, even in the simplest of systems. While robust methodologies and software exist for the analysis of binary PPIs and also for the single protein structure refinement using cross-linking-derived constraints, undertaking a proteome-wide cross-linking study is highly complex. Difficulties include i) identifying cross-linkers of the right length and selectivity that could capture interactions of interest; ii) enrichment of the cross-linked species; iii) identification and validation of the cross-linked peptides and cross-linked sites. In this review we examine existing literature aimed at the large-scale protein cross-linking and discuss possible paths for improvement. We also discuss short-length cross-linkers of broad specificity such as formaldehyde and diazirine-based photo-cross-linkers. These cross-linkers could potentially capture many types of interactions, without strict requirement for a particular amino-acid to be present at a given protein-protein interface. How these shortlength, broad specificity cross-linkers be applied to proteome-wide studies? We will suggest specific advances in methodology, instrumentation and software that are needed to

  4. Neohemoglobins and Cross-Linked Hemoglobins as Blood Substitute.

    DTIC Science & Technology

    1982-12-01

    normal SFH. For bovine hemoglobi, cross-linking of the oxy and carboxy derivatives increased substantially the oxygen affinity and eliminated the oxygen...hemoglobins were prepared by the filtration method. The respective heme-free proteins (apohemoglobins) were prepared by extraction with methyl ...protein. Recombined neohemoglogins and cross-linked hemoglobins were purified by chromatography on CM cellulose using a linear gradient formed by equal

  5. A New Cross-Link for an Old Cross-Linking Drug: The Nitrogen Mustard Anticancer Agent Mechlorethamine Generates Cross-Links Derived from Abasic Sites in Addition to the Expected Drug-Bridged Cross-Links.

    PubMed

    Nejad, Maryam Imani; Johnson, Kevin M; Price, Nathan E; Gates, Kent S

    2016-12-20

    Nitrogen mustard anticancer drugs generate highly reactive aziridinium ions that alkylate DNA. Monoadducts arising from reaction with position N7 of guanine residues are the major DNA adducts generated by these agents. Interstrand cross-links in which the drug bridges position N7 of two guanine residues are formed in low yields relative to those of the monoadducts but are generally thought to be central to medicinal activity. The N7-alkylguanine residues generated by nitrogen mustards are depurinated to yield abasic (Ap) sites in duplex DNA. Here, we show that Ap sites generated by the nitrogen mustard mechlorethamine lead to interstrand cross-links of a type not previously associated with this drug. Gel electrophoretic data were consistent with early evolution of the expected drug-bridged cross-links, followed by the appearance of Ap-derived cross-links. The evidence is further consistent with a reaction pathway involving alkylation of a guanine residue in a 5'-GT sequence, followed by depurination to generate the Ap site, and cross-link formation via reaction of the Ap aldehyde residue with the opposing adenine residue at this site [Price, N. E., Johnson, K. M., Wang, J., Fekry, M. I., Wang, Y., and Gates, K. S. (2014) J. Am. Chem. Soc. 136, 3483-3490]. The monofunctional DNA-alkylating agents 2-chloro-N,N-diethylethanamine 5, (2-chloroethyl)ethylsulfide 6, and natural product leinamycin similarly were found to induce the formation of Ap-derived cross-links in duplex DNA. This work provides the first characterization of Ap-derived cross-links at sequences in which a cytosine residue is located directly opposing the Ap site. Cross-linking processes of this type could be relevant in medicine and biology because Ap sites with directly opposing cytosine residues occur frequently in genomic DNA via spontaneous or enzymatic depurination of guanine and N7-alkylguanine residues.

  6. Cross-linked polyelectrolyte multilayers for marine antifouling applications.

    PubMed

    Zhu, Xiaoying; Jańczewski, Dominik; Lee, Serina Siew Chen; Teo, Serena Lay-Ming; Vancso, G Julius

    2013-07-10

    A polyionic multilayer film was fabricated by layer-by-layer (LbL) sequential deposition followed by cross-linking under mild conditions on a substrate surface to inhibit marine fouling. A novel polyanion, featuring methyl ester groups for an easy cross-linking was used as a generic solution for stabilization of LbL films in a harsh environment. Covalent cross-linking was confirmed by FTIR and XPS spectroscopy. AFM was used to observe film morphology and its variation because of cross-linking, as well as to measure the thickness of the LbL films. Cross-linking improved the stability of the LbL film when it was immersed in artificial seawater, natural seawater, and in a polar organic solvent (DMSO). No changes in the thickness and topography of the film were observed in these media. The LbL films prevented settlement of Amphibalanus amphitrite barnacle cyprids and reduced adhesion of the benthic diatom Amphora coffeaeformis. Assay results indicated that the cross-linking process did not weaken the antifouling effect of LbL films. The high stability and low degree of fouling make these coatings potentially promising candidates in marine applications.

  7. Cross-linking and the molecular packing of corneal collagen

    NASA Technical Reports Server (NTRS)

    Yamauchi, M.; Chandler, G. S.; Tanzawa, H.; Katz, E. P.

    1996-01-01

    We have quantitatively characterized, for the first time, the cross-linking in bovine cornea collagen as a function of age. The major iminium reducible cross-links were dehydro-hydroxylysinonorleucine (deH-HLNL) and dehydro-histidinohydroxymerodesmosine (deH-HHMD). The former rapidly diminished after birth; however, the latter persisted in mature animals at a level of 0.3 - 0.4 moles/mole of collagen. A nonreducible cross-link, histidinohydroxylysinonorleucine (HHL), previously found only in skin, was also found to be a major mature cross-link in cornea. The presence of HHL indicates that cornea fibrils have a molecular packing similar to skin collagen. However, like deH-HHMD, the HHL content in corneal fibrils only reaches a maximum value with time about half that of skin. These data suggest that the corneal fibrils are comprised of discrete filaments that are internally stabilized by HHL and deH-HHMD cross-links. This pattern of intermolecular cross-linking would facilitate the special collagen swelling property required for corneal transparency.

  8. Plant Actin-Depolymerizing Factors Possess Opposing Biochemical Properties Arising from Key Amino Acid Changes throughout Evolution.

    PubMed

    Nan, Qiong; Qian, Dong; Niu, Yue; He, Yongxing; Tong, Shaofei; Niu, Zhimin; Ma, Jianchao; Yang, Yang; An, Lizhe; Wan, Dongshi; Xiang, Yun

    2017-02-01

    Functional divergence in paralogs is an important genetic source of evolutionary innovation. Actin-depolymerizing factors (ADFs) are among the most important actin binding proteins and are involved in generating and remodeling actin cytoskeletal architecture via their conserved F-actin severing or depolymerizing activity. In plants, ADFs coevolved with actin, but their biochemical properties are diverse. Unfortunately, the biochemical function of most plant ADFs and the potential mechanisms of their functional divergence remain unclear. Here, in vitro biochemical analyses demonstrated that all 11 ADF genes in Arabidopsis thaliana exhibit opposing biochemical properties. Subclass III ADFs evolved F-actin bundling (B-type) function from conserved F-actin depolymerizing (D-type) function, and subclass I ADFs have enhanced D-type function. By tracking historical mutation sites on ancestral proteins, several fundamental amino acid residues affecting the biochemical functions of these proteins were identified in Arabidopsis and various plants, suggesting that the biochemical divergence of ADFs has been conserved during the evolution of angiosperm plants. Importantly, N-terminal extensions on subclass III ADFs that arose from intron-sliding events are indispensable for the alteration of D-type to B-type function. We conclude that the evolution of these N-terminal extensions and several conserved mutations produced the diverse biochemical functions of plant ADFs from a putative ancestor.

  9. Plant Actin-Depolymerizing Factors Possess Opposing Biochemical Properties Arising from Key Amino Acid Changes throughout Evolution[OPEN

    PubMed Central

    Nan, Qiong; Niu, Yue; He, Yongxing; Tong, Shaofei; Niu, Zhimin; Ma, Jianchao; Yang, Yang; An, Lizhe; Wan, Dongshi

    2017-01-01

    Functional divergence in paralogs is an important genetic source of evolutionary innovation. Actin-depolymerizing factors (ADFs) are among the most important actin binding proteins and are involved in generating and remodeling actin cytoskeletal architecture via their conserved F-actin severing or depolymerizing activity. In plants, ADFs coevolved with actin, but their biochemical properties are diverse. Unfortunately, the biochemical function of most plant ADFs and the potential mechanisms of their functional divergence remain unclear. Here, in vitro biochemical analyses demonstrated that all 11 ADF genes in Arabidopsis thaliana exhibit opposing biochemical properties. Subclass III ADFs evolved F-actin bundling (B-type) function from conserved F-actin depolymerizing (D-type) function, and subclass I ADFs have enhanced D-type function. By tracking historical mutation sites on ancestral proteins, several fundamental amino acid residues affecting the biochemical functions of these proteins were identified in Arabidopsis and various plants, suggesting that the biochemical divergence of ADFs has been conserved during the evolution of angiosperm plants. Importantly, N-terminal extensions on subclass III ADFs that arose from intron-sliding events are indispensable for the alteration of D-type to B-type function. We conclude that the evolution of these N-terminal extensions and several conserved mutations produced the diverse biochemical functions of plant ADFs from a putative ancestor. PMID:28123105

  10. Influence of cross-linking degree of a biodegradable genipin-cross-linked gelatin guide on peripheral nerve regeneration.

    PubMed

    Lu, Ming-Chin; Hsiang, Shih-Wei; Lai, Tung-Yuan; Yao, Chun-Hsu; Lin, Li-Yu; Chen, Yueh-Sheng

    2007-01-01

    We evaluated peripheral nerve regeneration using biodegradable genipin-cross-linked gelatin nerve conduits (GGCs) with three different cross-linking degrees, 24, 36 and 51%. Biocompatibility and biodegradability of the GGC and its efficiency as a guidance channel were examined based on the repair process of a 10-mm gap in the rat sciatic nerve. From this pilot study we concluded that GGCs with a mean cross-linking degree of 36% can ensure nerve regeneration with a more mature structure, as demonstrated by better developed epineural and perineural organisation and axonal development, as well as better-recovered electrophysiology with a relatively positive sciatic functional index and a shorter latency of the muscle action potential curve. Regenerated nerves in the GGCs with mean cross-linking degrees of 24 and 51% were less favourable, due to irritation caused by degradation material and compression by the remaining tube walls, respectively.

  11. Cross-linked smart poly(dimethylsiloxane) membranes for removal of volatile organic compounds in water

    NASA Astrophysics Data System (ADS)

    Ohshima, Tadahiro; Miyata, Takashi; Uragami, Tadashi; Berghmens, Hugo

    2005-04-01

    This paper focuses on the effects of fluorine cross-linker of the cross-linked poly(dimethylsiloxane) membranes from polydimethylsiloxane dimethylmethacrylate macromonomer (PDMSDMMA) and divinyl perfluoro- n-hexane (DVF) on the pervaporation characteristics of the removal of benzene from an aqueous solution of dilute benzene. When an aqueous solution of 0.05 wt% benzene was permeated through the cross-linked PDMSDMMA (PDMSDMMA-DVF) membranes, they showed a high benzene permselectivity and permeability of these membranes was enhanced with increasing DVF content significantly. The best normalized permeation rate, separation factor for benzene permselectivity, and pervaporation separation index (PSI) of a PDMSDMMA-DVF membrane were 1.72×10 -5 kg m/m 2 h, 4316, and 7423, respectively. The best normalized permeation rate of a PDMSDMMA-DVF membrane was approximately same as the PDMSDMMA membranes cross-linked with other divinyl compounds, but the separation factor and PSI of the former membrane were greater than those of the latter ones. These pervaporation characteristics are discussed from the viewpoint of chemical and physical structure of the cross-linked PDMSDMMA-DVF membranes in detail.

  12. 1,2,3,4-Diepoxybutane-Induced DNA-Protein Cross-Linking in Human Fibrosarcoma (HT1080) Cells

    PubMed Central

    Gherezghiher, Teshome B.; Ming, Xun; Villalta, Peter; Campbell, Colin; Tretyakova, Natalia Y.

    2013-01-01

    1,2,3,4-diepoxybutane (DEB) is the key carcinogenic metabolite of 1,3-butadiene (BD), an important industrial and environmental chemical present in urban air and in cigarette smoke. DEB is a genotoxic bis-electrophile capable of cross-linking cellular biomolecules to form DNA-DNA and DNA-protein cross-links (DPCs). In the present work, mass spectrometry-based proteomics was employed to characterize DEB-mediated DNA-protein cross-linking in human fibrosarcoma (HT1080) cells. Over 150 proteins including histones, high mobility group proteins, transcription factors, splicing factors, and tubulins were found among those covalently cross-linked to chromosomal DNA in the presence of DEB. A large portion of the cross-linked proteins are known factors involved in DNA binding, transcriptional regulation, cell signaling, DNA repair, and DNA damage response. HPLC-ESI+-MS/MS analysis of total proteolytic digests revealed the presence of 1-(S-cysteinyl)-4-(guan-7-yl)-2,3-butanediol conjugates, confirming that DEB forms DPCs between cysteine thiols within proteins and the N-7 guanine positions within DNA. However, relatively high concentrations of DEB were required to achieve significant DPC formation, indicating that it is a poor cross-linking agent as compared to antitumor nitrogen mustards and platinum compounds. PMID:23506368

  13. Synthesis and enzymatic degradation of epichlorohydrin cross-linked pectins.

    PubMed

    Semdé, Rasmané; Moës, André J; Devleeschouwer, Michel J; Amighi, Karim

    2003-02-01

    The water solubility of pectin was successfully decreased by cross-linking with increasing amounts of epichlorohydrin in the reaction media. The initial molar ratios of epichlorohydrin/ galacturonic acid monomer in the reaction mixtures were 0, 0.37, 0.56, 0.74, 1.00, 1.47, and 2.44. The resulting epichlorohydrin cross-linked pectins were thus referred to as C-LP0, C-LP37, C-LP56, C-LP75, C-LP100, C-LP150, and C-LP250, respectively. Methoxylation degrees ranged from 60.5 +/- 0.9% to 68.0 +/- 0.6%, and the effective cross-linking degrees, determined by quantification of the hydroxyl anions consumed during the reaction, were 0, 17.8, 26.0, 38.3, 46.5, 53.5, and 58.7%. respectively. After incubating the different cross-linked pectins (0.5% w/v) in 25 mL of 0.05 M acetate-phosphate buffer (pH 4.5), containing 50 microL of Pectinex Ultra SP-L (pectinolytic enzymes), between 60 and 80% of the pectin osidic bounds were broken in less than 1 hr. Moreover, increasing the cross-linking degree only resulted in a weak slowing on the enzymatic degradation velocity.

  14. Mapping of psoralen cross-linked nucleotides in RNA.

    PubMed Central

    Garrett-Wheeler, E; Lockard, R E; Kumar, A

    1984-01-01

    A method is described for using the cross-linking reagent 4'-(hydroxy-methyl)-4,5',8-trimethylpsoralen (HMT) to map base paired regions and higher-order structure within RNA molecules. Applying this method to yeast tRNAPhe, we have specifically identified cross-links within the acceptor stem between U6 X U68, in the D-stem between C11 X C25, and in the T psi-stem between U50 X C63 and U52 X C63. We have also identified a unique cross-link between U8 X C48 which are trans pyrimidines in the core region due to tertiary interactions between U8:A14 and C48:G15. The precise point of cross-linking was deduced in every case by using purine-specific U2 ribonuclease along with cytidine-specific CL3 ribonuclease which will anomalously cleave after photoreversed pyrimidines. The ability to map the precise point of cross-linking should prove invaluable in identifying nucleotides in close proximity within the tertiary structure of other RNA molecules. Images PMID:6425802

  15. Spectroscopic characterization of collagen cross-links in bone

    NASA Technical Reports Server (NTRS)

    Paschalis, E. P.; Verdelis, K.; Doty, S. B.; Boskey, A. L.; Mendelsohn, R.; Yamauchi, M.

    2001-01-01

    Collagen is the most abundant protein of the organic matrix in mineralizing tissues. One of its most critical properties is its cross-linking pattern. The intermolecular cross-linking provides the fibrillar matrices with mechanical properties such as tensile strength and viscoelasticity. In this study, Fourier transform infrared (FTIR) spectroscopy and FTIR imaging (FTIRI) analyses were performed in a series of biochemically characterized samples including purified collagen cross-linked peptides, demineralized bovine bone collagen from animals of different ages, collagen from vitamin B6-deficient chick homogenized bone and their age- and sex-matched controls, and histologically stained thin sections from normal human iliac crest biopsy specimens. One region of the FTIR spectrum of particular interest (the amide I spectral region) was resolved into its underlying components. Of these components, the relative percent area ratio of two subbands at approximately 1660 cm(-1) and approximately 1690 cm(-1) was related to collagen cross-links that are abundant in mineralized tissues (i.e., pyridinoline [Pyr] and dehydrodihydroxylysinonorleucine [deH-DHLNL]). This study shows that it is feasible to monitor Pyr and DHLNL collagen cross-links spatial distribution in mineralized tissues. The spectroscopic parameter established in this study may be used in FTIRI analyses, thus enabling the calculation of relative Pyr/DHLNL amounts in thin (approximately 5 microm) calcified tissue sections with a spatial resolution of approximately 7 microm.

  16. Mixed-Isotope Labeling with LC-IMS-MS for Characterization of Protein–Protein Interactions by Chemical Cross-Linking

    SciTech Connect

    Merkley, Eric D.; Baker, Erin S.; Crowell, Kevin L.; Orton, Daniel J.; Taverner, Thomas; Ansong, Charles; Ibrahim, Yehia M.; Burnet, Meagan C.; Cort, John R.; Anderson, Gordon A.; Smith, Richard D.; Adkins, Joshua N.

    2013-02-20

    Chemical cross-linking of proteins followed by proteolysis and mass spectrometric analysis of the resulting cross-linked peptides can provide insights into protein structure and protein-protein interactions. However, cross-linked peptides are by necessity of low stoichometry and have different physicochemical properties than linear peptides, routine unambiguous identification of the cross-linked peptides has remained difficult. To address this challenge, we demonstrated the use of liquid chromatography and ion mobility separations coupled with mass spectrometry in combination with a heavy-isotope labeling method. The combination of mixed-isotope cross-linking and ion mobility provided unique and easily interpretable spectral multiplet features for the intermolecular cross-linked peptides. Application of the method to two different homodimeric proteins - SrfN, a virulence factor from Salmonella Typhimurium and SO_2176, a protein of unknown function from Shewanella oneidensis- revealed several cross-linked peptides from both proteins that were identified with a low false discovery rate (estimated using a decoy approach). A greater number of cross-linked peptides were identified using ion mobility drift time information in the analysis than when the data were summed across the drift time dimension before analysis. The identified cross-linked peptides migrated more quickly in the ion mobility drift tube than the unmodified peptides.

  17. Extreme dryness and DNA-protein cross-links

    NASA Astrophysics Data System (ADS)

    Bieger-Dose, A.; Dose, K.; Meffert, R.; Mehler, M.; Risi, S.

    Exposure of fungal conidia (Aspergillus ochraceus) or spores of Bacillus subtilis to extreme dryness or vacuum induces DNA lesions, including strand breaks and the formation of DNA-protein cross-links. In wet cells only a small amount of protein is bound to DNA, but exposure to conditions of lowered water activity results in an increasing number of cross-links between DNA and proteins. In fungal conidia these cross-links are detected after selective iodination (125J) of the DNA-bound proteins followed by gel electrophoresis and subsequent autoradiography. Another approach is the labelling of DNA with 32p by means of nick translation and the detection of differences in the electrophoretic mobility of DNA before and after digestion with proteinase K of proteins bound to DNA.

  18. Functional polymer laminates from hyperthermal hydrogen induced cross-linking.

    PubMed

    Thompson, David B; Trebicky, Tomas; Crewdson, Patrick; McEachran, Matthew J; Stojcevic, Goran; Arsenault, Gilles; Lau, Woon M; Gillies, Elizabeth R

    2011-12-20

    The use of a hyperthermal hydrogen induced cross-linking process to prepare laminates comprising polypropylene, poly(isobutylene-co-isoprene), and poly(vinyl acetate) is described. In this new, milder alternative to conventional plasma techniques, neutral molecular hydrogen projectiles were used to create carbon radicals on impacted surfaces by collision-induced dissociation of C-H bonds, and this process was used to cross-link polymers on a polypropylene surface. It was demonstrated that multiple layers of cross-linked materials could be added, creating polymer laminates with each layer introducing new functionalities and properties. In particular, the present work shows that the process is largely nondestructive toward ester functionalities. First, the esters were grafted to become nonleachable. Then, the esters were subsequently hydrolyzed to convert the surface from hydrophobic to hydrophilic. Afterward, the esters could be recovered by simple esterification demonstrating that further chemical transformations were possible.

  19. FTIR Spectroscopic Studies on Cross Linking of SU-8 Photoresist

    NASA Astrophysics Data System (ADS)

    Kalaiselvi, S. M. P.; Tan, T. L.; Rawat, R. S.; Lee, P.; Heussler, S. P.; Breese, M. B. H.

    2013-11-01

    The usage of chemically-amplified, negative tone SU-8 photoresist is numerous, spanning industrial, scientific and medical fields. Hence, in this study, some preliminary studies were conducted to understand the dosage and heat treatment requirements of the SU-8 photoresist essential for pattern generation using X-ray lithography. In this work, using Synchrotron as the X-ray source, SU-8 photoresist was characterized for X-ray lithography in terms of its process parameters such as X-ray exposure dose, post exposure bake (PEB) time and temperature for various photoresist thicknesses which is considered worthwhile in view of applications of SU-8 for the fabrication of very high aspect ratio micro structures. The process parameters were varied and the resultant cross linking of the molecular chains of the photoresist was accurately monitored using a Fourier Transform Infra-Red (FTIR) spectrometer and the results are discussed. The infrared absorption peak at 914 cm-1 in the spectrum of the SU-8 photoresist was found to be a useful indicator for the completion of cross linking in the SU-8 photoresist. Results show that the cross linking of the SU-8 photoresist is at a higher rate from 0 J/cm3 to 30 J/cm3 after which the peak almost saturates regardless of the PEB time. It is a good evidence for the validation of dosage requirement of SU-8 photoresist for effective completion of cross linking, which in turn is a requirement for efficient fabrication of micro and nano structures. An analogous behavior was also observed between the extent of cross linking and the PEB time and temperature. The rate of cross linking declines after a certain period of PEB time regardless of PEB temperature. The obtained results also show a definite relation between variation of the absorbance area of the peak at 914 cm-1 and the X-ray exposure dose.

  20. Positronium yields in amorphous, cross-linked and conductive polystyrene

    NASA Astrophysics Data System (ADS)

    Procházka, Ivan; Čížek, Jakub; Motyčka, Václav

    2007-02-01

    Variations in positronium yields due to positron irradiation of specimens during experiment were investigated on the three commercially available modifications of polystyrene (Goodfellow): amorphous, cross-linked and conductive. Positron lifetime technique was employed. The variations of the positronium yields were expressed as changes of the ortho-positronium intensity as functions of the irradiation time. It was found that the positronium yield curves obtained for the amorphous and cross-linked polystyrene cannot be represented as a simple single-exponential relaxation towards a steady state and at least one additional component or a modified shape of the relaxation curve should be considered.

  1. Cross-Linked Nanotube Materials with Variable Stiffness Tethers

    NASA Technical Reports Server (NTRS)

    Frankland, Sarah-Jane V.; Odegard, Gregory M.; Herzog, Matthew N.; Gates, Thomas S.; Fay, Catherine C.

    2004-01-01

    The constitutive properties of a cross-linked single-walled carbon nanotube material are predicted with a multi-scale model. The material is modeled as a transversely isotropic solid using concepts from equivalent-continuum modeling. The elastic constants are determined using molecular dynamics simulation. Some parameters of the molecular force field are determined specifically for the cross-linker from ab initio calculations. A demonstration of how the cross-linked nanotubes may affect the properties of a nanotube/polyimide composite is included using a micromechanical analysis.

  2. A gel network constituted by rigid schizophyllan chains and nonpermanent cross-links.

    PubMed

    Fang, Yapeng; Takahashi, Rheo; Nishinari, Katsuyoshi

    2004-01-01

    This work reports a gel network formed by rigid schizophyllan (SPG) chains with Borax as a cross-linking agent. The formed cross-links are non-permanent and somewhat dynamic in nature because the cross-linking reaction is governed by a complexation equilibrium. Gelation processes are traced by dynamic viscoelastic measurements to examine the effects of Borax content, SPG concentration, temperature, salt concentration, salt type, and strain. The first-order kinetic model containing three parameters, t(0) (induction time), 1/tau(c) (gelation rate), and (saturated storage modulus), is successfully applied to describe the gelation of the SPG-Borax system. Gelation occurs faster at higher Borax content, higher SPG concentration, higher salt concentration, or lower temperature. Moreover the gelation is cation-type-specific. Storage modulus is a linear function of both Borax content and SPG concentration. The linear relationship between storage modulus and Borax content can be explained by a modified ideal rubber elasticity theory with a front factor alpha to take into account the presence of ineffective cross-links and the effect of SPG chain rigidity. On the other hand, the linear dependence of storage modulus on SPG concentration could be explained on the basis of chain-chain contacting behavior of extended SPG chains. Apparent activation energy and cross-linking enthalpy are calculated to be -74.5 and -32.4 kJ/mol for the present system. Strain sweep measurements manifest that the elasticity behavior of this gel starts to deviate from Gaussian-chain network at a small strain of 10%.

  3. Triglycidylamine Cross-linking Combined with Ethanol Inhibits Bioprosthetic Heart Valve Calcification

    PubMed Central

    Connolly, Jeanne M.; Bakay, Marina A.; Alferiev, Ivan S.; Gorman, Robert C.; Gorman, Joseph H.; Kruth, Howard S.; Ashworth, Paul E.; Kutty, Jaishankar K.; Schoen, Frederick J.; Bianco, Richard W.; Levy, Robert J.

    2012-01-01

    Background One of the most important factors responsible for the calcific failure of bioprosthetic heart valves is glutaraldehyde cross-linking. Ethanol (EtOH) incubation after glutaraldehyde cross-linking has previously been reported to confer anti-calcification efficacy for bioprostheses. The present studies investigated the anticalcification efficacy in vivo of the novel cross-linking agent, triglycidyl amine (TGA), with or without EtOH incubation, in comparison to glutaraldehyde. Methods TGA cross-linking (+/− EtOH) was used to prepare porcine aortic valves for both rat subdermal implants and sheep mitral valve replacements, for comparisons with glutaraldehyde-fixed controls. Thermal denaturation temperature (Ts), an index of cross-linking, cholesterol extraction, and hydrodynamic properties were quantified. Explant endpoints included quantitative and morphologic assessment of calcification. Results Ts after TGA were intermediate between unfixed and glutaraldehyde-fixed. EtOH incubation resulted in almost complete extraction of cholesterol from TGA or glutaraldehyde-fixed cusps. Rat subdermal explants (90days) demonstrated that TGA-EtOH resulted in a significantly greater level of inhibition of calcification than other conditions. Thus, TGA-ethanol stent mounted porcine aortic valve bioprostheses were fabricated for comparisons with glutaraldehyde-pretreated controls. In hydrodynamic studies, TGA-EtOH bioprostheses had lower pressure gradients than glutaraldehyde-fixed. TGA-ethanol bioprostheses used as mitral valve replacements in juvenile sheep (150 days) demonstrated significantly lower calcium levels in both explanted porcine aortic cusp and aortic wall samples compared to glutaraldehyde-fixed controls. However, TGA-EtOH sheep explants also demonstrated isolated calcific nodules and intracuspal hematomas. Conclusions TGA-EtOH pretreatment of porcine aortic valves confers significant calcification resistance in both rat subdermal and sheep circulatory

  4. Biocompatibility of chemically cross-linked gelatin hydrogels for ophthalmic use.

    PubMed

    Lai, Jui-Yang

    2010-06-01

    Biocompatibility is a major requirement for the development of functional biomaterials for ophthalmic applications. In this study, we investigated the effect of cross-linker functionality on ocular biocompatibility of chemically modified gelatin hydrogels. The test materials were cross-linked with glutaraldehyde (GTA) or 1-ethyl-3-(3-dimethyl aminopropyl)carbodiimide (EDC), and were analyzed using in vitro and in vivo assays. Primary rat iris pigment epithelial cultures were incubated with various gelatin discs for 2 days, and the cellular responses were monitored by cell proliferation, viability, and pro-inflammatory gene and cytokine expression. The results demonstrated that the cells exposed to EDC cross-linked gelatins had relatively lower lactate dehydrogenase activity, cytotoxicity, and interleukin-1beta and tumor necrosis factor-alpha levels than did those to GTA treated samples. In addition, the gelatin implants were inserted in the anterior chamber of rabbit eyes for 12 weeks and characterized by clinical observations and scanning electron microscopy studies. The EDC cross-linked gelatin hydrogels exhibited good biocompatibility and were well tolerated without causing toxicity and adverse effects. However, a significant inflammatory reaction was elicited by the presence of GTA treated materials. It was noted that, despite its biocompatibility, the potential application of non-cross-linked gelatin for local delivery of cell and drug therapeutics would be limited due to rapid dissolution in aqueous environments. In conclusion, these findings suggest ocular cell/tissue response to changes in cross-linker properties. In comparison to GTA treatment, the EDC cross-linking is more suitable for preparation of chemically modified gelatin hydrogels for ophthalmic use.

  5. Translation elongation factor EF-Tu modulates filament formation of actin-like MreB protein in vitro.

    PubMed

    Defeu Soufo, Hervé Joël; Reimold, Christian; Breddermann, Hannes; Mannherz, Hans G; Graumann, Peter L

    2015-04-24

    EF-Tu has been shown to interact with actin-like protein MreB and to affect its localization in Escherichia coli and in Bacillus subtilis cells. We have purified YFP-MreB in an active form, which forms filaments on glass slides in vitro and was active in dynamic light-scattering assays, polymerizing in milliseconds after addition of magnesium. Purified EF-Tu enhanced the amount of MreB filaments, as seen by sedimentation assays, the speed of filament formation and the length of MreB filaments in vitro. EF-Tu had the strongest impact on MreB filaments in a 1:1 ratio, and EF-Tu co-sedimented with MreB filaments, revealing a stoichiometric interaction between both proteins. This was supported by cross-linking assays where 1:1 species were well detectable. When expressed in E. coli cells, B. subtilis MreB formed filaments and induced the formation of co-localizing B. subtilis EF-Tu structures, indicating that MreB can direct the positioning of EF-Tu structures in a heterologous cell system. Fluorescence recovery after photobleaching analysis showed that MreB filaments have a higher turnover in B. subtilis cells than in E. coli cells, indicating different filament kinetics in homologous or heterologous cell systems. The data show that MreB can direct the localization of EF-Tu in vivo, which in turn positively affects the formation and dynamics of MreB filaments. Thus, EF-Tu is a modulator of the activity of a bacterial actin-like protein.

  6. The composition and role of cross links in mechanoelectrical transduction in vertebrate sensory hair cells.

    PubMed

    Hackney, Carole M; Furness, David N

    2013-04-15

    The key components of acousticolateralis systems (lateral line, hearing and balance) are sensory hair cells. At their apex, these cells have a bundle of specialized cellular protrusions, which are modified actin-containing microvilli, connected together by extracellular filaments called cross links. Stereociliary deflections open nonselective cation channels allowing ions from the extracellular environment into the cell, a process called mechanoelectrical transduction. This produces a receptor potential that causes the release of the excitatory neurotransmitter glutamate onto the terminals of the sensory nerve fibres, which connect to the cell base, causing nerve signals to be sent to the brain. Identification of the cellular mechanisms underlying mechanoelectrical transduction and of some of the proteins involved has been assisted by research into the genetics of deafness, molecular biology and mechanical measurements of function. It is thought that one type of cross link, the tip link, is composed of cadherin 23 and protocadherin 15, and gates the transduction channel when the bundle is deflected. Another type of link, called lateral (or horizontal) links, maintains optimal bundle cohesion and stiffness for transduction. This Commentary summarizes the information currently available about the structure, function and composition of the links and how they might be relevant to human hearing impairment.

  7. The Interplay between Viscoelastic and Thermodynamic Properties Determines the Birefringence of F-Actin Gels

    PubMed Central

    Helfer, Emmanuèle; Panine, Pierre; Carlier, Marie-France; Davidson, Patrick

    2005-01-01

    F-actin gels of increasing concentrations (25–300 μM) display in vitro a progressive onset of birefringence due to orientational ordering of actin filaments. At F-actin concentrations <100 μM, this birefringence can be erased and restored at will by sonication and gentle flow, respectively. Hence, the orientational ordering does not result from a thermodynamic transition to a nematic phase but instead is due to mechanical stresses stored in the gels. In contrast, at F-actin concentrations ≥100 μM, gels display spontaneous birefringence recovery, at rest, which is the sign of true nematic ordering, in good agreement with statistical physics models of the isotropic/nematic transition. Well-aligned samples of F-actin gels could be produced and their small-angle x-ray scattering patterns are quite anisotropic. These patterns show no sign of filament positional short-range order and could be modeled by averaging the form factor with the Maier-Saupe nematic distribution function. The derived nematic order parameter S of the gels ranged from S = 0.7 at 300 μM to S = 0.4 at 25 μM. Both birefringence and small-angle x-ray scattering data indicate that, even in absence of cross-linking proteins, spontaneous cooperative alignment of actin filaments may arise in motile regions of living cells where F-actin concentrations can reach values of a few 100 μM. PMID:15863487

  8. In vitro expression of the alpha-smooth muscle actin isoform by rat lung mesenchymal cells: regulation by culture condition and transforming growth factor-beta.

    PubMed

    Mitchell, J J; Woodcock-Mitchell, J L; Perry, L; Zhao, J; Low, R B; Baldor, L; Absher, P M

    1993-07-01

    alpha-Smooth muscle actin (alpha SM actin)-containing cells recently have been demonstrated in intraalveolar lesions in both rat and human tissues following lung injury. In order to develop model systems for the study of such cells, we examined cultured lung cell lines for this phenotype. The adult rat lung fibroblast-like "RL" cell lines were found to express alpha SM actin mRNA and protein and to organize this actin into stress fiber-like structures. Immunocytochemical staining of subclones of the RL87 line demonstrated the presence in the cultures of at least four cell phenotypes, one that fails to express alpha SM actin and three distinct morphologic types that do express alpha SM actin. The proportion of cellular actin that is the alpha-isoform was modulated by the culture conditions. RL cells growing at low density expressed minimal alpha SM actin. On reaching confluent densities, however, alpha SM actin increased to at least 20% of the total actin content. This effect, combined with the observation that the most immunoreactive cells were those that displayed overlapping cell processes in culture, suggests that cell-cell contact may be involved in actin isoform regulation in these cells. Similar to the response of some smooth muscle cell lines, alpha SM actin expression in RL cells also was promoted by conditions, e.g., maintenance in low serum medium, which minimize cell division. alpha SM actin expression was modulated in RL cells by the growth factor transforming growth factor-beta. Addition of this cytokine to growing cells substantially elevated the proportion of alpha SM actin protein.(ABSTRACT TRUNCATED AT 250 WORDS)

  9. Polyimide Aerogels with Three-Dimensional Cross-Linked Structure

    NASA Technical Reports Server (NTRS)

    Meador, Mary Ann B. (Inventor)

    2016-01-01

    A method for creating a three dimensional cross-linked polyimide structure includes dissolving a diamine, a dianhydride, and a triamine in a solvent, imidizing a polyamic acid gel by heating the gel, extracting the gel in a second solvent, supercritically drying the gel, and removing the solvent to create a polyimide aerogel.

  10. Porous Cross-Linked Polyimide-Urea Networks

    NASA Technical Reports Server (NTRS)

    Meador, Mary Ann B. (Inventor); Nguyen, Baochau N. (Inventor)

    2015-01-01

    Porous cross-linked polyimide-urea networks are provided. The networks comprise a subunit comprising two anhydride end-capped polyamic acid oligomers in direct connection via a urea linkage. The oligomers (a) each comprise a repeating unit of a dianhydride and a diamine and a terminal anhydride group and (b) are formulated with 2 to 15 of the repeating units. The subunit was formed by reaction of the diamine and a diisocyanate to form a diamine-urea linkage-diamine group, followed by reaction of the diamine-urea linkage-diamine group with the dianhydride and the diamine to form the subunit. The subunit has been cross-linked via a cross-linking agent, comprising three or more amine groups, at a balanced stoichiometry of the amine groups to the terminal anhydride groups. The subunit has been chemically imidized to yield the porous cross-linked polyimide-urea network. Also provided are wet gels, aerogels, and thin films comprising the networks, and methods of making the networks.

  11. Potential Effects of Corneal Cross-Linking upon the Limbus

    PubMed Central

    2016-01-01

    Corneal cross-linking is nowadays the most used strategy for the treatment of keratoconus and recently it has been exploited for an increasing number of different corneal pathologies, from other ectatic disorders to keratitis. The safety of this technique has been widely assessed, but clinical complications still occur. The potential effects of cross-linking treatment upon the limbus are incompletely understood; it is important therefore to investigate the effect of UV exposure upon the limbal niche, particularly as UV is known to be mutagenic to cellular DNA and the limbus is where ocular surface tumors can develop. The risk of early induction of ocular surface cancer is undoubtedly rare and has to date not been published other than in one case after cross-linking. Nevertheless it is important to further assess, understand, and reduce where possible any potential risk. The aim of this review is to summarize all the reported cases of a pathological consequence for the limbal cells, possibly induced by cross-linking UV exposure, the studies done in vitro or ex vivo, the theoretical bases for the risks due to UV exposure, and which aspects of the clinical treatment may produce higher risk, along with what possible mechanisms could be utilized to protect the limbus and the delicate stem cells present within it. PMID:27689081

  12. Cross-linking of dithiols by mitomycin C.

    PubMed

    Paz, Manuel M

    2010-08-16

    Upon reduction, the antitumor drug mitomycin C undergoes a cascade of reactions to give a bis-electrophile that alkylates cellular nucleophiles. We recently reported that dithiols activate mitomycin C by reduction, and we report here that dithiols, after executing the reductive activation of mitomycin C, are bis-alkylated by the activated drug to form S,S'-cross-links as the predominant end products. The diastereomeric pair of adducts formed by 1,3-propanedithiol has been fully characterized by UV, HRMS, CD, and NMR experiments. Racemic dithiol (+/-)-dithiothreitol gave four diastereomeric cross-links, and (+/-)-dihydrolipoic acid gave eight cross-links (two regioisomers with four diastereomers each) that were partially characterized by UV and MS. The observed dependence of cross-link formation on dithiol concentration indicated the requirement of a second reduction step by dithiol, prior to the alkylation of the second arm of the dithiol. The existence of unidentified reaction pathways was manifested by the formation of unexpected intermediates during the course of the reaction of mitomycin C with dithiols and by the formation of unsoluble mitosene derivatives in the reaction between equimolar amounts of dithiol and mitomycin C. Mechanistic details of the reaction are addressed in light of these results. Finally, we discuss the potential relevance of our findings for the interaction of mitomycin C with dithiol-containing proteins.

  13. Citric-acid-derived photo-cross-linked biodegradable elastomers.

    PubMed

    Gyawali, Dipendra; Tran, Richard T; Guleserian, Kristine J; Tang, Liping; Yang, Jian

    2010-01-01

    Citric-acid-derived thermally cross-linked biodegradable elastomers (CABEs) have recently received significant attention in various biomedical applications, including tissue-engineering orthopedic devices, bioimaging and implant coatings. However, citric-acid-derived photo-cross-linked biodegradable elastomers are rarely reported. Herein, we report a novel photo-cross-linked biodegradable elastomer, referred to as poly(octamethylene maleate citrate) (POMC), which preserves pendant hydroxyl and carboxylic functionalities after cross-linking for the potential conjugation of biologically active molecules. Pre-POMC is a low-molecular-mass pre-polymer with an average molecular mass between 701 and 1291 Da. POMC networks are soft and elastic with an initial modulus of 0.07 to 1.3 MPa and an elongation-at-break between 38 and 382%. FT-IR-ATR results confirmed the successful surface immobilization of type-I collagen onto POMC films, which enhanced in vitro cellular attachment and proliferation. Photo-polymerized POMC films implanted subcutaneously into Sprague-Dawley rats demonstrated minimal in vivo inflammatory responses. The development of POMC enriches the family of citric-acid-derived biodegradable elastomers and expands the available biodegradable polymers for versatile needs in biomedical applications.

  14. Viscoelastic Nanomechanics of Ionically Cross-linked Polyelectrolyte Networks

    NASA Astrophysics Data System (ADS)

    Han, Biao; Lee, Daeyeon; Han, Lin

    2015-03-01

    Understanding the mechanics of ionic polyelectrolyte networks is critical for applications where nm-to-um mechanics is the key to success. This study aims to reveal the roles of ionic cross-links and fixed charges in the viscoelasticity of layer-by-layer poly(allylamine hydrochloride)/poly(acrylic acid) microfilms, PAH/PAA, a complex held by pH-sensitive amine-carboxyl links. AFM-nanoindentation and force relaxation (tip R =12.5um) was performed at ionic strength(IS) =0.01-1.0M, pH =5.5-2.0 (pKa of PAA =2.3). When pH changes from 5.5 to 2.0, the films swell for 4x from densely linked, net neutral state to loosely linked, positively charged one. A >100x reduction in indentation modulus was observed at all IS, suggesting the dominance of decrease in cross-link density. In most states, more than 90% force relaxation was observed, where cross-link breaking/reformation likely dominates viscoelasticity. However, at pH =2.5 and IS =0.01M, when electrical double layer repulsion is important (Debye length =3nm), relaxation was about 60%, highlighting the contribution of fixed charges. In summary, this study revealed unique viscoelastic behaviors of PAH/PAA due to the pH- and IS-dependent cross-link and charge densities.

  15. Femtosecond laser collagen cross-linking without traditional photosensitizers

    NASA Astrophysics Data System (ADS)

    Guo, Yizang; Wang, Chao; Celi, Nicola; Vukelic, Sinisa

    2015-03-01

    Collagen cross-linking in cornea has the capability of enhancing its mechanical properties and thereby providing an alternative treatment for eye diseases such as keratoconus. Currently, riboflavin assisted UVA light irradiation is a method of choice for cross-link induction in eyes. However, ultrafast pulsed laser interactions may be a powerful alternative enabling in-depth treatment while simultaneously diminishing harmful side effects such as, keratocyte apoptosis. In this study, femtosecond laser is utilized for treatment of bovine cornea slices. It is hypothesized that nonlinear absorption of femtosecond laser pulses plays a major role in the maturation of immature cross-links and the promotion of their growth. Targeted irradiation with tightly focused laser pulses allows for the absence of a photosensitizing agent. Inflation test was conducted on half treated porcine cornea to identify the changes of mechanical properties due to laser treatment. Raman spectroscopy was utilized to study subtle changes in the chemical composition of treated cornea. The effects of treatment are analyzed by observing shifts in Amide I and Amide III bands, which suggest deformation of the collagen structure in cornea due to presence of newly formed cross-links.

  16. 21 CFR 177.1211 - Cross-linked polyacrylate copolymers.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... weight of aqueous sodium chloride solution at 20 °C for 24 hours. The low molecular weight extractives... applied mass). The solvent used shall be at least 60 milliliters aqueous sodium chloride solution per gram... polyacrylate copolymers consist of: (1) The grafted copolymer of cross-linked sodium polyacrylate identified...

  17. 21 CFR 177.1211 - Cross-linked polyacrylate copolymers.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ...). The solvent used shall be at least 60 milliliters aqueous sodium chloride solution per gram of... grafted copolymer of cross-linked sodium polyacrylate identified as 2-propenoic acid, polymers with N,N-di-2-propenyl-2-propen-1-amine and hydrolyzed polyvinyl acetate, sodium salts, graft (CAS Reg....

  18. 21 CFR 177.1211 - Cross-linked polyacrylate copolymers.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... weight of aqueous sodium chloride solution at 20 °C for 24 hours. The low molecular weight extractives... applied mass). The solvent used shall be at least 60 milliliters aqueous sodium chloride solution per gram... polyacrylate copolymers consist of: (1) The grafted copolymer of cross-linked sodium polyacrylate identified...

  19. 21 CFR 177.1211 - Cross-linked polyacrylate copolymers.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... weight of aqueous sodium chloride solution at 20 °C for 24 hours. The low molecular weight extractives... applied mass). The solvent used shall be at least 60 milliliters aqueous sodium chloride solution per gram... polyacrylate copolymers consist of: (1) The grafted copolymer of cross-linked sodium polyacrylate identified...

  20. 21 CFR 177.1211 - Cross-linked polyacrylate copolymers.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... weight of aqueous sodium chloride solution at 20 °C for 24 hours. The low molecular weight extractives... applied mass). The solvent used shall be at least 60 milliliters aqueous sodium chloride solution per gram... polyacrylate copolymers consist of: (1) The grafted copolymer of cross-linked sodium polyacrylate identified...

  1. Simulation of Fracture Nucleation in Cross-Linked Polymer Networks

    NASA Astrophysics Data System (ADS)

    Moller, J. C.; Barr, S. A.; Schultz, E. J.; Breitzman, T. D.; Berry, R. J.

    2013-02-01

    A novel atomistic simulation method is developed whereby polymer systems can undergo strain-rate-controlled deformation while bond scission is enabled. The aim is to provide insight into the nanoscale origins of fracture. Various highly cross-linked epoxy systems including various resin chain lengths and levels of nonreactive dilution were examined. Consistent with the results of physical experiments, cured resin strength increased and ductility decreased with increasing cross-link density. An analysis of dihedral angle activity shows the locations in the molecular network that are most absorptive of mechanical energy. Bond scission occurred principally at cross-link sites as well as between phenyl rings in the bisphenol moiety. Scissions typically occurred well after yield and were accompanied by steady increases in void size and dihedral angle motion between bisphenol moieties and at cross-link sites. The methods developed here could be more broadly applied to explore and compare the atomistic nature of deformation for various polymers such that mechanical and fracture properties could be tuned in a rational way. This method and its results could become part of a solution system that spans multiple length and time scales and that could more completely represent such mechanical events as fracture.

  2. Clinical fracture of cross-linked UHMWPE acetabular liners.

    PubMed

    Furmanski, Jevan; Anderson, Martin; Bal, Sonny; Greenwald, A Seth; Halley, David; Penenberg, Brad; Ries, Michael; Pruitt, Lisa

    2009-10-01

    Highly cross-linked ultrahigh molecular weight polyethylene (UHMWPE) is increasingly used as a bearing material in total hip replacements. Cross-linking of UHMWPE has been shown to increase wear resistance but decrease its fracture resistance. We analyzed the clinical fracture failure of four cross-linked UHMWPE total hip replacement components of four different designs via microscopic observation of the fracture surfaces, and found that in all cases fractures initiated at stress concentrations in an unsupported region of the component (termed the elevated rim). Finite element analyses (FEA) of each individual implant design were then conducted. Results from this analysis demonstrated that the predicted magnitude and orientation of maximum principal stress due to mechanical loading of the elevated rim was sufficient to propagate initiated fatigue cracks in each case. FEA also predicted that cracks may arrest after some amount of growth due to a steep stress gradient near the initiation site. Further, while anatomical positioning of the implant and material properties affect the risk of fracture, we examined whether these failures are strongly related to the notched elevated rim design feature that is common to the four failed cases presented here. We believe that cross-linked UHMWPE remains an excellent bearing material for total hip replacements but that designs employing this material should mitigate stress concentrations or other design features that increase the risk of fracture.

  3. Cross-Linked Protein Crystals for Vaccine Delivery

    NASA Astrophysics Data System (ADS)

    St. Clair, Nancy; Shenoy, Bhami; Jacob, Lawrence D.; Margolin, Alexey L.

    1999-08-01

    The progress toward subunit vaccines has been limited by their poor immunogenicity and limited stability. To enhance the immune response, subunit vaccines universally require improved adjuvants and delivery vehicles. In the present paper, we propose the use of cross-linked protein crystals (CLPCs) as antigens. We compare the immunogenicity of CLPCs of human serum albumin with that of soluble protein and conclude that there are marked differences in the immune response to the different forms of human serum albumin. Relative to the soluble protein, crystalline forms induce and sustain over almost a 6-month study a 6- to 10-fold increase in antibody titer for highly cross-linked crystals and an approximately 30-fold increase for lightly cross-linked crystals. We hypothesize that the depot effect, the particulate structure of CLPCs, and highly repetitive nature of protein crystals may play roles in the enhanced production of circulating antibodies. Several features of CLPCs, such as their remarkable stability, purity, biodegradability, and ease of manufacturing, make them highly attractive for vaccine formulations. This work paves the way for a systematic study of protein crystallinity and cross-linking on enhancement of humoral and T cell responses.

  4. Molecular mechanisms in deformation of cross-linked hydrogel nanocomposite.

    PubMed

    Mathesan, Santhosh; Rath, Amrita; Ghosh, Pijush

    2016-02-01

    The self-folding behavior in response to external stimuli observed in hydrogels is potentially used in biomedical applications. However, the use of hydrogels is limited because of its reduced mechanical properties. These properties are enhanced when the hydrogels are cross-linked and reinforced with nanoparticles. In this work, molecular dynamics (MD) simulation is applied to perform uniaxial tension and pull out tests to understand the mechanism contributing towards the enhanced mechanical properties. Also, nanomechanical characterization is performed using quasi static nanoindentation experiments to determine the Young's modulus of hydrogels in the presence of nanoparticles. The stress-strain responses for chitosan (CS), chitosan reinforced with hydroxyapatite (HAP) and cross-linked chitosan are obtained from uniaxial tension test. It is observed that the Young's modulus and maximum stress increase as the HAP content increases and also with cross-linking process. Load displacement plot from pullout test is compared for uncross-linked and cross-linked chitosan chains on hydroxyapatite surface. MD simulation reveals that the variation in the dihedral conformation of chitosan chains and the evolution of internal structural variables are associated with mechanical properties. Additional results reveal that the formation of hydrogen bonds and electrostatic interactions is responsible for the above variations in different systems.

  5. Cross-linked protein crystals for vaccine delivery

    PubMed Central

    St. Clair, Nancy; Shenoy, Bhami; Jacob, Lawrence D.; Margolin, Alexey L.

    1999-01-01

    The progress toward subunit vaccines has been limited by their poor immunogenicity and limited stability. To enhance the immune response, subunit vaccines universally require improved adjuvants and delivery vehicles. In the present paper, we propose the use of cross-linked protein crystals (CLPCs) as antigens. We compare the immunogenicity of CLPCs of human serum albumin with that of soluble protein and conclude that there are marked differences in the immune response to the different forms of human serum albumin. Relative to the soluble protein, crystalline forms induce and sustain over almost a 6-month study a 6- to 10-fold increase in antibody titer for highly cross-linked crystals and an approximately 30-fold increase for lightly cross-linked crystals. We hypothesize that the depot effect, the particulate structure of CLPCs, and highly repetitive nature of protein crystals may play roles in the enhanced production of circulating antibodies. Several features of CLPCs, such as their remarkable stability, purity, biodegradability, and ease of manufacturing, make them highly attractive for vaccine formulations. This work paves the way for a systematic study of protein crystallinity and cross-linking on enhancement of humoral and T cell responses. PMID:10449716

  6. Connective tissue growth factor modulates podocyte actin cytoskeleton and extracellular matrix synthesis and is induced in podocytes upon injury.

    PubMed

    Fuchshofer, Rudolf; Ullmann, Sabrina; Zeilbeck, Ludwig F; Baumann, Matti; Junglas, Benjamin; Tamm, Ernst R

    2011-09-01

    Structural changes of podocytes and retraction of their foot processes are a critical factor in the pathogenesis of minimal change nephritis and glomerulosclerosis. Here we tested, if connective tissue growth factor (CTGF) is involved in podocyte injury during acute and chronic puromycin aminonucleoside nephrosis (PAN) as animal models of minimal change nephritis, and focal segmental glomerulosclerosis, respectively. Rats were treated once (acute PAN) or for 13 weeks (chronic PAN). In both experimental conditions, CTGF and its mRNA were found to be highly upregulated in podocytes. The upregulation correlated with onset and duration of proteinuria in acute PAN, and glomerulosclerosis and high expression of glomerular fibronectin, and collagens I, III, and IV in chronic PAN. In vitro, treatment of podocytes with recombinant CTGF increased amount and density of actin stress fibers, the expression of actin-associated molecules such as podocalyxin, synaptopodin, ezrin, and actinin-4, and activation of focal adhesion kinase (FAK) and extracellular signal-regulated kinase (ERK). Moreover, we observed increased podocyte expression of mRNA for transforming growth factor (TGF)-β2, TGF-β receptor II, fibronectin, and collagens I, III, and IV. Treatment of cultured podocytes with puromycin aminonucleoside resulted in loss of actin stress fibers and cell death, effects that were partially prevented when CTGF was added to the culture medium. Depletion of CTGF mRNA in cultured podocytes by RNA interference reduced both the number of actin stress fibers and the expression of actin-associated molecules. We propose that the expression of CTGF is acutely upregulated in podocytes as part of a cellular attempt to repair structural changes of the actin cytoskeleton. When the damaging effects on podocyte structure and function persist chronically, continuous CTGF expression in podocytes is a critical factor that promotes progressive accumulation of glomerular extracellular matrix and

  7. Identification of a novel regulatory sequence of actin nucleation promoting factor encoded by Autographa californica multiple nucleopolyhedrovirus.

    PubMed

    Wang, Yun; Zhang, Yongli; Han, Shili; Hu, Xue; Zhou, Yuan; Mu, Jingfang; Pei, Rongjuan; Wu, Chunchen; Chen, Xinwen

    2015-04-10

    Actin polymerization induced by nucleation promoting factors (NPFs) is one of the most fundamental biological processes in eukaryotic cells. NPFs contain a conserved output domain (VCA domain) near the C terminus, which interacts with and activates the cellular actin-related protein 2/3 complex (Arp2/3) to induce actin polymerization and a diverse regulatory domain near the N terminus. Autographa californica multiple nucleopolyhedrovirus (AcMNPV) nucleocapsid protein P78/83 is a virus-encoded NPF that contains a C-terminal VCA domain and induces actin polymerization in virus-infected cells. However, there is no similarity between the N terminus of P78/83 and that of other identified NPFs, suggesting that P78/83 may possess a unique regulatory mechanism. In this study, we identified a multifunctional regulatory sequence (MRS) located near the N terminus of P78/83 and determined that one of its functions is to serve as a degron to mediate P78/83 degradation in a proteasome-dependent manner. In AcMNPV-infected cells, the MRS also binds to another nucleocapsid protein, BV/ODV-C42, which stabilizes P78/83 and modulates the P78/83-Arp2/3 interaction to orchestrate actin polymerization. In addition, the MRS is also essential for the incorporation of P78/83 into the nucleocapsid, ensuring virion mobility powered by P78/83-induced actin polymerization. The triple functions of the MRS enable P78/83 to serve as an essential viral protein in the AcMNPV replication cycle, and the possible roles of the MRS in orchestrating the virus-induced actin polymerization and viral genome decapsidation are discussed.

  8. Nuclear alpha spectrin: Critical roles in DNA interstrand cross-link repair and genomic stability

    PubMed Central

    2016-01-01

    Non-erythroid alpha spectrin (αIISp) is a structural protein which we have shown is present in the nucleus of human cells. It interacts with a number of nuclear proteins such as actin, lamin, emerin, chromatin remodeling factors, and DNA repair proteins. αIISp’s interaction with DNA repair proteins has been extensively studied. We have demonstrated that nuclear αIISp is critical in DNA interstrand cross-link (ICL) repair in S phase, in both genomic (non-telomeric) and telomeric DNA, and in maintenance of genomic stability following ICL damage to DNA. We have proposed that αIISp acts as a scaffold aiding to recruit repair proteins to sites of damage. This involvement of αIISp in ICL repair and telomere maintenance after ICL damage represents new and critical functions for αIISp. These studies have led to development of a model for the role of αIISp in DNA ICL repair. They have been aided by examination of cells from patients with Fanconi anemia (FA), a repair-deficient genetic disorder in which a deficiency in αIISp leads to defective ICL repair in genomic and telomeric DNA, telomere dysfunction, and chromosome instability following DNA ICL damage. We have shown that loss of αIISp in FA cells is due to increased breakdown by the protease, µ-calpain. Importantly, we have demonstrated that this deficiency can be corrected by knockdown of µ-calpain and restoring αIISp levels to normal. This corrects a number of the phenotypic deficiencies in FA after ICL damage. These studies suggest a new and unexplored direction for therapeutically restoring genomic stability in FA cells and for correcting numerous phenotypic deficiencies occurring after ICL damage. Developing a more in-depth understanding of the importance of the interaction of αIISp with other nuclear proteins could significantly enhance our knowledge of the consequences of loss of αIISp on critical nuclear processes. PMID:27480253

  9. Three cotton genes preferentially expressed in flower tissues encode actin-depolymerizing factors which are involved in F-actin dynamics in cells.

    PubMed

    Li, Xue-Bao; Xu, Dan; Wang, Xiu-Lan; Huang, Geng-Qing; Luo, Juan; Li, Deng-Di; Zhang, Ze-Ting; Xu, Wen-Liang

    2010-01-01

    To investigate whether the high expression levels of actin-depolymerizing factor genes are related to pollen development, three GhADF genes (cDNAs) were isolated and characterized in cotton. Among them, GhADF6 and GhADF8 were preferentially expressed in petals, whereas GhADF7 displayed the highest level of expression in anthers, revealing its anther specificity. The GhADF7 transcripts in anthers reached its peak value at flowering, suggesting that its expression is developmentally-regulated in anthers. The GhADF7 gene including the promoter region was isolated from the cotton genome. To demonstrate the specificity of the GhADF7 promoter, the 5'-flanking region, including the promoter and 5'-untranslated region, was fused with the GUS gene. Histochemical assays demonstrated that the GhADF7:GUS gene was specifically expressed in pollen grains. When pollen grains germinated, very strong GUS staining was detected in the elongating pollen tube. Furthermore, overexpression of GhADF7 gene in Arabidopsis thaliana reduced the viable pollen grains and, consequently, transgenic plants were partially male-sterile. Overexpression of GhADF7 in fission yeast (Schizosaccharomyces pombe) altered the balance of actin depolymerization and polymerization, leading to the defective cytokinesis and multinucleate formation in the cells. Given all the above results together, it is proposed that the GhADF7 gene may play an important role in pollen development and germination.

  10. Wear of PEEK-OPTIMA® and PEEK-OPTIMA®-Wear Performance articulating against highly cross-linked polyethylene.

    PubMed

    East, Rebecca H; Briscoe, Adam; Unsworth, Anthony

    2015-03-01

    The idea of all polymer artificial joints, particularly for the knee and finger, has been raised several times in the past 20 years. This is partly because of weight but also to reduce stress shielding in the bone when stiffer materials such as metals or ceramics are used. With this in mind, pin-on-plate studies of various polyetheretherketone preparations against highly cross-linked polyethylene were conducted to investigate the possibility of using such a combination in the design of a new generation of artificial joints. PEEK-OPTIMA(®) (no fibre) against highly cross-linked polyethylene gave very low wear factors of 0.0384 × 10(-6) mm(3)/N m for the polyetheretherketone pins and -0.025 × 10(-6) mm(3)/N m for the highly cross-linked polyethylene plates. The carbon-fibre-reinforced polyetheretherketone (PEEK-OPTIMA(®)-Wear Performance) also produced very low wear rates in the polyetheretherketone pins but produced very high wear in the highly cross-linked polyethylene, as might have been predicted since the carbon fibres are quite abrasive. When the fibres were predominantly tangential to the sliding plane, the mean wear factor was 0.052 × 10(-6) mm(3)/N m for the pins and 49.3 × 10(-6) mm(3)/N m for the highly cross-linked polyethylene plates; a half of that when the fibres ran axially in the pins (0.138 × 10(-6) mm(3)/N m for the pins and 97.5 × 10(-6) mm/ N m for the cross-linked polyethylene plates). PEEK-OPTIMA(®) against highly cross-linked polyethylene merits further investigation.

  11. Nucleotide exchange factor GEF-H1 mediates cross-talk between microtubules and the actin cytoskeleton.

    PubMed

    Krendel, Mira; Zenke, Frank T; Bokoch, Gary M

    2002-04-01

    Regulation of the actin cytoskeleton by microtubules is mediated by the Rho family GTPases. However, the molecular mechanisms that link microtubule dynamics to Rho GTPases have not, as yet, been identified. Here we show that the Rho guanine nucleotide exchange factor (GEF)-H1 is regulated by an interaction with microtubules. GEF-H1 mutants that are deficient in microtubule binding have higher activity levels than microtubule-bound forms. These mutants also induce Rho-dependent changes in cell morphology and actin organization. Furthermore, drug-induced microtubule depolymerization induces changes in cell morphology and gene expression that are similar to the changes induced by the expression of active forms of GEF-H1. Furthermore, these effects are inhibited by dominant-negative versions of GEF-H1. Thus, GEF-H1 links changes in microtubule integrity to Rho-dependent regulation of the actin cytoskeleton.

  12. Transforming growth factor-beta 1 induces alpha-smooth muscle actin expression in granulation tissue myofibroblasts and in quiescent and growing cultured fibroblasts

    PubMed Central

    1993-01-01

    Granulation tissue fibroblasts (myofibroblasts) develop several ultrastructural and biochemical features of smooth muscle (SM) cells, including the presence of microfilament bundles and the expression of alpha-SM actin, the actin isoform typical of vascular SM cells. Myofibroblasts have been proposed to play a role in wound contraction and in retractile phenomena observed during fibrotic diseases. We show here that the subcutaneous administration of transforming growth factor- beta 1 (TGF beta 1) to rats results in the formation of a granulation tissue in which alpha-SM actin expressing myofibroblasts are particularly abundant. Other cytokines and growth factors, such as platelet-derived growth factor and tumor necrosis factor-alpha, despite their profibrotic activity, do not induce alpha-SM actin in myofibroblasts. In situ hybridization with an alpha-SM actin probe shows a high level of alpha-SM actin mRNA expression in myofibroblasts of TGF beta 1-induced granulation tissue. Moreover, TGF beta 1 induces alpha-SM actin protein and mRNA expression in growing and quiescent cultured fibroblasts and preincubation of culture medium containing whole blood serum with neutralizing antibodies to TGF beta 1 results in a decrease of alpha-SM actin expression by fibroblasts in replicative and non-replicative conditions. These results suggest that TGF beta 1 plays an important role in myofibroblast differentiation during wound healing and fibrocontractive diseases by regulating the expression of alpha-SM actin in these cells. PMID:8314838

  13. Equilibrium and kinetic modeling of adsorption of reactive dye on cross-linked chitosan beads.

    PubMed

    Chiou, Ming Shen; Li, Hsing Ya

    2002-07-22

    The adsorption of reactive dye (Reactive Red 189) from aqueous solutions on cross-linked chitosan beads was studied in a batch system. The equilibrium isotherms at different particle sizes (2.3-2.5, 2.5-2.7 and 3.5-3.8mm) and the kinetics of adsorption with respect to the initial dye concentration (4320, 5760 and 7286 g/m(3)), temperature (30, 40 and 50 degrees C), pH (1.0, 3.0, 6.0 and 9.0), and cross-linking ratio (cross-linking agent/chitosan weight ratio: 0.2, 0.5, 0.7 and 1.0) were investigated. Langmuir and Freundlich adsorption models were applied to describe the experimental isotherms and isotherm constants. Equilibrium data fitted very well to the Langmuir model in the entire saturation concentration range (0-1800 g/m(3)). The maximum monolayer adsorption capacities obtained from the Langmuir model are very large, which are 1936, 1686 and 1642 g/kg for small, mediumand large particle sizes, respectively, at pH 3.0, 30 degrees C, and the cross-linking ratio of 0.2. The pseudo first- and second-order kinetic models were used to describe the kinetic data, and the rate constants were evaluated. The experimental data fitted well to the second-order kinetic model, which indicates that the chemical sorption is the rate-limiting step, instead of mass transfer. The initial dye concentration and the solution pH both significantly affect the adsorption capacity, but the temperature and the cross-linking ratio are relatively minor factors. An increase in initial dye concentration results in the increase of adsorption capacity, which also increases with decreasing pH. The activation energy is 43.0 kJ/mol for the adsorption of the dye on the cross-linked chitosan beads at pH 3.0 and initial dye concentration 3768 g/m(3).

  14. The actin cytoskeleton in endothelial cell phenotypes

    PubMed Central

    Prasain, Nutan; Stevens, Troy

    2009-01-01

    Endothelium forms a semi-permeable barrier that separates blood from the underlying tissue. Barrier function is largely determined by cell-cell and cell-matrix adhesions that define the limits of cell borders. Yet, such cell-cell and cell-matrix tethering is critically reliant upon the nature of adherence within the cell itself. Indeed, the actin cytoskeleton fulfills this essential function, to provide a strong, dynamic intracellular scaffold that organizes integral membrane proteins with the cell’s interior, and responds to environmental cues to orchestrate appropriate cell shape. The actin cytoskeleton is comprised of three distinct, but interrelated structures, including actin cross-linking of spectrin within the membrane skeleton, the cortical actin rim, and actomyosin-based stress fibers. This review addresses each of these actin-based structures, and discusses cellular signals that control the disposition of actin in different endothelial cell phenotypes. PMID:19028505

  15. Model selection for athermal cross-linked fiber networks.

    PubMed

    Shahsavari, A; Picu, R C

    2012-07-01

    Athermal random fiber networks are usually modeled by representing each fiber as a truss, a Euler-Bernoulli or a Timoshenko beam, and, in the case of cross-linked networks, each cross-link as a pinned, rotating, or welded joint. In this work we study the effect of these various modeling options on the dependence of the overall network stiffness on system parameters. We conclude that Timoshenko beams can be used for the entire range of density and beam stiffness parameters, while the Euler-Bernoulli model can be used only at relatively low network densities. In the high density-high bending stiffness range, strain energy is stored predominantly in the axial and shear deformation modes, while in the other extreme range of parameters, the energy is stored in the bending mode. The effect of the model size on the network stiffness is also discussed.

  16. Transparent Humidity Sensor Using Cross-Linked Polyelectrolyte Membrane

    SciTech Connect

    Zhang, Q.; Smith, James R.; Saraf, Laxmikant V.; Hua, Feng

    2009-07-02

    This paper describes the fabrication of a porous cross-linked polyelectrolyte membrane and the characterization of its humidity sensitivity performance. Electrostatic self-assembly, combined with acid treatment, and post-deposition annealing produced the membrane. The fabrication process offers the ability to control the thickness of the membrane, as well as enabling the engineering of the humidity sensitivity properties. A transparent humidity sensor was fabricated by integrating the membrane between two parallel electrodes. In order to improve the moisture absorption and diffusion, both the polyelectrolyte layer and the electrode were made porous. The membrane was cross-linked to enhance the durability in high humid environments. Such a polyelectrolyte membrane showed high sensitivity to relative humidity variation over a range of 25%–99%. The see-through property of the structure adds extra features and benefits to the sensor.

  17. Cross-linking reveals laminin coiled-coil architecture

    PubMed Central

    Armony, Gad; Jacob, Etai; Moran, Toot; Levin, Yishai; Mehlman, Tevie; Levy, Yaakov; Fass, Deborah

    2016-01-01

    Laminin, an ∼800-kDa heterotrimeric protein, is a major functional component of the extracellular matrix, contributing to tissue development and maintenance. The unique architecture of laminin is not currently amenable to determination at high resolution, as its flexible and narrow segments complicate both crystallization and single-particle reconstruction by electron microscopy. Therefore, we used cross-linking and MS, evaluated using computational methods, to address key questions regarding laminin quaternary structure. This approach was particularly well suited to the ∼750-Å coiled coil that mediates trimer assembly, and our results support revision of the subunit order typically presented in laminin schematics. Furthermore, information on the subunit register in the coiled coil and cross-links to downstream domains provide insights into the self-assembly required for interaction with other extracellular matrix and cell surface proteins. PMID:27815530

  18. Reversible PH Lability of Cross-Linked Vault Nanocapsules

    SciTech Connect

    Yu, M.; Ng, B.C.; Rome, L.H.; Tolbert, S.H.; Monbouquette, H.G.

    2009-05-28

    Vaults are ubiquitous, self-assembled protein nanocapsules with dimension in the sub-100 nm range that are conserved across diverse phyla from worms to humans. Their normal presence in humans at a copy number of over 10 000/cell makes them attractive as potential drug delivery vehicles. Toward this goal, bifunctional amine-reactive reagents are shown to be useful for the reversible cross-linking of recombinant vaults such that they may be closed and opened in a controllable manner.

  19. Magnetic Characterization of Iron Oxide Cross Linked Hydro gels

    NASA Astrophysics Data System (ADS)

    Senaratne, U.; Powell, N.; Kroll, E.; Tsoi, G.; Naik, R.; Naik, V.; Vaishnava, P. P.; Wenger, L. E.

    2004-03-01

    Magnetic hydro gels have potential applications in drug delivery, cells sorting, sensors, and actuating technologies. Iron oxide alginate nanocomposites were synthesized following the method of Kroll et al^1 by cross linking sodium alginate with Fe^2+ and Fe^3+ in methanol: water. The ion-cross linked alginate hydro gels are oxidized in an alkaline solution. The resulting hydro gel consists of iron oxide cross linked alginate. The alginate hydro gels are inert to the reaction conditions and therefore the reaction sequence can be repeated. The multiple loadings result in an increase in the amount of iron oxide and the size of the iron oxide nanoparticles in the cross linked hydro gels. The third and sixth loaded iron oxide alginate hydro gels were dried and characterized by X-ray diffraction (XRD), Transmission Electron Microscopy (TEM), and Superconducting Quantum Interference Device (SQUID) magnetometry. The XRD patterns have characteristic features of γ- Fe_2O3 or Fe_3O4 phases. The average particle size, calculated from the XRD peaks, for third loaded iron oxide alginate was 2 nm. The zero-field-cooled and field-cooled SQUID measurements show the iron oxide nanoparticles are superparamagnetic with blocking temperature (T_B) of approximately 35 K. Above the blocking temperature, the inverse susceptibility versus temperature relationship does not follow the Curie-Weiss law, indicating strong inter-particle interactions. The M vs. H data above the blocking temperature was fitted with a modified Langevin function to obtain additional information about the iron oxide particle size. Details of the relationship between coercive field and temperature as well as the particle size distribution obtained from XRD and TEM measurements will be presented. *Research supported by NSF grant # DGE ˜980720 **Supported by NSF REU grant # EEC-0097736 ^1E. Kroll, F.M. Winnik, and R.F. Ziolo, Chem. Mater, 8, 1594 (1996).

  20. Estimating the Degree of Cross-Linking in Rubber

    NASA Technical Reports Server (NTRS)

    Fedors, R. F.

    1983-01-01

    Degree of cross-linking or network chain concentration of rubber estimated with aid of new method. Quantity is needed in studies of mechanical behavior of rubber. New method is based on finding rubber follows different stress/ strain relationships in extension and retraction. When rubber specimen is stretched to given extension ration and released. Stress-vs-strain curve follows two paths: one for extension and other for retraction.

  1. Physicochemical properties of collagen solutions cross-linked by glutaraldehyde.

    PubMed

    Tian, Zhenhua; Li, Conghu; Duan, Lian; Li, Guoying

    2014-06-01

    The physicochemical properties of collagen solutions (5 mg/ml) cross-linked by various amounts of glutaraldehyde (GTA) [GTA/collagen (w/w) = 0-0.5] under acidic condition (pH 4.00) were examined. Based on the results of the determination of residual amino group content, sodium dodecyl sulphate-polyacrylamide gel electrophoresis, dynamic rheological measurements, differential scanning calorimetry and atomic force microscopy (AFM), it was proved that the collagen solutions possessed strikingly different physicochemical properties depending on the amount of GTA. At low GTA amounts [GTA/collagen (w/w) ≤ 0.1], the residual amino group contents of the cross-linked collagens decreased largely from 100% to 32.76%, accompanied by an increase in the molecular weight. Additionally, increases of the fiber diameter and the values of G', G″ and η* were measured, while the thermal denaturation temperature (Td) did not change visibly and the fluidity of collagen samples was still retained with increasing the GTA amount. When the ratio of GTA to collagen exceeded 0.1, although the residual amino group content only decreased by ~8.2%, the cross-linked collagen solution [GTA/collagen (w/w) = 0.3] displayed a clear loss of flow and a sudden rise (~2.0 °C) of the Td value compared to the uncross-linked collagen solution, probably illustrating that the collagen solution was converted into a gel with mature network structure-containing nuclei observed in AFM image. It was conjectured that the physicochemical properties of the collagen solutions might be in connection with the cross-linking between collagen molecules from the same aggregate or different aggregates.

  2. Nanoscale segregation of actin nucleation and elongation factors determines dendritic spine protrusion

    PubMed Central

    Chazeau, Anaël; Mehidi, Amine; Nair, Deepak; Gautier, Jérémie J; Leduc, Cécile; Chamma, Ingrid; Kage, Frieda; Kechkar, Adel; Thoumine, Olivier; Rottner, Klemens; Choquet, Daniel; Gautreau, Alexis; Sibarita, Jean-Baptiste; Giannone, Grégory

    2014-01-01

    Actin dynamics drive morphological remodeling of neuronal dendritic spines and changes in synaptic transmission. Yet, the spatiotemporal coordination of actin regulators in spines is unknown. Using single protein tracking and super-resolution imaging, we revealed the nanoscale organization and dynamics of branched F-actin regulators in spines. Branched F-actin nucleation occurs at the PSD vicinity, while elongation occurs at the tip of finger-like protrusions. This spatial segregation differs from lamellipodia where both branched F-actin nucleation and elongation occur at protrusion tips. The PSD is a persistent confinement zone for IRSp53 and the WAVE complex, an activator of the Arp2/3 complex. In contrast, filament elongators like VASP and formin-like protein-2 move outwards from the PSD with protrusion tips. Accordingly, Arp2/3 complexes associated with F-actin are immobile and surround the PSD. Arp2/3 and Rac1 GTPase converge to the PSD, respectively, by cytosolic and free-diffusion on the membrane. Enhanced Rac1 activation and Shank3 over-expression, both associated with spine enlargement, induce delocalization of the WAVE complex from the PSD. Thus, the specific localization of branched F-actin regulators in spines might be reorganized during spine morphological remodeling often associated with synaptic plasticity. PMID:25293574

  3. Homogeneous UVA system for corneal cross-linking treatment

    NASA Astrophysics Data System (ADS)

    Ayres Pereira, Fernando R.; Stefani, Mario A.; Otoboni, José A.; Richter, Eduardo H.; Ventura, Liliane

    2010-02-01

    The treatment of keratoconus and corneal ulcers by collagen cross-linking using ultraviolet type A irradiation, combined with photo-sensitizer Riboflavin (vitamin B2), is a promising technique. The standard protocol suggests instilling Riboflavin in the pre-scratched cornea every 5min for 30min, during the UVA irradiation of the cornea at 3mW/cm2 for 30 min. This process leads to an increase of the biomechanical strength of the cornea, stopping the progression, or sometimes, even reversing Keratoconus. The collagen cross-linking can be achieved by many methods, but the utilization of UVA light, for this purpose, is ideal because of its possibility of a homogeneous treatment leading to an equal result along the treated area. We have developed a system, to be clinically used for treatment of unhealthy corneas using the cross-linking technique, which consists of an UVA emitting delivery device controlled by a closed loop system with high homogeneity. The system is tunable and delivers 3-5 mW/cm2, at 365nm, for three spots (6mm, 8mm and 10mm in diameter). The electronics close loop presents 1% of precision, leading to an overall error, after the calibration, of less than 10% and approximately 96% of homogeneity.

  4. Fiber optic immunosensor for cross-linked fibrin concentration

    NASA Astrophysics Data System (ADS)

    Moskowitz, Samuel E.

    2000-08-01

    Working with calcium ions in the blood, platelets produce thromboplastin which transforms prothrombin into thrombin. Removing peptides, thrombin changes fibrinogen into fibrin. Cross-linked insoluble fibrin polymers are solubilized by enzyme plasmin found in blood plasma. Resulting D-dimers are elevated in patients with intravascular coagulation, deep venous thrombosis, pulmonary embolism, myocardial infarction, multiple trauma, cancer, impaired renal and liver functions, and sepsis. Consisting principally of a NIR 780 nm GaAlAs laser diode and a 800 nm avalanche photodiode (APD), the fiber-optic immunosensor can determined D-dimer concentration to levels <0.1 ng/ml. A capture monoclonal antibody to the antigen soluble cross-linked fibrin is employed. Immobilized at the tip of an optical fiber by avidin-biotin, the captured antigen is detected by a second antibody which is labeled with NN 382 fluorescent dye. An evanescent wave traveling on an excitation optical fiber excites the antibody-antigen fluorophore complex. Concentration of cross-linked fibrin is directly proportional to the APD measured intensity of fluorescence. NIR fluorescence has advantages of low background interference, short fluorescence lifetime, and large difference between excitation and emission peaks. Competitive ELISA test for D-dimer concentration requires trained personnel performing a time consuming operation.

  5. Electrochemical Characterization of Ultrathin Cross-Linked Metal Nanoparticle Films.

    PubMed

    Han, Chu; Percival, Stephen J; Zhang, Bo

    2016-09-06

    Here we report the preparation, characterization, and electrochemical study of conductive, ultrathin films of cross-linked metal nanoparticles (NPs). Nanoporous films ranging from 40 to 200 nm in thickness composed of gold and platinum NPs of ∼5 nm were fabricated via a powerful layer-by-layer spin coating process. This process allows preparation of uniform NP films as large as 2 × 2 cm(2) with precise control over thickness, structure, and electrochemical and electrocatalytic properties. Gold, platinum, and bimetallic NP films were fabricated and characterized using cyclic voltammetry, scanning electron microscopy, and conductance measurements. Their electrocatalytic activity toward the oxygen reduction reaction (ORR) was investigated. Our results show that the electrochemical activity of such NP films is initially hindered by the presence of dense thiolate cross-linking ligands. Both electrochemical cycling and oxygen plasma cleaning are effective means in restoring their electrochemical activity. Gold NP films have higher electric conductivity than platinum possibly due to more uniform film structure and closer particle-particle distance. The electrochemical and electrocatalytic performance of platinum NP films can be greatly enhanced by the incorporation of gold NPs. This work focuses on electrochemical characterization of cross-linked NP films and demonstrates several unique properties. These include quick and easy preparation, ultrathin and uniform film thickness, tunable structure and composition, and transferability to many other substrates.

  6. Supersaturated lysozyme solution structure studied by chemical cross-linking.

    PubMed

    Hall, Clayton L; Clemens, John R; Brown, Amanda M; Wilson, Lori J

    2005-06-01

    Glutaraldehyde cross-linking followed by separation has been used to detect aggregates of chicken egg-white lysozyme (CEWL) in supersaturated solutions. In solutions of varying NaCl content, the number of aggregates was found to be related to the ionic strength of the solution. Separation by SDS-PAGE showed that percentage of dimer in solution ranged from 25.3% for no NaCl to 27.1% at 15% NaCl, and the aggregates larger than dimer increased from 1.9% for no NaCl to 36.8% at 15% NaCl. Conversely, the percentage of monomers decreased from 72.8% without NaCl to 36.1% at 15% NaCl. Molecular weights by capillary electrophoresis (SDS-CE) were found to be multiples of the monomer molecular weights, with the exception of trimer, which indicates a very compact structure. Native separation was accomplished using size-exclusion chromatography (SEC) and gave a lower monomer concentration and higher aggregate concentration than SDS-CE, which is a denaturing separation method. Most noticeably, trimers were absent in the SEC separation. The number of aggregates did not change with increased time between addition of NaCl and addition of cross-linking agent when separated by gel electrophoresis (SDS-PAGE). The results suggest that high ionic strength CEWL solutions are highly aggregated and that denaturing separation methods disrupt cross-linked products.

  7. Relative toxicities of DNA cross-links and monoadducts: new insights from studies of decarbamoyl mitomycin C and mitomycin C.

    PubMed

    Palom, Yolanda; Suresh Kumar, Gopinatha; Tang, Li-Qian; Paz, Manuel M; Musser, Steven M; Rockwell, Sara; Tomasz, Maria

    2002-11-01

    Mitomycin C (MC), a cytotoxic anticancer drug and bifunctional DNA DNA alkylating agent, induces cross-linking of the complementary strands of DNA. The DNA interstrand cross-links (ICLs) are thought to be the critical cytotoxic lesions produced by MC. Decarbamoyl mitomycin C (DMC) has been regarded as a monofunctional mitomycin, incapable of causing ICLs. Paradoxically, DMC is slightly more toxic than MC to hypoxic EMT6 mouse mammary tumor cells as well as to CHO cells. To resolve this paradox, EMT6 cells were treated with MC or DMC under hypoxia at equimolar concentrations and the resulting DNA adducts were analyzed using HPLC and UV detection. MC treatment generated both intrastrand and interstrand cross-link adducts and four monoadducts, as shown previously. DMC generated two stereoisomeric monoadducts and two stereoisomeric ICL adducts, all of which were structurally characterized; one was identical with that formed with MC, the other was new and unique to DMC. Overall, adduct frequencies were strikingly higher (20-30-fold) with DMC than with MC. Although DMC monoadducts greatly exceeded DMC cross-link adducts ( approximately 10:1 ratio), the latter were equal or higher in number than the cross-link adducts from MC. DMC displayed a much higher monoadduct:cross-link ratio than MC. The similar cytotoxicities of the two drug show a correlation with their similar DNA cross-link adduct frequencies, but not with their total adduct or monoadduct frequencies. This provides specific experimental evidence that the ICLs rather than the monoadducts are critical factors in the cell death induced by MC. In vitro, overall alkylation of calf thymus DNA by DMC was much less efficient than by MC. Nevertheless, ICLs formed with DMC were clearly detectable. The chemical pathway of the cross-linking was shown to be analogous to that occurring with MC. These results also suggest that the differential sensitivity of Fanconi's Anemia cells to MC and DMC is related to factors other

  8. Wear Analysis of Second-generation Highly Cross-Linked Polyethylene in Primary Total Hip Arthroplasty.

    PubMed

    Samujh, Christopher; Bhimani, Samrath; Smith, Langan; Malkani, Arthur L

    2016-11-01

    A major limiting factor in the longevity of total hip replacement is the wear rate of the hip bearing. As manufacturing technology has improved during the past several decades, much attention has been focused on developing newer generations of polyethylene that have lower rates of wear while minimizing free radical formation and subsequent osteolysis. The turning point for the manufacture of polyethylene was moving from gamma irradiation in air to irradiation in a low oxygen environment, which reduced free radical formation while increasing the wear resistance. New polyethylene manufacturing methods, including multiple cycles of irradiation and annealing, have resulted in greater wear resistance. Wear analysis studies are essential to determine if these new liners actually show a benefit from prior generations of polyethylene and, more importantly, if they are safe to use. This study involved a single center retrospective review of 60 patients with a mean follow-up of 5.5 years who underwent primary total hip arthroplasty with a second-generation highly cross-linked polyethylene manufactured by 3 cycles of sequential irradiation and annealing. Linear and volumetric wear rates were determined from digitized radiographs using contemporary wear analysis software. The mean linear wear rate for the entire group was 0.025 millimeters per year (mm/y). This value represents a linear wear rate 2.7 times less than that of a first-generation highly cross-linked polyethylene and 4.2 times less than that of a conventional polyethylene. At an average of 5 years, compared with a first-generation highly cross-linked polyethylene, a second-generation highly cross-linked polyethylene appears to show significant improvement regarding wear. [Orthopedics. 2016; 39(6):e1178-e1182.].

  9. Corneal Cross-Linking: Evaluating the Potential for a Lower Power, Shorter Duration Treatment

    PubMed Central

    Caruso, Ciro; Barbaro, Gaetano; Tronino, Diana; Ostacolo, Carmine; Sacchi, Antonia; Pacente, Luigi; Del Prete, Antonio; Sala, Marina; Troisi, Salvatore

    2016-01-01

    Purpose: To determine the cross-linking effect of a riboflavin ultraviolet-A (UV-A) corneal cross-linking treatment that is both shorter and has lower energy than the Dresden protocol. Methods: In a first experiment, 12 human corneas were presoaked with riboflavin and then irradiated with UV-A at 3 mW/cm2 after clearing the surface of riboflavin, with no added riboflavin during irradiation. Percent UV-A transmission through the corneas was measured at intervals up to 30 minutes. A second experiment involved 24 porcine corneas. Eight were de-epithelialized, presoaked in riboflavin for 30 minutes, and irradiated at 1.5 mW/cm2 for 10 minutes. An additional 8 were riboflavin treated and similarly irradiated, but with epithelium intact and a final 8 corneas were not treated. Young modulus was measured in all 24 corneas at the end of the experiment. Results: The first experiment showed essentially complete riboflavin oxidation after only 10 minutes. Based on these results, a shortened UV-A exposure cross-linking experiment was designed using a reduced UV-A fluence of 1.5 mW/cm2, an endothelial exposure within safety limits in humans. With this protocol Young modulus was the same in the irradiated porcine corneas but with epithelium intact as in the untreated corneas. In contrast, Young modulus increased by a factor of 1.99 in the UV-A cross-linked corneas at 1.5 mW/cm2 for 10 minutes with the epithelium removed. Conclusions: A shorter, lower energy protocol than the Dresden protocol seems to provide a significant increase in Young modulus, similar to published results with higher energy, longer exposure protocols. PMID:26989958

  10. Low cross-linked molecularly imprinted monolithic column prepared in molecular crowding conditions.

    PubMed

    Mu, Li-Na; Wang, Xian-Hua; Zhao, Liang; Huang, Yan-Ping; Liu, Zhao-Sheng

    2011-12-23

    Molecular crowding is a new approach to promoting molecular imprinting more efficiently. In this work, this concept was applied to the preparation of low cross-linked imprinted polymers in the presence of an immobilised template for stabilizing binding sites and improving molecular recognition. An imprinted monolithic column was synthesized using a mixture of 2,4-diamino-6-methyl-1,3,5-triazine (template), 2,4-diamino-6-(methacryloyloxy) ethyl-1,3,5-triazine (polymerisable template), methacrylic acid, ethylene glycol dimethacrylate, and polystyrene (molecular crowding agent). Some polymerization factors, such as template-monomer molar ratio, the composition of the porogen and crosslinking density, on the imprinting effect of resulting MIP monolith were systematically investigated. The results indicated that the imprinted monolithic columns prepared in the presence of molecular crowding agent retained affinity and specificity for template even when prepared with a level of cross-linker as low as 9%. Moreover, a stoichiometric displacement model for retention was successfully applied to evaluate the interaction between the solute and the stationary phase. Compared with the low cross-linked MIP prepared by conventional polymerization, the molecular crowding-based low cross-linked monolithic MIPs showed higher selectivity. The results suggested that molecular crowding is a powerful strategy to increase the effect of molecular imprinting at a low level of crosslinker.

  11. Fracture mechanics of collagen fibrils: influence of natural cross-links.

    PubMed

    Svensson, Rene B; Mulder, Hindrik; Kovanen, Vuokko; Magnusson, S Peter

    2013-06-04

    Tendons are important load-bearing structures, which are frequently injured in both sports and work. Type I collagen fibrils are the primary components of tendons and carry most of the mechanical loads experienced by the tissue, however, knowledge of how load is transmitted between and within fibrils is limited. The presence of covalent enzymatic cross-links between collagen molecules is an important factor that has been shown to influence mechanical behavior of the tendons. To improve our understanding of how molecular bonds translate into tendon mechanics, we used an atomic force microscopy technique to measure the mechanical behavior of individual collagen fibrils loaded to failure. Fibrils from human patellar tendons, rat-tail tendons (RTTs), NaBH₄ reduced RTTs, and tail tendons of Zucker diabetic fat rats were tested. We found a characteristic three-phase stress-strain behavior in the human collagen fibrils. There was an initial rise in modulus followed by a plateau with reduced modulus, which was finally followed by an even greater increase in stress and modulus before failure. The RTTs also displayed the initial increase and plateau phase, but the third region was virtually absent and the plateau continued until failure. The importance of cross-link lability was investigated by NaBH₄ reduction of the rat-tail fibrils, which did not alter their behavior. These findings shed light on the function of cross-links at the fibril level, but further studies will be required to establish the underlying mechanisms.

  12. Cross linking molecular systems to form ultrathin dielectric layers

    NASA Astrophysics Data System (ADS)

    Feng, Danqin

    Dehydrogenation leads to cross linking of polymer or polymer like formation in very different systems: self-assembled monolayers and in closo -carboranes leading to the formation of semiconducting and dielectric boron carbide. We find evidence of intermolecular interactions for a self-assembled monolayer (SAM) formed from a large molecular adsorbate, [1,1';4',1"-terphenyl]-4,4"-dimethanethiol, from the dispersion of the molecular orbitals with changing the wave vector k and from the changes with temperature. With the formation self assembled molecular (SAM) layer, the molecular orbitals hybridize to electronic bands, with indications of significant band dispersion of the unoccupied molecular orbitals. Although organic adsorbates and thin films are generally regarded as "soft" materials, the effective Debye temperature, indicative of the dynamic motion of the lattice normal to the surface, can be very high, e.g. in the multilayer film formed from [1,1'-biphenyl]-4,4'-dimethanethiol (BPDMT). Depending on molecular orientation, the effective Debye temperature can be comparable to that of graphite due to the 'stiffness' of the benzene rings, but follows the expected Debye-Waller behavior for the core level photoemission intensities with temperature. This is not always the case. We find that a monomolecular film formed from [1,1';4',1"-terphenyl]-4,4"-dimethanethiol deviates from Debye-Waller temperature behavior and is likely caused by temperature dependent changes in molecular orientation. We also find evidence for the increase in dielectric character with polymerization (cross-linking) in spite of the decrease in the HOMO-LUMO gap upon irradiation of TPDMT. The changes in the HOMO-LUMO gap, with cross-linking, are roughly consistent with the band dispersion. The decomposition and cross-linking processes are also accompanied by changes in molecular orientation. The energetics of the three isomeric carborane cage compounds [ closo-1,2-orthocarborane, closo-1

  13. A copper sulfate and hydroxylysine treatment regimen for enhancing collagen cross-linking and biomechanical properties in engineered neocartilage.

    PubMed

    Makris, Eleftherios A; MacBarb, Regina F; Responte, Donald J; Hu, Jerry C; Athanasiou, Kyriacos A

    2013-06-01

    The objective of this study was to improve the biomechanical properties of engineered neotissues through promoting the development of collagen cross-links. It was hypothesized that supplementing medium with copper sulfate and the amino acid hydroxylysine would enhance the activity of lysyl oxidase enzyme to form collagen cross-links, increasing the strength and integrity of the neotissue. Neocartilage constructs were generated using a scaffoldless, self-assembling process and treated with copper sulfate and hydroxylysine, either alone or in combination, following a 2-factor, full-factorial study design. Following a 6-wk culture period, the biomechanical and biochemical properties of the constructs were measured. Results found copper sulfate to significantly increase pyridinoline (PYR) cross-links in all copper sulfate-containing groups over controls. When copper sulfate and hydroxylysine were combined, the result was synergistic, with a 10-fold increase in PYR content over controls. This increase in PYR cross-links manifested in a 3.3-fold significant increase in the tensile properties of the copper sulfate + hydroxylysine group. In addition, an 123% increase over control values was detected in the copper sulfate group in terms of the aggregate modulus. These data elucidate the role of copper sulfate and hydroxylysine toward improving the biomechanical properties of neotissues through collagen cross-linking enhancement.

  14. Characterization of the Raptor/4E-BP1 interaction by chemical cross-linking coupled with mass spectrometry analysis.

    PubMed

    Coffman, Kimberly; Yang, Bing; Lu, Jie; Tetlow, Ashley L; Pelliccio, Emelia; Lu, Shan; Guo, Da-Chuan; Tang, Chun; Dong, Meng-Qiu; Tamanoi, Fuyuhiko

    2014-02-21

    mTORC1 plays critical roles in the regulation of protein synthesis, growth, and proliferation in response to nutrients, growth factors, and energy conditions. One of the substrates of mTORC1 is 4E-BP1, whose phosphorylation by mTORC1 reverses its inhibitory action on eIF4E, resulting in the promotion of protein synthesis. Raptor in mTOR complex 1 is believed to recruit 4E-BP1, facilitating phosphorylation of 4E-BP1 by the kinase mTOR. We applied chemical cross-linking coupled with mass spectrometry analysis to gain insight into interactions between mTORC1 and 4E-BP1. Using the cross-linking reagent bis[sulfosuccinimidyl] suberate, we showed that Raptor can be cross-linked with 4E-BP1. Mass spectrometric analysis of cross-linked Raptor-4E-BP1 led to the identification of several cross-linked peptide pairs. Compilation of these peptides revealed that the most N-terminal Raptor N-terminal conserved domain (in particular residues from 89 to 180) of Raptor is the major site of interaction with 4E-BP1. On 4E-BP1, we found that cross-links with Raptor were clustered in the central region (amino acid residues 56-72) we call RCR (Raptor cross-linking region). Intramolecular cross-links of Raptor suggest the presence of two structured regions of Raptor: one in the N-terminal region and the other in the C-terminal region. In support of the idea that the Raptor N-terminal conserved domain and the 4E-BP1 central region are closely located, we found that peptides that encompass the RCR of 4E-BP1 inhibit cross-linking and interaction of 4E-BP1 with Raptor. Furthermore, mutations of residues in the RCR decrease the ability of 4E-BP1 to serve as a substrate for mTORC1 in vitro and in vivo.

  15. Predisposing factors of actinic keratosis in a North-West German population.

    PubMed

    Hensen, Peter; Müller, Marcel L; Haschemi, Ramin; Ständer, Hartmut; Luger, Thomas A; Sunderkötter, Cord; Schiller, Meinhard

    2009-01-01

    The growing incident rates of skin cancer and their corresponding precursor lesions, e.g. actinic keratosis (AK), among Caucasians have become an important public health problem. A multicenter case-control study was conducted to identify the risk factors of AK of a prototypical Central European population. The study population comprised a total of 331 cases and 383 controls. Using multivariate analysis we identified ten independent variables predicting the AK risk. The five most crucial were age (OR 1.11; 95% CI 1.08-1,14), gender (OR 3.92; 95% CI 2.42-6.36), history of previous skin malignancies (OR 6.47; 95% CI 3.21-13.03), pale skin phototype (OR 2.5; 95% CI 1.53-4.06), and sun exposure for occupational reasons (OR 1.72; 95% CI 1.01-2.92). Additionally, sun exposure for recreational reasons, denial of the use of sunscreens, painful sunburn episodes before the age of 20, and a familial history of skin malignancies are also significant independent correlates of AK. Our epidemiological data suggest that constitutional susceptibility and sunlight exposure are equally involved in the onset of AK. Additional prophylactic and educational efforts should focus on increasing sun protection policies and educational programs especially aimed at outdoor workers, men, fair skinned individuals and patients with a history of previous skin malignancies. These measures should be able to reduce the excessive incidence rates of AK among Caucasians in Central Europe.

  16. [Expression of elongation factor-1 alpha-A and beta-actin promoters in embryos of transgenic Medaka (Oryzias latipes)].

    PubMed

    Long, Hua

    2003-06-01

    Two expression vectors with the promoter of either Medaka (Oryzias latipes) elongation factor gene or beta-actin gene were constructed based on pBluescript SK+. Both of them are linked with green-fluorescent protein (GFP) gene. And they are named as pB-EF and pB-BA, respectively. The microinjection experiments were conducted with fertilized Medaka eggs at one-cell stage. The expression of two vectors, pB-EF and pB-BA, was observed under stereo-fluorescence microscope. The detection results showed that both EF-1 alpha-A promoter and beta-actin promoter are strong. In the process of embryo development, the activity of beta-actin promoter became stronger while that of EF-1 alpha-A promoter weaker gradually. beta-actin promoter was but EF-1 alpha-A promoter distributed throughout fish body uniformly. The expression rate of two vectors, pB-EF and pB-BA, are 8.23% and 6.10%, respectively.

  17. Autoclavable highly cross-linked polyurethane networks in ophthalmology.

    PubMed

    Bruin, P; Meeuwsen, E A; van Andel, M V; Worst, J G; Pennings, A J

    1993-11-01

    Highly cross-linked aliphatic polyurethane networks have been prepared by the bulk step reaction of low molecular weight polyols and hexamethylenediisocyanate (HDI). These polyurethane networks are optically transparent, colourless and autoclavable amorphous glassy thermosets, which are suited for use in ophthalmic applications such as intraocular lenses and keratoprostheses. The properties of these glassy polyurethanes, obtained from the reaction of the low molecular weight polyols triisopropanolamine (TIPA) or tetrakis (2-hydroxypropyl)ethylenediamine (Quadrol) and HDI in stoichiometric proportions, have been investigated in more detail. The glassy Quadrol/HDI-based polyurethane exhibits a reduction in ultimate glass transition temperature from 85 to 48 degrees C by uptake of 1% of water, and good ultimate mechanical properties (tensile strength 80-85 MPa, elongation at break ca 15%, modulus ca 1.5 GPa). IR spectra of these hydrophobic polyurethane networks revealed the absence of an isocyanate absorption, indicating that all isocyanates, apparently, had reacted during the cross-linking reaction. The biocompatibility could be increased by grafting tethered polyacrylamide chains onto the surface during network formation. These transparent cross-linked polyurethanes did not transmit UV light up to 400 nm, by incorporation of a small amount of the UV absorbing chromophore Coumarin 102, and could be sterilized simply by autoclaving. They were implanted in rabbit eyes, either in the form of small circular disks or in the form of a keratoprosthesis (artificial cornea). It was shown that the material was well tolerated by the rabbit eyes. Serious opacification of the cornea, a direct result of an adverse reaction to the implant, was never seen. Even 1 yr after implantation of a polyurethane keratoprosthesis the eye was still 'quiet'.

  18. Macrophage response to cross-linked and conventional UHMWPE.

    PubMed

    Sethi, Rajiv K; Neavyn, Mark J; Rubash, Harry E; Shanbhag, Arun S

    2003-07-01

    To prevent wear debris-induced osteolysis and aseptic loosening, cross-linked ultra-high molecular weight polyethylene's (UHMWPE) with improved wear resistance have been developed. Hip simulator studies have demonstrated very low wear rates with these new materials leading to their widespread clinical use. However, the biocompatibility of this material is not known. We studied the macrophage response to cross-linked UHMWPE (XLPE) and compared it to conventional UHMWPE (CPE) as well as other clinically used orthopaedic materials such as titanium-alloy (TiAlV) and cobalt-chrome alloy (CoCr). Human peripheral blood monocytes and murine macrophages, as surrogates for cells mediating peri-implant inflammation, were cultured onto custom designed lipped disks fabricated from the test materials to isolate cells. Culture supernatants were collected at 24 and 48h and analyzed for cytokines such as IL-1alpha, IL-1beta, TNF-alpha and IL-6. Total RNA was extracted from adherent cells and gene expression was analyzed using qualitative RT-PCR. In both in vitro models, macrophages cultured on cross-linked and conventional polyethylene released similar levels of cytokines, which were also similar to levels on control tissue culture dishes. Macrophages cultured on TiAlV and CoCr-alloy released significantly higher levels of cytokines. Human monocytes from all donors varied in the magnitude of cytokines released when cultured on identical surfaces. The variability in individual donor responses to TiAlV and CoCr surfaces may reflect how individuals respond differently to similar stimuli and perhaps reveal a predisposed sensitivity to particular materials.

  19. Polyimide Aerogels with Three-Dimensional Cross-Linked Structure

    NASA Technical Reports Server (NTRS)

    Panek, John

    2010-01-01

    Polyimide aerogels with three-dimensional cross-linked structure are made using linear oligomeric segments of polyimide, and linked with one of the following into a 3D structure: trifunctional aliphatic or aromatic amines, latent reactive end caps such as nadic anhydride or phenylethynylphenyl amine, and silica or silsesquioxane cage structures decorated with amine. Drying the gels supercritically maintains the solid structure of the gel, creating a polyimide aerogel with improved mechanical properties over linear polyimide aerogels. Lightweight, low-density structures are desired for acoustic and thermal insulation for aerospace structures, habitats, astronaut equipment, and aeronautic applications. Aerogels are a unique material for providing such properties because of their extremely low density and small pore sizes. However, plain silica aerogels are brittle. Reinforcing the aerogel structure with a polymer (X-Aerogel) provides vast improvements in strength while maintaining low density and pore structure. However, degradation of polymers used in cross-linking tends to limit use temperatures to below 150 C. Organic aerogels made from linear polyimide have been demonstrated, but gels shrink substantially during supercritical fluid extraction and may have lower use temperature due to lower glass transition temperatures. The purpose of this innovation is to raise the glass transition temperature of all organic polyimide aerogel by use of tri-, tetra-, or poly-functional units in the structure to create a 3D covalently bonded network. Such cross-linked polyimides typically have higher glass transition temperatures in excess of 300 400 C. In addition, the reinforcement provided by a 3D network should improve mechanical stability, and prevent shrinkage on supercritical fluid extraction. The use of tri-functional aromatic or aliphatic amine groups in the polyimide backbone will provide such a 3D structure.

  20. A new model for the interaction of dystrophin with F-actin

    PubMed Central

    1996-01-01

    The F-actin binding and cross-linking properties of skeletal muscle dystrophin-glycoprotein complex were examined using high and low speed cosedimentation assays, microcapillary falling ball viscometry, and electron microscopy. Dystrophin-glycoprotein complex binding to F-actin saturated near 0.042 +/- 0.005 mol/ mol, which corresponds to one dystrophin per 24 actin monomers. Dystrophin-glycoprotein complex bound to F-actin with an average apparent Kd for dystrophin of 0.5 microM. These results demonstrate that native, full-length dystrophin in the glycoprotein complex binds F-actin with some properties similar to those measured for several members of the actin cross-linking super- family of proteins. However, we failed to observe dystrophin- glycoprotein complex-induced cross-linking of F-actin by three different methods, each positively controlled with alpha-actinin. Furthermore, high speed cosedimentation analysis of dystrophin- glycoprotein complex digested with calpain revealed a novel F-actin binding site located near the middle of the dystrophin rod domain. Recombinant dystrophin fragments corresponding to the novel actin binding site and the first 246 amino acids of dystrophin both bound F- actin but with significantly lower affinity and higher capacity than was observed with purified dystrophin-glycoprotein complex. Finally, dystrophin-glycoprotein complex was observed to significantly slow the depolymerization of F-actin, Suggesting that dystrophin may lie along side an actin filament through interaction with multiple actin monomers. These data suggest that although dystrophin is most closely related to the actin cross-linking superfamily based on sequence homology, dystrophin binds F-actin in a manner more analogous to actin side-binding proteins. PMID:8909541

  1. Novel magnetic cross-linked lipase aggregates for improving the resolution of (R, S)-2-octanol.

    PubMed

    Liu, Ying; Guo, Chen; Liu, Chun-Zhao

    2015-03-01

    Novel magnetic cross-linked lipase aggregates were fabricated by immobilizing the cross-linked lipase aggregates onto magnetic particles with a high number of -NH2 terminal groups using p-benzoquinone as the cross-linking agent. At the optimal fabrication conditions, 100% of immobilization efficiency and 139% of activity recovery of the magnetic cross-linked lipase aggregates were achieved. The magnetic cross-linked lipase aggregates were able to efficiently resolve (R, S)-2-octanol, and retained 100% activity and 100% enantioselectivity after 10 cycles of reuse, whereas the cross-linked lipase aggregates only retained about 50% activity and 70% enantioselectivity due to insufficient cross-linking. These results provide a great potential for industrial applications of the magnetic cross-linked lipase aggregates.

  2. Actinic keratosis

    MedlinePlus

    Solar keratosis; Sun-induced skin changes - keratosis; Keratosis - actinic (solar); Skin lesion - actinic keratosis ... likely to develop it if you: Have fair skin, blue or green eyes, or blond or red ...

  3. Reduced 4,5',8-trimethylpsoralen cross-linking of left-handed Z-DNA stabilized by DNA supercoiling

    SciTech Connect

    Sinden, R.R.; Kochel, T.J.

    1987-03-10

    Z-DNA-forming sequences, (GT)/sub 21/, (GT)/sub 12/ATGT, and (CG)/sub 6/TA(CG)/sub 6/, were cloned into plasmids. These sequences formed left-handed Z-DNA conformations under torsional tension from negative supercoiling of DNA. (/sup 3/H)4,5',8-Trimethylpsoralen, on absorption of 360-nm light, forms monoadducts and interstrand cross-links in DNA that exists in the B-helical conformation. Trimethylpsoralen cross-links were introduced into the potential Z-DNA-forming sequences in relaxed DNA when these sequences existed as B-form DNA. In supercoiled DNA when these sequences existed in the Z conformation, the rate of cross-linking was greatly reduced, and trimethylpsoralen did not form monoadducts appreciably to Z-DNA. As an internal control in these experiments, the rates of cross-linking of the Z-DNA-forming sequences were measured relative to that of an adjacent, cloned sequence that could not adopt a Z conformation. The initial relative rates of cross-linking to Z-DNA-forming sequences were dependent on the superhelical density of the DNA, and the rates were ultimately reduced by factors of 10-15 for Z-DNA in highly supercoiled plasmids. This differential rate of cross-linking provides a novel assay for Z-DNA. Initial application of this assay in vivo suggests that a substantial fraction of (CG)/sub 6/TA(CG)/sub 6/, which existed as Z-DNA in plasmid molecules purified from cells, existed in the B conformation in vivo.

  4. Clinical safety and wear resistance of the phospholipid polymer-grafted highly cross-linked polyethylene liner.

    PubMed

    Moro, Toru; Takatori, Yoshio; Tanaka, Sakae; Ishihara, Kazuhiko; Oda, Hiromi; Kim, Yoon Taek; Umeyama, Takashige; Fukatani, Eisei; Ito, Hideya; Kyomoto, Masayuki; Oshima, Hirofumi; Tanaka, Takeyuki; Kawaguchi, Hiroshi; Nakamura, Kozo

    2016-11-03

    To reduce the production of wear particles and subsequent aseptic loosening, we created a human articular cartilage-mimicked surface for a highly cross-linked polyethylene liner, whose surface grafted layer consisted of a biocompatible phospholipid polymer, poly(2-methacryloyloxyethyl phosphorylcholine). Although our previous in vitro findings showed that poly(2-methacryloyloxyethyl phosphorylcholine)-grafted particles were biologically inert and caused no subsequent bone resorptive responses, and poly(2-methacryloyloxyethyl phosphorylcholine) grafting markedly decreased wear in hip joint simulator tests, the clinical safety, and in vivo wear resistance of poly(2-methacryloyloxyethyl phosphorylcholine)-grafted highly cross-linked polyethylene liners remained open to question. Therefore, in the present study, we evaluated clinical and radiographic outcomes of poly(2-methacryloyloxyethyl phosphorylcholine)-grafted highly cross-linked polyethylene liners 5 years subsequent to total hip replacement in 68 consecutive patients. No reoperation was required for any reason, and no adverse events were associated with the implanted liners. The average Harris Hip Score increased from 38.6 preoperatively to 96.5 5 years postoperatively, and health-related quality of life, as indicated by the Short Form 36 Health Survey, improved. Radiographic analyses showed no periprosthetic osteolysis or implant migration. Between 1 and 5 years postoperatively, the mean steady-state wear rate was 0.002 mm/year, which represented a marked reduction relative to other highly cross-linked polyethylene liners, and appeared to be unaffected by patient-related or surgical factors. Although longer follow up is required, poly(2-methacryloyloxyethyl phosphorylcholine)-grafted highly cross-linked polyethylene liners improved mid-term clinical outcomes. The clinical safety and wear-resistance results are encouraging with respect to the improvement of long-term clinical outcomes with poly(2

  5. F-actin sequesters elongation factor 1alpha from interaction with aminoacyl-tRNA in a pH-dependent reaction

    PubMed Central

    1996-01-01

    The machinery of eukaryotic protein synthesis is found in association with the actin cytoskeleton. A major component of this translational apparatus, which is involved in the shuttling of aa-tRNA, is the actin- binding protein elongation factor 1alpha (EF-1alpha). To investigate the consequences for translation of the interaction of EF-1alpha with F- actin, we have studied the effect of F-actin on the ability of EF- 1alpha to bind to aa-tRNA. We demonstrate that binding of EF-1alpha:GTP to aa-tRNA is not pH sensitive with a constant binding affinity of approximately 0.2 microM over the physiological range of pH. However, the sharp pH dependence of binding of EF-1alpha to F-actin is sufficient to shift the binding of EF-1alpha from F-actin to aa-tRNA as pH increases. The ability of EF-1alpha to bind either F-actin or aa- tRNA in competition binding experiments is also consistent with the observation that EF-1alpha's binding to F-actin and aa-tRNA is mutually exclusive. Two pH-sensitive actin-binding sequences in EF-1alpha are identified and are predicted to overlap with the aa-tRNA-binding sites. Our results suggest that pH-regulated recruitment and release of EF- 1alpha from actin filaments in vivo will supply a high local concentration of EF-1alpha to facilitate polypeptide elongation by the F-actin-associated translational apparatus. PMID:8922379

  6. Damage and fatigue in cross-linked rubbers

    NASA Astrophysics Data System (ADS)

    Melnikov, Alexei

    Damage and fatigue of elastomers have not been fundamentally understood because of the complex nature of these materials. All currently existing models are completely phenomenological. Therefore two problems have been investigated in this research to address those fundamental issues. The first problem was creating an innovative concept with a mathematical modeling, which would be able to describe the damage using molecular characteristics of elastomers. The second problem is developing new approaches to study fatigue, and especially impact fatigue of elastomers. The following results have been obtained in this research. A theoretical model of damage has been developed which involves the basic molecular characteristics of cross-linked elastomers and takes into account the effects of viscoelasticity and stress-induced crystallization. This model was found very reliable and successful in description of numerous quasi-static simple extension experiments for monotonous and repeating loadings. It also roughly predicts in molecular terms the failure of elastomers with various degrees of cross-linking. Quasi-impact fatigue tests with different geometry of an indenter have also been performed. Some microscopic features of rubber damage have been investigated using optical microscopy and SEM. In particular, the accumulation of a completely de-vulcanized, liquid-like substance was observed under intense, multi-cycle impacts. All the findings discovered in quasi-impact experiments are consistent with the damage model predictions.

  7. Tea Derived Galloylated Polyphenols Cross-Link Purified Gastrointestinal Mucins

    PubMed Central

    Georgiades, Pantelis; Pudney, Paul D. A.; Rogers, Sarah; Thornton, David J.; Waigh, Thomas A.

    2014-01-01

    Polyphenols derived from tea are thought to be important for human health. We show using a combination of particle tracking microrheology and small-angle neutron scattering that polyphenols acts as cross-linkers for purified gastrointestinal mucin, derived from the stomach and the duodenum. Both naturally derived purified polyphenols, and green and black tea extracts are shown to act as cross-linkers. The main active cross-linking component is found to be the galloylated forms of catechins. The viscosity, elasticity and relaxation time of the mucin solutions experience an order of magnitude change in value upon addition of the polyphenol cross-linkers. Similarly small-angle neutron scattering experiments demonstrate a sol-gel transition with the addition of polyphenols, with a large increase in the scattering at low angles, which is attributed to the formation of large scale (>10 nm) heterogeneities during gelation. Cross-linking of mucins by polyphenols is thus expected to have an impact on the physicochemical environment of both the stomach and duodenum; polyphenols are expected to modulate the barrier properties of mucus, nutrient absorption through mucus and the viscoelastic microenvironments of intestinal bacteria. PMID:25162539

  8. Supermacroporous chemically cross-linked poly(aspartic acid) hydrogels.

    PubMed

    Gyarmati, Benjámin; Mészár, E Zsuzsanna; Kiss, Lóránd; Deli, Mária A; László, Krisztina; Szilágyi, András

    2015-08-01

    Chemically cross-linked poly(aspartic acid) (PASP) gels were prepared by a solid-liquid phase separation technique, cryogelation, to achieve a supermacroporous interconnected pore structure. The precursor polymer of PASP, polysuccinimide (PSI) was cross-linked below the freezing point of the solvent and the forming crystals acted as templates for the pores. Dimethyl sulfoxide was chosen as solvent instead of the more commonly used water. Thus larger temperatures could be utilized for the preparation and the drawback of increase in specific volume of water upon freezing could be eliminated. The morphology of the hydrogels was characterized by scanning electron microscopy and interconnectivity of the pores was proven by the small flow resistance of the gels. Compression tests also confirmed the interconnected porous structure and the complete re-swelling and shape recovery of the supermacroporous PASP hydrogels. The prepared hydrogels are of interest for several biomedical applications as scaffolding materials because of their cytocompatibility, controllable morphology and pH-responsive character.

  9. Preparation and characterization of cross-linked composite polymer electrolytes

    SciTech Connect

    Hou, J.; Baker, G.L.

    1998-11-01

    Cross-linkable composite electrolytes were prepared from poly(ethylene glycol) dimethyl ether (PEGDME)-500, LiClO{sub 4}, fumed silica, and 10 wt % methyl, butyl, or octyl methacrylate. The silicas used were chemically modified by attaching methacrylate groups to the silica surface through C{sub 8} and C{sub 3} tethers. Before cross-linking, the electrolytes were thixotropic and had ionic conductivities of >2 {times} 10{sup {minus}4} S/cm. After ultraviolet (UV)-induced cross-linking, the electrolytes were rubbery and dimensionally stable, and the conductivities were unchanged. Conductivity, extraction, and thermal analysis data all support a model where the added methacrylate monomer and growing polymer chains phase separate from the electrolyte phase during photopolymerization to yield a methacrylate-rich silica/polymer phase and little or no polymer in the PEGDME-500 phase. Thus, the mechanical properties of the composite electrolyte and its ionic conductivity are decoupled and can be optimized independently.

  10. Site-directed photo-cross-linking of rRNA transcription initiation complexes.

    PubMed Central

    Gong, X; Radebaugh, C A; Geiss, G K; Simon, M N; Paule, M R

    1995-01-01

    Site-specific photo-cross-linking of the rRNA committed transcription complex was carried out by using 5-[N-(p-azidobenzoyl)-3-aminoallyl]-dUMP-derivatized promoter DNA. Putative TAFIs of 145, 99, 96, and 91 kDa, as well as TATA-binding protein (TBP), were found to specifically photo-cross-link to different positions along the promoter. These had been identified as potential subunits of the fundamental transcription initiation factor TIF-IB (also known as SL1, factor D, and TFID) from Acanthamoeba castellanii by purification to apparent homogeneity. No other polypeptides attributable to the rRNA architectural transcription factor UBF were identified, suggesting that this protein is not part of the committed complex. Scanning transmission electron microscopy of the complexes was used to estimate the mass of the complex and the contour length of the DNA in the complex. This showed that a single molecule of TIF-IB is in each committed complex and that the DNA is not looped around the protein, as would be expected if UBF were in the complex. A circular permutation analysis of DNA bending resulting from TIF-IB binding revealed a 45 +/- 3.1 degrees (n = 14) bend centered 23 bp upstream of the transcription initiation site. This degree of bending and the position of the bend relative to the site of TBP photo-cross-linking are consistent with earlier data showing that the TBP TATA box-binding domain is not utilized in the assembly of the rRNA committed complex (C. A. Radebaugh, J. L. Mathews, G. K. Geiss, F. Liu, J. Wong, E. Bateman, S. Camier, A. Sentenac, and M. R. Paule, Mol. Cell. Biol. 14:597-605, 1994). PMID:7651413

  11. Site-directed photo-cross-linking of rRNA transcription initiation complexes.

    PubMed

    Gong, X; Radebaugh, C A; Geiss, G K; Simon, M N; Paule, M R

    1995-09-01

    Site-specific photo-cross-linking of the rRNA committed transcription complex was carried out by using 5-[N-(p-azidobenzoyl)-3-aminoallyl]-dUMP-derivatized promoter DNA. Putative TAFIs of 145, 99, 96, and 91 kDa, as well as TATA-binding protein (TBP), were found to specifically photo-cross-link to different positions along the promoter. These had been identified as potential subunits of the fundamental transcription initiation factor TIF-IB (also known as SL1, factor D, and TFID) from Acanthamoeba castellanii by purification to apparent homogeneity. No other polypeptides attributable to the rRNA architectural transcription factor UBF were identified, suggesting that this protein is not part of the committed complex. Scanning transmission electron microscopy of the complexes was used to estimate the mass of the complex and the contour length of the DNA in the complex. This showed that a single molecule of TIF-IB is in each committed complex and that the DNA is not looped around the protein, as would be expected if UBF were in the complex. A circular permutation analysis of DNA bending resulting from TIF-IB binding revealed a 45 +/- 3.1 degrees (n = 14) bend centered 23 bp upstream of the transcription initiation site. This degree of bending and the position of the bend relative to the site of TBP photo-cross-linking are consistent with earlier data showing that the TBP TATA box-binding domain is not utilized in the assembly of the rRNA committed complex (C. A. Radebaugh, J. L. Mathews, G. K. Geiss, F. Liu, J. Wong, E. Bateman, S. Camier, A. Sentenac, and M. R. Paule, Mol. Cell. Biol. 14:597-605, 1994).

  12. Carbonylation and disassembly of the F-actin cytoskeleton in oxidant induced barrier dysfunction and its prevention by epidermal growth factor and transforming growth factor α in a human colonic cell line

    PubMed Central

    Banan, A; Zhang, Y; Losurdo, J; Keshavarzian, A

    2000-01-01

    BACKGROUND—Intestinal barrier dysfunction concomitant with high levels of reactive oxygen metabolites (ROM) in the inflamed mucosa have been observed in inflammatory bowel disease (IBD). The cytoskeletal network has been suggested to be involved in the regulation of barrier function. Growth factors (epidermal growth factor (EGF) and transforming growth factor α (TGF-α)) protect gastrointestinal barrier integrity against a variety of noxious agents. However, the underlying mechanisms of oxidant induced disruption and growth factor mediated protection remain elusive.
AIMS—To determine: (1) if oxidation and disassembly of actin (a key cytoskeletal component) plays a major role in ROM induced epithelial monolayer barrier dysfunction; and (2) if growth factor mediated protection involves prevention of theses alterations.
METHODS—Caco-2 monolayers were preincubated with EGF, TGF-α, or vehicle before incubation with ROM (H2O2 or HOCl). Effects on cell integrity, barrier function, and G- and F-actin (oxidation, disassembly, and assembly) were determined.
RESULTS—ROM dose dependently and significantly increased F- and G-actin oxidation (carbonylation), decreased the stable F-actin fraction (index of stability), and increased the monomeric G-actin fraction (index of disassembly). Concomitant with these changes were disruption of the actin cytoskeleton and loss of the monolayer barrier function. In contrast, growth factor pretreatment decreased actin oxidation and enhanced the stable F-actin, while in concert prevented actin disruption and restored normal barrier function of monolayers exposed to ROM. Cytochalasin-D, an inhibitor of actin assembly, not only caused actin disassembly and barrier dysfunction but also abolished the protective action of growth factors. Moreover, an actin stabilising agent, phalloidin, mimicked the protective actions of the growth factors.
CONCLUSIONS—Oxidation, disassembly, and instability of the actin cytoskeleton appears to

  13. The influence of thermal aging on absorption phenomena in cross-linked polyethylene cable insulation

    NASA Astrophysics Data System (ADS)

    Borisova, M. E.; Osina, Yu. K.

    2017-01-01

    Absorption-controlled charge and discharge currents in cross-linked polyethylene (XPE) cable insulation before and after thermal aging have been measured. The experimental dependences were analyzed in terms of the equivalent Voigt scheme. Known parameters of the Voigt scheme were used to calculate the frequency dependences of relative dielectric permittivity, loss factor, and loss tangent of XPE films in the region of low frequencies (ω = 10-3-1 s-1) at high temperatures. Results of analysis of the absorption characteristics of XPE were applied to modeling of the process of thermal aging of cable insulation.

  14. Acoustic Radiation Force for Noninvasive Evaluation of Corneal Biomechanical Changes Induced by Cross-linking Therapy

    PubMed Central

    Urs, Raksha; Lloyd, Harriet O.; Silverman, Ronald H.

    2015-01-01

    Objectives To noninvasively measure changes in corneal biomechanical properties induced by ultraviolet-activated riboflavin cross-linking therapy using acoustic radiation force (ARF). Methods Cross-linking was performed on the right eyes of 6 rabbits, with the left eyes serving as controls. Acoustic radiation force was used to assess corneal stiffness before treatment and weekly for 4 weeks after treatment. Acoustic power levels were within US Food and Drug Administration guidelines for ophthalmic safety. Strain, determined from ARF-induced displacement of the front and back surfaces of the cornea, was fit to the Kelvin-Voigt model to determine the elastic modulus (E) and coefficient of viscosity (η). The stiffness factor, the ratio of E after treatment to E before treatment, was calculated for treated and control eyes. At the end of 4 weeks, ex vivo thermal shrinkage temperature analysis was performed for comparison with in vivo stiffness measurements. One-way analysis of variance and Student t tests were performed to test for differences in E, η, the stiffness factor, and corneal thickness. Results Biomechanical stiffening was immediately evident in cross-linking–treated corneas. At 4 weeks after treatment, treated corneas were 1.3 times stiffer and showed significant changes in E(P= .006) and η (P= .007), with no significant effect in controls. Corneal thickness increased immediately after treatment but did not differ significantly from the pretreatment value at 4 weeks. Conclusions Our findings demonstrate a statistically significant increase in stiffness in cross-linking–treated rabbit corneas based on in vivo axial stress/strain measurements obtained using ARF. The capacity to noninvasively monitor corneal stiffness offers the potential for clinical monitoring of cross-linking therapy. PMID:25063407

  15. CRP2, a new invadopodia actin bundling factor critically promotes breast cancer cell invasion and metastasis

    PubMed Central

    Dieterle, Monika; Moreau, Flora; Al Absi, Antoun; Steinmetz, André; Oudin, Anaïs; Berchem, Guy; Janji, Bassam; Thomas, Clément

    2016-01-01

    A critical process underlying cancer metastasis is the acquisition by tumor cells of an invasive phenotype. At the subcellular level, invasion is facilitated by actin-rich protrusions termed invadopodia, which direct extracellular matrix (ECM) degradation. Here, we report the identification of a new cytoskeletal component of breast cancer cell invadopodia, namely cysteine-rich protein 2 (CRP2). We found that CRP2 was not or only weakly expressed in epithelial breast cancer cells whereas it was up-regulated in mesenchymal/invasive breast cancer cells. In addition, high expression of the CRP2 encoding gene CSRP2 was associated with significantly increased risk of metastasis in basal-like breast cancer patients. CRP2 knockdown significantly reduced the invasive potential of aggressive breast cancer cells, whereas it did not impair 2D cell migration. In keeping with this, CRP2-depleted breast cancer cells exhibited a reduced capacity to promote ECM degradation, and to secrete and express MMP-9, a matrix metalloproteinase repeatedly associated with cancer progression and metastasis. In turn, ectopic expression of CRP2 in weakly invasive cells was sufficient to stimulate cell invasion. Both GFP-fused and endogenous CRP2 localized to the extended actin core of invadopodia, a structure primarily made of actin bundles. Purified recombinant CRP2 autonomously crosslinked actin filaments into thick bundles, suggesting that CRP2 contributes to the formation/maintenance of the actin core. Finally, CRP2 depletion significantly reduced the incidence of lung metastatic lesions in two xenograft mouse models of breast cancer. Collectively, our data identify CRP2 as a new cytoskeletal component of invadopodia that critically promotes breast cancer cell invasion and metastasis. PMID:26883198

  16. Multiple crystal structures of actin dimers and their implications for interactions in the actin filament

    PubMed Central

    Sawaya, Michael R.; Kudryashov, D. S.; Pashkov, Inna; Adisetiyo, Helty; Reisler, Emil; Yeates, Todd O.

    2008-01-01

    The structure of actin in its monomeric form is known at high resolution, while the structure of filamentous F-actin is only understood at considerably lower resolution. Knowing pre­cisely how the monomers of actin fit together would lead to a deeper understanding of the dynamic behavior of the actin filament. Here, a series of crystal structures of actin dimers are reported which were prepared by cross-linking in either the longitudinal or the lateral direction in the filament state. Laterally cross-linked dimers, comprised of monomers belonging to different protofilaments, are found to adopt configurations in crystals that are not related to the native structure of filamentous actin. In contrast, multiple structures of longitudinal dimers consistently reveal the same interface between monomers within a single protofilament. The re­appearance of the same longitudinal interface in multiple crystal structures adds weight to arguments that the interface visualized is similar to that in actin filaments. Highly conserved atomic interactions involving residues 199–205 and 287–291 are highlighted. PMID:18391412

  17. Leptospira Immunoglobulin-Like Protein B (LigB) Binds to Both the C-Terminal 23 Amino Acids of Fibrinogen αC Domain and Factor XIII: Insight into the Mechanism of LigB-Mediated Blockage of Fibrinogen α Chain Cross-Linking

    PubMed Central

    Hsieh, Ching-Lin; Chang, Eric; Tseng, Andrew; Ptak, Christopher; Wu, Li-Chen; Su, Chun-Li; McDonough, Sean P.; Lin, Yi-Pin; Chang, Yung-Fu

    2016-01-01

    The coagulation system provides a primitive but effective defense against hemorrhage. Soluble fibrinogen (Fg) monomers, composed of α, β and γ chains, are recruited to provide structural support for the formation of a hemostatic plug. Fg binds to platelets and is processed into a cross-linked fibrin polymer by the enzymatic clotting factors, thrombin and Factor XIII (FXIII). The newly formed fibrin-platelet clot can act as barrier to protect against pathogens from entering the bloodstream. Further, injuries caused by bacterial infections can be confined to the initial wound site. Many pathogenic bacteria have Fg-binding adhesins that can circumvent the coagulation pathway and allow the bacteria to sidestep containment. Fg expression is upregulated during lung infection providing an attachment surface for bacteria with the ability to produce Fg-binding adhesins. Fg binding by leptospira might play a crucial factor in Leptospira-associated pulmonary hemorrhage, the main factor contributing to lethality in severe cases of leptospirosis. The 12th domain of Leptospira immunoglobulin-like protein B (LigB12), a leptospiral adhesin, interacts with the C-terminus of FgαC (FgαCC). In this study, the binding site for LigB12 was mapped to the final 23 amino acids at the C-terminal end of FgαCC (FgαCC8). The association of FgαCC8 with LigB12 (ELISA, KD = 0.76 μM; SPR, KD = 0.96 μM) was reduced by mutations of both charged residues (R608, R611 and H614 from FgαCC8; D1061 from LigB12) and hydrophobic residues (I613 from FgαCC8; F1054 and A1065 from LigB12). Additionally, LigB12 bound strongly to FXIII and also inhibited fibrin formation, suggesting that LigB can disrupt coagulation by suppressing FXIII activity. Here, the detailed binding mechanism of a leptospiral adhesin to a host hemostatic factor is characterized for the first time and should provide better insight into the pathogenesis of leptospirosis. PMID:27622634

  18. Phylogenetic Patterns of Codon Evolution in the ACTIN-DEPOLYMERIZING FACTOR/COFILIN (ADF/CFL) Gene Family.

    PubMed

    Roy-Zokan, Eileen M; Dyer, Kelly A; Meagher, Richard B

    2015-01-01

    The actin-depolymerizing factor/cofilin (ADF/CFL) gene family encodes a diverse group of relatively small proteins. Once known strictly as modulators of actin filament dynamics, recent research has demonstrated that these proteins are involved in a variety of cellular processes, from signal transduction to the cytonuclear trafficking of actin. In both plant and animal lineages, expression patterns of paralogs in the ADF/CFL gene family vary among tissue types and developmental stages. In this study we use computational approaches to investigate the evolutionary forces responsible for the diversification of the ADF/CFL gene family. Estimating the rate of non-synonymous to synonymous mutations (dN/dS) across phylogenetic lineages revealed that the majority of ADF/CFL codon positions were under strong purifying selection, with rare episodic events of accelerated protein evolution. In both plants and animals these instances of accelerated evolution were ADF/CFL subclass specific, and all of the sites under selection were located in regions of the protein that could serve in new functional roles. We suggest these sites may have been important in the functional diversification of ADF/CFL proteins.

  19. Yield and Failure Behavior Investigated for Cross-Linked Phenolic Resins Using Molecular Dynamics

    NASA Technical Reports Server (NTRS)

    Monk, Joshua D.; Lawson, John W.

    2016-01-01

    Molecular dynamics simulations were conducted to fundamentally evaluate the yield and failure behavior of cross-linked phenolic resins at temperatures below the glass transition. Yield stress was investigated at various temperatures, strain rates, and degrees of cross-linking. The onset of non-linear behavior in the cross-linked phenolic structures was caused by localized irreversible molecular rearrangements through the rotation of methylene linkers followed by the formation or annihilation of neighboring hydrogen bonds. The yield stress results, with respect to temperature and strain rate, could be fit by existing models used to describe yield behavior of amorphous glasses. The degree of cross-linking only indirectly influences the maximum yield stress through its influence on glass transition temperature (Tg), however there is a strong relationship between the degree of cross-linking and the failure mechanism. Low cross-linked samples were able to separate through void formation, whereas the highly cross-linked structures exhibited bond scission.

  20. Adding chemical cross-links to a physical hydrogel.

    PubMed

    Paradossi, Gaio; Finelli, Ivana; Cerroni, Barbara; Chiessi, Ester

    2009-09-17

    Synergistic hydrogels are often encountered in polysaccharide mixtures widely used in food and biopharma products. The xanthan and konjac glucomannan pair provides one of the most studied synergistic hydrogels. Recently we showed that the junction zones stabilizing the 3D structure of this gel are present as macromolecular complexes in solution formed by the partially depolymerised polysaccharidic chains. The non-covalent interactions stabilizing the structure of the polysaccharidic complex cause the melting of the ordered structure of the complex in the solution and of the hydrogels. Introduction of chemical cross-links in the 3D structure of the synergistic hydrogel removes this behaviour, adding new features to the swelling and to the viscoelastic properties of the cured hydrogel. The use of epichlorohydrin as low molecular weight cross-linker does not impact unfavourably on the viability of NIH 3T3 fibroblasts.

  1. Encapsulation of cobalt nanoparticles in cross-linked-polymer cages

    NASA Astrophysics Data System (ADS)

    Hatamie, Shadie; Dhole, S. D.; Ding, J.; Kale, S. N.

    2009-07-01

    Nanoparticles embedded in polymeric cages give rise to interesting applications ranging from nanocatalysis to drug-delivery systems. In this context, we report on synthesis of cobalt (Co) nanoparticles trapped in polyvinyl alcohol (PVA) matrix to yield self-supporting magnetic films in PVA slime. A 20 nm, Co formed in FCC geometry encapsulated with a weak citrate coat when caged in PVA matrix exhibited persistence of magnetism and good radio-frequency response. Cross-linking of PVA chains to form cage-like structures to arrest Co nanoparticles therein, is believed to be the reason for oxide-free nature of Co, promising applications in biomedicine as well as in radio-frequency shielding.

  2. Photoinduced Plasticity in Cross-Linked Liquid Crystalline Networks.

    PubMed

    McBride, Matthew K; Hendrikx, Matthew; Liu, Danqing; Worrell, Brady T; Broer, Dirk J; Bowman, Christopher N

    2017-02-24

    Photoactivated reversible addition fragmentation chain transfer (RAFT)-based dynamic covalent chemistry is incorporated into liquid crystalline networks (LCNs) to facilitate spatiotemporal control of alignment, domain structure, and birefringence. The RAFT-based bond exchange process, which leads to stress relaxation, is used in a variety of conditions, to enable the LCN to achieve a near-equilibrium structure and orientation upon irradiation. Once formed, and in the absence of subsequent triggering of the RAFT process, the (dis)order in the LCN and its associated birefringence are evidenced at all temperatures. Using this approach, the birefringence, including the formation of spatially patterned birefringent elements and surface-active topographical features, is selectively tuned by adjusting the light dose, temperature, and cross-linking density.

  3. Conventional Versus Cross-Linked Polyethylene for Total Hip Arthroplasty.

    PubMed

    Surace, Michele F; Monestier, Luca; Vulcano, Ettore; Harwin, Steven F; Cherubino, Paolo

    2015-09-01

    The clinical and radiographic outcomes of 88 patients who underwent primary total hip arthroplasty with either conventional polyethylene or cross-linked polyethylene (XLPE) from the same manufacturer were compared. There were no significant differences between the 2 subpopulations regarding average age, gender, side affected, or prosthetic stem and cup size. The average follow-up was 104 months (range, 55 to 131 months). To the authors' knowledge, this is the longest follow-up for this particular insert. Clinical and radiographic evaluations were performed at 1, 3, 6, and 12 months and then annually. Results showed that XLPE has a significantly greater wear reduction than that of standard polyethylene in primary total hip arthroplasty. At the longest available follow-up for these specific inserts, XLPE proved to be effective in reducing wear.

  4. Cross-linked network development in compatibilized alkyd/acrylic hybrid latex films for the creation of hard coatings.

    PubMed

    Wang, Tao; de las Heras Alarcón, Carolina; Goikoetxea, Monika; Beristain, Itxaso; Paulis, Maria; Barandiaran, Maria J; Asua, José M; Keddie, Joseph L

    2010-09-07

    Hybrids made from an alkyd resin and an acrylic copolymer can potentially combine the desired properties of each component. Alkyd/acrylic hybrid latex particles were synthesized via miniemulsion polymerization and used to create films at room temperature. Comparisons of the alkyd auto-oxidative cross-linking rates and the associated network development are made between two alkyd resins (with differing levels of hydrophilicity as measured by their acid numbers). The effects of increasing the compatibilization between the alkyd and the acrylic phase via functionalization with glycidyl methacrylate (GMA) are investigated. Magnetic resonance profiling and microindentation measurements reveal that film hardening occurs much faster in a GMA-functionalized alkyd hybrid than in the standard hybrid. The film's hardness increases by a factor of 4 over a 5-day period. The rate of cross-linking is significantly slower in nonfunctionalized alkyd hybrid films and when the more hydrophilic alkyd resin is used. Tensile deformation of the hybrid latex films reveals the effects of GMA functionalization and drier concentration in creating a denser cross-linked network. Modeling of the tensile deformation behavior of the hybrid films used a combination of the upper convected Maxwell model (to describe the viscoelastic component) and the Gent model (to describe the elastic component). The modeling provides a correlation between the cross-linked network formation and the resulting mechanical properties.

  5. [A Method for Protein Photo-cross-linking in Living Cells Facilitating Analysis of Physiological Interactions of Proteins].

    PubMed

    Hino, Nobumasa

    2015-01-01

    In living cells, most proteins form complexes with other proteins to exert their functions. Since protein functions are regulated in response to changes in the cellular environment, the components of the complexes can vary; therefore, proteins often interact in a weak and transient manner. To capture such labile protein interactions, we have developed a method for photo-cross-linking of proteins directly interacting in mammalian cells; this method involves expansion of the genetic code and site-specific incorporation of photoreactive amino acids into proteins. Upon cross-linking, protein complexes are stabilized by a covalent bond and can be readily isolated from cell extracts without the problems usually associated with simple affinity purification methods such as co-immunoprecipitation. Photo-cross-linkers have another benefit: they react exclusively with molecules within a range defined by the linker length. This property becomes useful for determining the binding interface of two proteins because the linkers can be introduced in a site-directed manner with our method. In this review, we first describe the expansion of the genetic code of mammalian cells for the incorporation of non-natural amino acids into proteins. Then, we introduce our recent applications and developments of the cross-linking method: identification of intracellular binding partners of the signaling protein growth factor receptor binding protein 2; analysis of the binding between membrane proteins on the cell surface; and a novel photoreactive amino acid that enables wide-ranging photo-cross-linking.

  6. Fabrication of cross-linked hydrazone covalent organic frameworks by click chemistry and application to solid phase microextraction.

    PubMed

    Wu, Mingxue; Chen, Gang; Ma, Jiutong; Liu, Ping; Jia, Qiong

    2016-12-01

    Covalent organic frameworks (COFs) are an emerging class of porous organic frameworks with diverse promising applications. Herein, we presented the first example of cross-linked hydrazone COFs (cross-linked COFs) coating via thiol-ene click chemistry for solid phase microextraction (SPME). Strong covalent bonds and interlayer of the prepared networks ensured the adsorption capacity and durability of the novel SPME fiber. π-π conjugated structure existed because of abundant phenyl rings and -C=N groups in the cross-lined COFs. A series of characterizations indicated that the cross-linked COFs possessed large surface areas, high porosities and stabilities as well as hydrophobicities. The fiber was applied to SPME of pesticide residues coupled with gas chromatography with an electron capture detector (GC-ECD). Under the optimum experimental conditions, enhancement factors in the range of 2190-10,998 were obtained, illustrating that the cross-linked COFs possessed remarkable preconcentration ability. The low detection limits of 0.0003-0.0023ngkg(-1) were achieved with relative standard deviations (RSDs) in the range of 3.4-7.6% (intra-batch) and 5.7-11.6% (inter-batch), respectively. Recovery values in the range of 78.2-107.0% were obtained when the SPME-GC method was applied to the analysis of pesticides in cucumber samples.

  7. Inactivation of Matrix-bound MMPs by Cross-linking Agents in Acid Etched Dentin

    PubMed Central

    Scheffel, Débora Lopes Salles; Hebling, Josimeri; Scheffel, Régis Henke; Agee, Kelly A.; Turco, Gianluca; de Souza Costa, Carlos Alberto; Pashley, David H.

    2014-01-01

    Objectives Published TEM analysis of in vivo resin-dentin bonds shows that in 44 months almost 70% of collagen fibrils from the hybrid layer disappear. Matrix metalloproteinases (MMPs) play an important role in that process and are thought to be the main factor responsible for the solubitization of dentin collagen. Therefore, this study aimed to evaluate the inactivation of matrix-bound MMPs by carbodiimide (EDC) or proanthocyanidin (PA) both cross-linking agents, or the MMP-inhibitor, chlorhexidine (CHX), on acid-etched dentin using a simplified MMP assay method. Methods Dentin beams (1×1×6mm) were obtained from mid-coronal dentin of sound third molars and randomly divided into 6 groups (G) according to the dentin treatment: G1: Deionized water (control), G2: 0.1M EDC, G3: 0.5M EDC, G4: 0.5M EDC+35% HEMA, G5: 5% Proanthocyanidin (PA) and G6: 2% CHX. The beams were etched for 15s with 37% phosphoric acid, rinsed and then immersed for 60s in one of the treatment solutions. The total MMP activity of dentin was analyzed for 1 h by colorimetric assay (Sensolyte). Data were submitted to Wilcoxon non-parametric test and Mann-Whitney tests (p>0.05). Results All experimental cross-linking solutions significantly reduced MMP activity compared to control, except 0.1M EDC (53.6% ±16.1). No difference was observed between cross-linking agents and 2% CHX 0.5M EDC + 35% HEMA (92.3% ±8.0) was similar to 0.5M EDC (89.1% ±6.4), 5% PA (100.8% ±10.9) and 2% CHX (83.4% ±10.9). Conclusion Dentin treatment with cross-linking agents is effective to significantly reduce MMP activity. Mixing 0.5M EDC and 35% HEMA did not influence EDC inhibitor potential. PMID:23786610

  8. Nuclear Function of Subclass I Actin-Depolymerizing Factor Contributes to Susceptibility in Arabidopsis to an Adapted Powdery Mildew Fungus.

    PubMed

    Inada, Noriko; Higaki, Takumi; Hasezawa, Seiichiro

    2016-03-01

    Actin-depolymerizing factors (ADFs) are conserved proteins that function in regulating the structure and dynamics of actin microfilaments in eukaryotes. In this study, we present evidence that Arabidopsis (Arabidopsis thaliana) subclass I ADFs, particularly ADF4, functions as a susceptibility factor for an adapted powdery mildew fungus. The null mutant of ADF4 significantly increased resistance against the adapted powdery mildew fungus Golovinomyces orontii. The degree of resistance was further enhanced in transgenic plants in which the expression of all subclass I ADFs (i.e. ADF1-ADF4) was suppressed. Microscopic observations revealed that the enhanced resistance of adf4 and ADF1-4 knockdown plants (ADF1-4Ri) was associated with the accumulation of hydrogen peroxide and cell death specific to G. orontii-infected cells. The increased resistance and accumulation of hydrogen peroxide in ADF1-4Ri were suppressed by the introduction of mutations in the salicylic acid- and jasmonic acid-signaling pathways but not by a mutation in the ethylene-signaling pathway. Quantification by microscopic images detected an increase in the level of actin microfilament bundling in ADF1-4Ri but not in adf4 at early G. orontii infection time points. Interestingly, complementation analysis revealed that nuclear localization of ADF4 was crucial for susceptibility to G. orontii. Based on its G. orontii-infected-cell-specific phenotype, we suggest that subclass I ADFs are susceptibility factors that function in a direct interaction between the host plant and the powdery mildew fungus.

  9. Biomechanical Analysis of a Pedicle Screw-Rod System with a Novel Cross-Link Configuration

    PubMed Central

    Hara, Masahito; Umebayashi, Daisuke; Haimoto, Shoichi; Yamamoto, Yu; Nishimura, Yusuke; Wakabayashi, Toshihiko

    2016-01-01

    Study Design The strength effects of a pedicle screw-rod system supplemented with a novel cross-link configuration were biomechanically evaluated in porcine spines. Purpose To assess the biomechanical differences between a conventional cross-link pedicle screw-rod system versus a novel cross-link instrumentation, and to determine the effect of the cross-links. Overview of Literature Transverse cross-link systems affect torsional rigidity, but are thought to have little impact on the sagittal motion of spinal constructs. We tested the strength effects in pullout and flexion-compression tests of novel cross-link pedicle screw constructs using porcine thoracic and lumbar vertebrae. Methods Five matched thoracic and lumbar vertebral segments from 15 porcine spines were instrumented with 5.0-mm pedicle screws, which were then connected with 6.0-mm rods after partial corpectomy in the middle vertebral body. The forces required for construct failure in pullout and flexion-compression tests were examined in a randomized manner for three different cross-link configurations: un-cross-link control, conventional cross-link, and cross-link passing through the base of the spinous process. Statistical comparisons of strength data were analyzed using Student's t-tests. Results The spinous process group required a significantly greater pullout force for construct failure than the control group (p=0.036). No difference was found between the control and cross-link groups, or the cross-link and spinous process groups in pullout testing. In flexion-compression testing, the spinous processes group required significantly greater forces for construct failure than the control and cross-link groups (p<0.001 and p=0.003, respectively). However, there was no difference between the control and cross-link groups. Conclusions A novel cross-link configuration that features cross-link devices passing through the base of the spinous processes increased the mechanical resistance in pullout and

  10. TaADF4, an actin-depolymerizing factor from wheat, is required for resistance to the stripe rust pathogen Puccinia striiformis f. sp. tritici.

    PubMed

    Zhang, Bing; Hua, Yuan; Wang, Juan; Huo, Yan; Shimono, Masaki; Day, Brad; Ma, Qing

    2017-03-01

    Actin filament assembly in plants is a dynamic process, requiring the activity of more than 75 actin-binding proteins. Central to the regulation of filament assembly and stability is the activity of a conserved family of actin-depolymerizing factors (ADFs), whose primarily function is to regulate the severing and depolymerization of actin filaments. In recent years, the activity of ADF proteins has been linked to a variety of cellular processes, including those associated with response to stress. Herein, a wheat ADF gene, TaADF4, was identified and characterized. TaADF4 encodes a 139-amino-acid protein containing five F-actin-binding sites and two G-actin-binding sites, and interacts with wheat (Triticum aestivum) Actin1 (TaACT1), in planta. Following treatment of wheat, separately, with jasmonic acid, abscisic acid or with the avirulent race, CYR23, of the stripe rust pathogen Puccinia striiformis f. sp. tritici, we observed a rapid induction in accumulation of TaADF4 mRNA. Interestingly, accumulation of TaADF4 mRNA was diminished in response to inoculation with a virulent race, CYR31. Silencing of TaADF4 resulted in enhanced susceptibility to CYR23, demonstrating a role for TaADF4 in defense signaling. Using a pharmacological-based approach, coupled with an analysis of host response to pathogen infection, we observed that treatment of plants with the actin-modifying agent latrunculin B enhanced resistance to CYR23, including increased production of reactive oxygen species and enhancement of localized hypersensitive cell death. Taken together, these data support the hypothesis that TaADF4 positively modulates plant immunity in wheat via the modulation of actin cytoskeletal organization.

  11. Controlled Release of Salicylic Acid from Biodegradable Cross-Linked Polyesters.

    PubMed

    Dasgupta, Queeny; Chatterjee, Kaushik; Madras, Giridhar

    2015-09-08

    The purpose of this work was to develop a family of cross-linked poly(xylitol adipate salicylate)s with a wide range of tunable release properties for delivering pharmacologically active salicylic acid. The synthesis parameters and release conditions were varied to modulate polyester properties and to understand the mechanism of release. Varying release rates were obtained upon longer curing (35% in the noncured polymer to 10% in the cured polymer in 7 days). Differential salicylic acid loading led to the synthesis of polymers with variable cross-linking and the release could be tuned (100% release for the lowest loading to 30% in the highest loading). Controlled release was monitored by changing various factors, and the release profiles were dependent on the stoichiometric composition, pH, curing time, and presence of enzyme. The polymer released a combination of salicylic acid and disalicylic acid, and the released products were found to be nontoxic. Minimal hemolysis and platelet activation indicated good blood compatibility. These polymers qualify as "bioactive" and "resorbable" and can, therefore, find applications as immunomodulatory resorbable biomaterials with tunable release properties.

  12. Neutron scattering and ab initio molecular dynamics study of cross-linking in biomedical phosphate glasses.

    PubMed

    Parsons, A J; Ahmed, I; Rudd, C D; Cuello, G J; Pellegrini, E; Richard, D; Johnson, M R

    2010-12-08

    Details of the microscopic structure of phosphate glasses destined for biomedical applications, which include sodium, magnesium and calcium cations, have been obtained from the static structure factor measured by means of neutron scattering. A complementary, molecular dynamics study has been performed on a range of phosphate glasses using density functional theory methods, which allow structural fluctuations, including bond breaking, in the liquid phase before quenching to the glass phase. Good agreement between experiment and simulation allows the molecular dynamics trajectories to be analysed in detail. In particular, attention is focused on the cross-linking of divalent cations in contrast with the structural aspects associated with monovalent cations. Magnesium cations are found equidistant and bridging between the phosphorus atoms of different phosphate chains, leading to a shorter phosphorus-phosphorus second neighbour distance (that is, a more compact packing of neighbouring phosphate chains) compared to the effect of sodium cations. Calcium cations show behaviour intermediate between those of magnesium and sodium. Molecular dynamics simulations give access to the cation mobility, which is lowest for magnesium, reflecting its structural, cross-linking role.

  13. The Effect of Crosslinking on the Microscale Stress Response and Molecular Deformations in Actin Networks

    NASA Astrophysics Data System (ADS)

    Gurmessa, Bekele; Fitzpatrick, Robert; Valdivia, Jonathon; Anderson, Rae M. R.

    Actin, the most abundant protein in eukaryotic cells, is a semi-flexible biopolymer in the cytoskeleton that plays a crucial structural and mechanical role in cell stability, motion and replication, as well as muscle contraction. Most of these mechanically driven structural changes in cells stem from the complex viscoelastic nature of entangled actin networks and the presence of a myriad of proteins that cross-link actin filaments. Despite their importance, the mechanical response of actin networks is not yet well understood, particularly at the molecular level. Here, we use optical trapping - coupled with fluorescence microscopy - to characterize the microscale stress response and induced filament deformations in entangled and cross-linked actin networks subject to localized mechanical perturbations. In particular, we actively drive a microsphere 10 microns through an entangled or cross- linked actin network at a constant speed and measure the resistive force that the deformed actin filaments exert on the bead during and following strain. We simultaneously visualize and track individual sparsely-labeled actin filaments to directly link force response to molecular deformations, and map the propagation of the initially localized perturbation field throughout the rest of the network (~100 um). By varying the concentration of actin and cross-linkers we directly determine the role of crosslinking and entanglements on the length and time scales of stress propagation, molecular deformation and relaxation mechanisms in actin networks.

  14. Fabrication of patterned calcium cross-linked alginate hydrogel films and coatings through reductive cation exchange.

    PubMed

    Bruchet, Marion; Melman, Artem

    2015-10-20

    Calcium cross-linked alginate hydrogels are widely used in targeted drug delivery, tissue engineering, wound treatment, and other biomedical applications. We developed a method for preparing homogeneous alginate hydrogels cross-linked with Ca(2+) cations using reductive cation exchange in homogeneous iron(III) cross-linked alginate hydrogels. Treatment of iron(III) cross-linked alginate hydrogels with calcium salts and sodium ascorbate results in reduction of iron(III) cations to iron(II) that are instantaneously replaced with Ca(2+) cations, producing homogeneous ionically cross-linking hydrogels. Alternatively, the cation exchange can be performed by photochemical reduction in the presence of calcium chloride using a sacrificial photoreductant. This approach allows fabrication of patterned calcium alginate hydrogels through photochemical patterning of iron(III) cross-linked alginate hydrogel followed by the photochemical reductive exchange of iron cations to calcium.

  15. Characterization of the deoxyguanosine-lysine cross-link of methylglyoxal.

    PubMed

    Petrova, Katya V; Millsap, Amy D; Stec, Donald F; Rizzo, Carmelo J

    2014-06-16

    Methylglyoxal is a mutagenic bis-electrophile that is produced endogenously from carbohydrate precursors. Methylglyoxal has been reported to induce DNA-protein cross-links (DPCs) in vitro and in cultured cells. Previous work suggests that these cross-links are formed between guanine and either lysine or cysteine side chains. However, the chemical nature of the methylglyoxal induced DPC have not been determined. We have examined the reaction of methylglyoxal, deoxyguanosine (dGuo), and Nα-acetyllysine (AcLys) and determined the structure of the cross-link to be the N2-ethyl-1-carboxamide with the lysine side chain amino group (1). The cross-link was identified by mass spectrometry and the structure confirmed by comparison to a synthetic sample. Further, the cross-link between methylglyoxal, dGuo, and a peptide (AcAVAGKAGAR) was also characterized. The mechanism of cross-link formation is likely to involve an Amadori rearrangement.

  16. Flanking sequences modulate diepoxide and mustard cross-linking efficiencies at the 5'-GNC site.

    PubMed

    Sawyer, Gregory A; Frederick, Elizabeth D; Millard, Julie T

    2004-08-01

    Diepoxybutane, diepoxyoctane, and mechlorethamine are cytotoxic agents that induce interstrand cross-links between the N7 positions of deoxyguanosine residues on opposite strands of the DNA duplex preferentially at 5'-GNC sequences. We have systematically varied the identity of either the base 5' to the cross-linked deoxyguanosine residues or the intervening base pair to determine flanking sequence effects on cross-linking efficiency. We used synthetic DNA oligomers containing four 5'-N(1)GN(2)C sites that varied either N(1) or N(2). Interstrand cross-links were purified through denaturing polyacrylamide gel electrophoresis and then subjected to piperidine cleavage. The amount of cleavage at each deoxyguanosine residue, representative of cross-linking efficiency at that site, was determined by sequencing gel analysis. Our data suggest that cross-linking efficiency varies with the identity of N(1) similarly (purines > pyrimidines) for diepoxybutane, diepoxyoctane, and mechlorethamine but that the effects of N(2) differ for the three compounds.

  17. Effect of glucose content on thermally cross-linked fibrous gelatin scaffolds for tissue engineering.

    PubMed

    Siimon, Kaido; Reemann, Paula; Põder, Annika; Pook, Martin; Kangur, Triin; Kingo, Külli; Jaks, Viljar; Mäeorg, Uno; Järvekülg, Martin

    2014-09-01

    Thermally cross-linked glucose-containing electrospun gelatin meshes were studied as possible cell substrate materials. FTIR analysis was used to study the effect of glucose on cross-linking reactions. It was found that the presence of glucose increases the extent of cross-linking of fibrous gelatin scaffolds, which in return determines scaffold properties and their usability in tissue engineering applications. Easy to handle fabric-like scaffolds were obtained from blends containing up to 15% glucose. Maximum extent of cross-linking was reached at nearly 20% glucose content. Cross-linking effectively resulted in decreased solubility and increased resistance to enzymatic degradation. Preliminary short-term cell culture experiments indicate that such thermally cross-linked gelatin-glucose scaffolds are suitable for tissue engineering applications.

  18. An Open Data Format for Visualization and Analysis of Cross-Linked Mass Spectrometry Results.

    PubMed

    Hoopmann, Michael R; Mendoza, Luis; Deutsch, Eric W; Shteynberg, David; Moritz, Robert L

    2016-11-01

    Protein-protein interactions are an important element in the understanding of protein function, and chemical cross-linking shotgun mass spectrometry is rapidly becoming a routine approach to identify these specific interfaces and topographical interactions. Protein cross-link data analysis is aided by dozens of algorithm choices, but hindered by a lack of a common format for representing results. Consequently, interoperability between algorithms and pipelines utilizing chemical cross-linking remains a challenge. pepXML is an open, widely-used format for representing spectral search algorithm results that has facilitated information exchange and pipeline development for typical shotgun mass spectrometry analyses. We describe an extension of this format to incorporate cross-linking spectral search results. We demonstrate application of the extension by representing results of multiple cross-linking search algorithms. In addition, we demonstrate adapting existing pepXML-supporting software pipelines to analyze protein cross-linking results formatted in pepXML. Graphical Abstract ᅟ.

  19. Energy harvesting from vibration with cross-linked polypropylene piezoelectrets

    SciTech Connect

    Zhang, Xiaoqing; Wu, Liming; Sessler, Gerhard M.

    2015-07-15

    Piezoelectret films are prepared by modification of the microstructure of polypropylene foam sheets cross-linked by electronic irradiation (IXPP), followed by proper corona charging. Young’s modulus, relative permittivity, and electromechanical coupling coefficient of the fabricated films, determined by dielectric resonance spectra, are about 0.7 MPa, 1.6, and 0.08, respectively. Dynamic piezoelectric d{sub 33} coefficients up to 650 pC/N at 200 Hz are achieved. The figure of merit (FOM, d{sub 33} ⋅ g{sub 33}) for a more typical d{sub 33} value of 400 pC/N is about 11.2 GPa{sup −1}. Vibration-based energy harvesting with one-layer and two-layer stacks of these films is investigated at various frequencies and load resistances. At an optimum load resistance of 9 MΩ and a resonance frequency of 800 Hz, a maximum output power of 120 μW, referred to the acceleration g due to gravity, is obtained for an energy harvester consisting of a one-layer IXPP film with an area of 3.14 cm{sup 2} and a seismic mass of 33.7 g. The output power can be further improved by using two-layer stacks of IXPP films in electric series. IXPP energy harvesters could be used to energize low-power electronic devices, such as wireless sensors and LED lights.

  20. Zinc cross-linked hydroxamated alginates for pulsed drug release

    PubMed Central

    Raut, Neha S; Deshmukh, Prasad R; Umekar, Milind J; Kotagale, Nandkishor R

    2013-01-01

    Introduction: Alginates can be tailored chemically to improve solubility, physicochemical, and biological properties and its complexation with metal ion is useful for controlling the drug release. Materials And Methods: Synthesized N,O-dimethyl, N-methyl, or N-Benzyl hydroxylamine derivatives of sodium alginate were subsequently complexed with zinc to form beads. Hydroxamation of sodium alginate was confirmed by Fourier transform infra-red spectroscopy (FTIR) and differential scanning calorimetry (DSC). Results: The synthesized polymeric material exhibited reduced aqueous, HCl and NaOH solubility. The hydroxamated derivatives demonstrated pulsed release where change in pH of the dissolution medium stimulated the atenolol release. Conclusion: Atenolol loaded Zn cross-linked polymeric beads demonstrated the sustained the plasma drug levels with increased half-life. Although the synthesized derivatives greatly altered the aqueous solubility of sodium alginate, no significant differences in in vitro and in vivo atenolol release behavior amongst the N,O-dimethyl, N-methyl, or N-Benzyl hydroxylamine derivatives of sodium alginate were observed. PMID:24350039

  1. Photosensitive cross-linked block copolymers with controllable release.

    PubMed

    Yu, Lili; Lv, Cong; Wu, LiZhu; Tung, ChenHo; Lv, WanLiang; Li, ZhongJin; Tang, XinJing

    2011-01-01

    We intend to form photosensitive block copolymer micelles for controllable release of encapsulated substances. Here, we designed and synthesized a new photocleavable cross-linker (2-nitrophenyl ethylene glycol dimethacrylate) for methyl methacrylate (MMA) atom transfer radical polymerization. Four different ratios (0:1, 1:26, 1:16, 1:8.8) of the photocleavable cross-linker to MMA monomer were used and four block copolymers (P0, P1, P2, P3) were synthesized with PEO-Br as the macroinitiator. Gel permeation chromatography and (1) H NMR studies showed that linear polymer molecules could be cross-linked by the photocleavable linker. The fluorescence studies of the encapsulated Nile Red (NR) showed that there were lower critical micelle concentrations for the polymer P1, P2 and P3 than polymer P0. And dynamic light scattering and SEM confirmed the formation of polymer micelles. Photolysis experiments demonstrated that NR encapsulated in the polymer micelles could be released upon UV irradiation (365 nm, 11 mW cm(-2)) due to the breakage of the photocleavable linker and the generation of more hydrophilic acid moieties, which destabilized polymer micelles. Our study shows a new strategy for the possibility of photocontrollable drug release for hydrophobic drugs.

  2. Synthesis and Properties of Cross-Linked Polyamide Aerogels

    NASA Technical Reports Server (NTRS)

    Williams, Jarrod C.; Meador, Mary Ann; McCorkle, Linda

    2015-01-01

    We report the first synthesis of cross-linked polyamide aerogels through step growth polymerization using a combination of diamines, diacid chloride and triacid chloride. Polyamide oligomers endcapped with amines are prepared as stable solutions in N-methylpyrrolidinone from several different diamine precursors and 1,3-benzenedicarbonyl dichloride. Addition of 1,3,5-benzenetricarbonyl trichloride yields gels which form in under five minutes according to the scheme shown. Solvent exchange of the gels into ethanol, followed by drying using supercritical CO2 extraction gives colorless aerogels with densities around 0.1 to 0.2 gcm3. Thicker monolithes of the polyamide aerogels are stiff and strong, while thin films of certain formulations are highly flexible, durable, and even translucent. These materials may have use as insulation for deployable space structures, rovers, habitats or extravehicular activity suits as well as in many terrestrial applications. Strucure property relationships of the aerogels, including surface area, mechanical properties, and thermal conductivity will be discussed.

  3. Automated determination of cross-linked fibrin derivatives in plasma.

    PubMed

    Elms, M J; Bundesen, P G; Rowbury, D; Goodall, S; Wakeham, N; Rowell, J A; Hillyard, C J; Rylatt, D B

    1993-02-01

    Automated assays for the measurement of cross-linked fibrin derivatives in plasma (XL-FbDP) have been developed utilizing latex beads coated with anti-D dimer monoclonal antibody (DD-3B6/22) for both the Cobas Fara Chemistry Centrifugal and the Cobas Mira analysers (Roche, Basle, Switzerland). The analysers were programmed to mix plasma and latex reagent simultaneously and analyse absorbance changes over a 10-15 min period. Results were interpolated by the analyser from a standard curve derived from a polymer of D-dimer. Both assays had high precision (< 5% CV) for values between 100 and 1000 ng/ml and provided clear discrimination between normal samples and samples from patients suffering from the thrombotic diseases, DVT/PE and DIC. The results obtained for XL-FbDP determination with both methods compared well with established methods: a high correlation was obtained with a semi-quantitative manual latex method for both the Fara (r = 0.92) and Mira (r = 0.83) and correlations (r) of 0.81 (Fara) and 0.84 (Mira) were obtained with an enzyme immunoassay (EIA). Correlation between the two automated procedures was high (r = 0.96). The automated method will enable laboratories to provide a rapid and accurate quantitation of XL-FbDP.

  4. Pyridinium cross-links in heritable disorders of collagen

    SciTech Connect

    Pasquali, M.; Still, M.J.; Dembure, P.P.

    1995-12-01

    Ehlers-Danlos syndrome (EDS) is a heterogeneous group of inherited disorders of collagen that is characterized by skin fragility, skin hyperextensibility, and joint hypermobility. EDS type VI is caused by impaired collagen lysyl hydroxylase (procollagen-lysine, 2-oxoglutarate 5-dioxygenase; E.C.1.14.11.4), the ascorbate-dependent enzyme that hydroxylates lysyl residues on collagen neopeptides. Different alterations in the gene for collagen lysyl hydroxylase have been reported in families with EDS type VI. In EDS type VI, impairment of collagen lysyl hydroxylase results in a low hydroxylysine content in mature collagen. Hydroxylysine is a precursor of the stable, covalent, intermolecular cross-links of collagen, pyridinoline (Pyr), and deoxypyridinoline (Dpyr). Elsewhere we reported in preliminary form that patients with EDS type VI had a distinctive alteration in the urinary excretion of Pyr and Dpyr. In the present study, we confirm that the increased Dpyr/Pyr ratio is specific for EDS type VI and is not observed in other inherited or acquired collagen disorders. In addition, we find that skin from patients with EDS type VI has reduced Pyr and increased Dpyr, which could account for the organ pathology. 19 refs., 1 tab.

  5. DNA-Protein Cross-links: Formation, Structural Identities, and Biological Outcomes

    PubMed Central

    Tretyakova, Natalia Y.; Groehler, Arnold; Ji, Shaofei

    2015-01-01

    CONSPECTUS Non-covalent DNA-protein interactions are at the heart of normal cell function. In eukaryotic cells, genomic DNA is wrapped around histone octamers to allow for chromosomal packaging in the nucleus. Binding of regulatory protein factors to DNA directs replication, controls transcription, and mediates cellular responses to DNA damage. Because of their fundamental significance in all cellular processes involving DNA, dynamic DNA-protein interactions are required for cell survival, and their disruption is likely to have serious biological consequences. DNA-protein cross-links (DPCs) form when cellular proteins become covalently trapped on DNA strands upon exposure to various endogenous, environmental and chemotherapeutic agents. DPCs progressively accumulate in the brain and heart tissues as a result of endogenous exposure to reactive oxygen species and lipid peroxidation products, as well as normal cellular metabolism. A range of structurally diverse DPCs are found following treatment with chemotherapeutic drugs, transition metal ions, and metabolically activated carcinogens. Because of their considerable size and their helix-distorting nature, DPCs interfere with the progression of replication and transcription machineries and hence hamper the faithful expression of genetic information, potentially contributing to mutagenesis and carcinogenesis. Mass spectrometry-based studies have identified hundreds of proteins that can become cross-linked to nuclear DNA in the presence of reactive oxygen species, carcinogen metabolites, and antitumor drugs. While many of these proteins including histones, transcription factors, and repair proteins are known DNA binding partners, other gene products with no documented affinity for DNA also participate in DPC formation. Furthermore, multiple sites within DNA can be targeted for cross-linking including the N7 of guanine, the C-5 methyl group of thymine, and the exocyclic amino groups of guanine, cytosine, and adenine

  6. Actinous enigma or enigmatic actin

    PubMed Central

    Povarova, Olga I; Uversky, Vladimir N; Kuznetsova, Irina M; Turoverov, Konstantin K

    2014-01-01

    Being the most abundant protein of the eukaryotic cell, actin continues to keep its secrets for more than 60 years. Everything about this protein, its structure, functions, and folding, is mysteriously counterintuitive, and this review represents an attempt to solve some of the riddles and conundrums commonly found in the field of actin research. In fact, actin is a promiscuous binder with a wide spectrum of biological activities. It can exist in at least three structural forms, globular, fibrillar, and inactive (G-, F-, and I-actin, respectively). G-actin represents a thermodynamically instable, quasi-stationary state, which is formed in vivo as a result of the energy-intensive, complex posttranslational folding events controlled and driven by cellular folding machinery. The G-actin structure is dependent on the ATP and Mg2+ binding (which in vitro is typically substituted by Ca2+) and protein is easily converted to the I-actin by the removal of metal ions and by action of various denaturing agents (pH, temperature, and chemical denaturants). I-actin cannot be converted back to the G-form. Foldable and “natively folded” forms of actin are always involved in interactions either with the specific protein partners, such as Hsp70 chaperone, prefoldin, and the CCT chaperonin during the actin folding in vivo or with Mg2+ and ATP as it takes place in the G-form. We emphasize that the solutions for the mysteries of actin multifunctionality, multistructurality, and trapped unfolding can be found in the quasi-stationary nature of this enigmatic protein, which clearly possesses many features attributed to both globular and intrinsically disordered proteins.

  7. Proteomics reveals dynamic assembly of repair complexes during bypass of DNA cross-links

    PubMed Central

    Räschle, Markus; Smeenk, Godelieve; Hansen, Rebecca K.; Temu, Tikira; Oka, Yasuyoshi; Hein, Marco Y.; Nagaraj, Nagarjuna; Long, David T.; Walter, Johannes C.; Hofmann, Kay; Storchova, Zuzana; Cox, Jürgen; Bekker-Jensen, Simon; Mailand, Niels; Mann, Matthias

    2017-01-01

    DNA interstrand cross-links (ICLs) block replication fork progression by inhibiting DNA strand separation. Repair of ICLs requires sequential incisions, translesion DNA synthesis, and homologous recombination, but the full set of factors involved in these transactions remains unknown. We devised a technique called chromatin mass spectrometry (CHROMASS) to study protein recruitment dynamics during perturbed DNA replication in Xenopus egg extracts. Using CHROMASS, we systematically monitored protein assembly and disassembly on ICL-containing chromatin. Among numerous prospective DNA repair factors, we identified SLF1 and SLF2, which form a complex with RAD18 and together define a pathway that suppresses genome instability by recruiting the SMC5/6 cohesion complex to DNA lesions. Our study provides a global analysis of an entire DNA repair pathway and reveals the mechanism of SMC5/6 relocalization to damaged DNA in vertebrate cells. PMID:25931565

  8. Methylglyoxal-induced DNA-protein cross-links and cytotoxicity in Chinese hamster ovary cells.

    PubMed

    Brambilla, G; Sciabà, L; Faggin, P; Finollo, R; Bassi, A M; Ferro, M; Marinari, U M

    1985-05-01

    The technique of alkaline elution was applied to study the capacity of methylglyoxal to induce DNA damage and repair in Chinese hamster ovary cells. DNA cross-linking was observed after a 90-min exposure to a subtoxic dose (1.5 mM), and the cross-links were fully repaired by 24 h. The cross-linking appeared to be DNA-protein in nature, since proteinase treatment removed the effect. When the same cells were exposed to methylglyoxal in the presence of a rat liver metabolic system, both cytotoxicity and cross-linking frequency were significantly reduced.

  9. Biologically relevant oxidants cause bound proteins to readily oxidatively cross-link at Guanine.

    PubMed

    Solivio, Morwena J; Nemera, Dessalegn B; Sallans, Larry; Merino, Edward J

    2012-02-20

    Oxidative DNA-protein cross-links have received less attention than other types of DNA damage and remain as one of the least understood types of oxidative lesion. A model system using ribonuclease A and a 27-nucleotide DNA was used to determine the propensity of oxidative cross-linking to occur in the presence of oxidants. Cross-link formation was examined using four different oxidation systems that generate singlet oxygen, superoxide, and metal-based Fenton reactions. It is shown that oxidative cross-linking occurs in yields ranging from 14% to a maximal yield of 61% in all oxidative systems when equivalent concentrations of DNA and protein are present. Because singlet oxygen is the most efficient oxidation system in generating DNA-protein cross-links, it was chosen for further analyses. Cross-linking occurred with single-stranded DNA binding protein and not with bovine serum albumin. Addition of salt lowered nonspecific binding affinity and lowered cross-link yield by up to 59%. The yield of cross-linking increased with increased ratios of protein compared with DNA. Cross-linking was highly dependent on the number of guanines in a DNA sequence. Loss of guanine content on the 27-nucleotide DNA led to nearly complete loss in cross-linking, while primer extension studies showed cross-links to predominantly occur at guanine base on a 100-nucleotide DNA. The chemical species generated were examined using two peptides derived from the ribonuclease A sequence, N-acetyl-AAAKF and N-acetyl-AYKTT, which were cross-linked to 2'-deoxyguanosine. The cross-link products were spiroiminodihydantoin, guanidinohydantoin, and tyrosyl-based adducts. Formation of tyrosine-based adducts may be competitive with the more well-studied lysine-based cross-links. We conclude that oxidative cross-links may be present at high levels in cells since the propensity to oxidatively cross-link is high and so much of the genomic DNA is coated with protein.

  10. Effect of cross-linking and enzymatic hydrolysis composite modification on the properties of rice starches.

    PubMed

    Xiao, Huaxi; Lin, Qinlu; Liu, Gao-Qiang

    2012-07-06

    Native rice starch lacks the versatility necessary to function adequately under rigorous industrial processing, so modified starches are needed to meet the functional properties required in food products. This work investigated the impact of enzymatic hydrolysis and cross-linking composite modification on the properties of rice starches. Rice starch was cross-linked with epichlorohydrin (EPI) with different concentrations (0.5%, 0.7%, 0.9% w/w, on a dry starch basis), affording cross-linked rice starches with the three different levels of cross-linking that were named R₁, R₂, and R₃, respectively. The cross-linked rice starches were hydrolyzed by α-amylase and native, hydrolyzed, and hydrolyzed cross-linked rice starches were comparatively studied. It was found that hydrolyzed cross-linked rice starches showed a lower the degree of amylase hydrolysis compared with hydrolyzed rice starch. The higher the degree of cross-linking, the higher the capacity to resist enzyme hydrolysis. Hydrolyzed cross-linked rice starches further increased the adsorptive capacities of starches for liquids and decreased the trend of retrogradation, and it also strengthened the capacity to resist shear compared to native and hydrolyzed rice starches.

  11. Xilmass: A New Approach toward the Identification of Cross-Linked Peptides.

    PubMed

    Yılmaz, Şule; Drepper, Friedel; Hulstaert, Niels; Černič, Maša; Gevaert, Kris; Economou, Anastassios; Warscheid, Bettina; Martens, Lennart; Vandermarliere, Elien

    2016-10-18

    Chemical cross-linking coupled with mass spectrometry plays an important role in unravelling protein interactions, especially weak and transient ones. Moreover, cross-linking complements several structural determination approaches such as cryo-EM. Although several computational approaches are available for the annotation of spectra obtained from cross-linked peptides, there remains room for improvement. Here, we present Xilmass, a novel algorithm to identify cross-linked peptides that introduces two new concepts: (i) the cross-linked peptides are represented in the search database such that the cross-linking sites are explicitly encoded, and (ii) the scoring function derived from the Andromeda algorithm was adapted to score against a theoretical tandem mass spectrometry (MS/MS) spectrum that contains the peaks from all possible fragment ions of a cross-linked peptide pair. The performance of Xilmass was evaluated against the recently published Kojak and the popular pLink algorithms on a calmodulin-plectin complex data set, as well as three additional, published data sets. The results show that Xilmass typically had the highest number of identified distinct cross-linked sites and also the highest number of predicted cross-linked sites.

  12. Effect of radiation cross-linking on the abrasive wear behaviour of polyethylenes

    NASA Astrophysics Data System (ADS)

    Gul, Rizwan M.; Khan, Tahir I.

    2014-06-01

    This study explores the differences in the dry abrasive wear behavior of different polyethylenes, and compares the effect of radiation cross-linking on the wear behavior. Four different types of polyethylenes: LDPE, LLDPE, HDPE and UHMWPE were studied. Cross-linking was carried out by high energy electron beam with radiation dose of 200 kGy. The results show that in unirradiated state UHMWPE has excellent wear resistance, with HDPE showing comparable wear properties; both LDPE and LLDPE exhibit high wear rate. Cross-linking improves wear rate of LDPE and UHMWPE, however, the wear rate of HDPE and LLDPE increases with cross-linking.

  13. The Nf-actin gene is an important factor for food-cup formation and cytotoxicity of pathogenic Naegleria fowleri.

    PubMed

    Sohn, Hae-Jin; Kim, Jong-Hyun; Shin, Myeong-Heon; Song, Kyoung-Ju; Shin, Ho-Joon

    2010-03-01

    Naegleria fowleri destroys target cells by trogocytosis, a phagocytosis mechanism, and a process of piecemeal ingestion of target cells by food-cups. Phagocytosis is an actin-dependent process that involves polymerization of monomeric G-actin into filamentous F-actin. However, despite the numerous studies concerning phagocytosis, its role in the N. fowleri food-cup formation related with trogocytosis has been poorly reported. In this study, we cloned and characterized an Nf-actin gene to elucidate the role of Nf-actin gene in N. fowleri pathogenesis. The Nf-actin gene is composed of 1,128-bp and produced a 54.1-kDa recombinant protein (Nf-actin). The sequence identity was 82% with nonpathogenic Naegleria gruberi but has no sequence identity with other mammals or human actin gene. Anti-Nf-actin polyclonal antibody was produced in BALB/c mice immunized with recombinant Nf-actin. The Nf-actin was localized on the cytoplasm, pseudopodia, and especially, food-cup structure (amoebastome) in N. fowleri trophozoites using immunofluorescence assay. When N. fowleri co-cultured with Chinese hamster ovary cells, Nf-actin was observed to localize around on phagocytic food-cups. We also observed that N. fowleri treated with cytochalasin D as actin polymerization inhibitor or transfected with antisense oligomer of Nf-actin gene had shown the reduced ability of food-cup formation and in vitro cytotoxicity. Finally, it suggests that Nf-actin plays an important role in phagocytic activity of pathogenic N. fowleri.

  14. Maleimide cross-linked bioactive PEG hydrogel exhibits improved reaction kinetics and cross-linking for cell encapsulation and in-situ delivery

    PubMed Central

    Phelps, Edward A.; Enemchukwu, Nduka O.; Fiore, Vincent F.; Sy, Jay C.; Murthy, Niren; Sulchek, Todd A.; Barker, Thomas H.

    2012-01-01

    Engineered polyethylene glycol-maleimide matrices for regenerative medicine exhibit improved reaction efficiency and wider range of Young’s moduli by utilizing maleimide cross-linking chemistry. This hydrogel chemistry is advantageous for cell delivery due to the mild reaction that occurs rapidly enough for in situ delivery, while easily lending itself to “plug-and-play” design variations such as incorporation of enzyme-cleavable cross-links and cell-adhesion peptides. PMID:22174081

  15. Alkaline battery containing a separator of a cross-linked copolymer of vinyl alcohol and unsaturated carboxylic acid

    NASA Technical Reports Server (NTRS)

    Hsu, L. C.; Philipp, W. H.; Sheibley, D. W.; Gonzalez-Sanabria, O. D. (Inventor)

    1985-01-01

    A battery separator for an alkaline battery is described. The separator comprises a cross linked copolymer of vinyl alcohol units and unsaturated carboxylic acid units. The cross linked copolymer is insoluble in water, has excellent zincate diffusion and oxygen gas barrier properties and a low electrical resistivity. Cross linking with a polyaldehyde cross linking agent is preferred.

  16. Integrated Cryogenic Satellite Communications Cross-Link Receiver Experiment

    NASA Technical Reports Server (NTRS)

    Romanofsky, R. R.; Bhasin, K. B.; Downey, A. N.; Jackson, C. J.; Silver, A. H.; Javadi, H. H. S.

    1995-01-01

    An experiment has been devised which will validate, in space, a miniature, high-performance receiver. The receiver blends three complementary technologies; high temperature superconductivity (HTS), pseudomorphic high electron mobility transistor (PHEMT) monolithic microwave integrated circuits (MMIC), and a miniature pulse tube cryogenic cooler. Specifically, an HTS band pass filter, InP MMIC low noise amplifier, HTS-sapphire resonator stabilized local oscillator (LO), and a miniature pulse tube cooler will be integrated into a complete 20 GHz receiver downconverter. This cooled downconverter will be interfaced with customized signal processing electronics and integrated onto the space shuttle's 'HitchHiker' carrier. A pseudorandom data sequence will be transmitted to the receiver, which is in low Earth orbit (LEO), via the Advanced Communication Technology Satellite (ACTS) on a 20 GHz carrier. The modulation format is QPSK and the data rate is 2.048 Mbps. The bit error rate (BER) will be measured in situ. The receiver is also equipped with a radiometer mode so that experiment success is not totally contingent upon the BER measurement. In this mode, the receiver uses the Earth and deep space as a hot and cold calibration source, respectively. The experiment closely simulates an actual cross-link scenario. Since the receiver performance depends on channel conditions, its true characteristics would be masked in a terrestrial measurement by atmospheric absorption and background radiation. Furthermore, the receiver's performance depends on its physical temperature, which is a sensitive function of platform environment, thermal design, and cryocooler performance. This empirical data is important for building confidence in the technology.

  17. Encoding Hydrogel Mechanics via Network Cross-Linking Structure

    PubMed Central

    2015-01-01

    The effects of mechanical cues on cell behaviors in 3D remain difficult to characterize as the ability to tune hydrogel mechanics often requires changes in the polymer density, potentially altering the material’s biochemical and physical characteristics. Additionally, with most PEG diacrylate (PEGDA) hydrogels, forming materials with compressive moduli less than ∼10 kPa has been virtually impossible. Here, we present a new method of controlling the mechanical properties of PEGDA hydrogels independent of polymer chain density through the incorporation of additional vinyl group moieties that interfere with the cross-linking of the network. This modification can tune hydrogel mechanics in a concentration dependent manner from <1 to 17 kPa, a more physiologically relevant range than previously possible with PEG-based hydrogels, without altering the hydrogel’s degradation and permeability. Across this range of mechanical properties, endothelial cells (ECs) encapsulated within MMP-2/MMP-9 degradable hydrogels with RGDS adhesive peptides revealed increased cell spreading as hydrogel stiffness decreased in contrast to behavior typically observed for cells on 2D surfaces. EC-pericyte cocultures exhibited vessel-like networks within 3 days in highly compliant hydrogels as compared to a week in stiffer hydrogels. These vessel networks persisted for at least 4 weeks and deposited laminin and collagen IV perivascularly. These results indicate that EC morphogenesis can be regulated using mechanical cues in 3D. Furthermore, controlling hydrogel compliance independent of density allows for the attainment of highly compliant mechanical regimes in materials that can act as customizable cell microenvironments. PMID:26082943

  18. Corneal cross-linking in 9 horses with ulcerative keratitis

    PubMed Central

    2013-01-01

    Background Corneal ulcers are one of the most common eye problems in the horse and can cause varying degrees of visual impairment. Secondary infection and protease activity causing melting of the corneal stroma are always concerns in patients with corneal ulcers. Corneal collagen cross-linking (CXL), induced by illumination of the corneal stroma with ultraviolet light (UVA) after instillation of riboflavin (vitamin B2) eye drops, introduces crosslinks which stabilize melting corneas, and has been used to successfully treat infectious ulcerative keratitis in human patients. Therefore we decided to study if CXL can be performed in sedated, standing horses with ulcerative keratitis with or without stromal melting. Results Nine horses, aged 1 month to 16 years (median 5 years) were treated with a combination of CXL and medical therapy. Two horses were diagnosed with mycotic, 5 with bacterial and 2 with aseptic ulcerative keratitis. A modified Dresden-protocol for CXL could readily be performed in all 9 horses after sedation. Stromal melting, diagnosed in 4 horses, stopped within 24 h. Eight of nine eyes became fluorescein negative in 13.5 days (median time; range 4–26 days) days after CXL. One horse developed a bacterial conjunctivitis the day after CXL, which was successfully treated with topical antibiotics. One horse with fungal ulcerative keratitis and severe uveitis was enucleated 4 days after treatment due to panophthalmitis. Conclusions CXL can be performed in standing, sedated horses. We did not observe any deleterious effects attributed to riboflavin or UVA irradiation per se during the follow-up, neither in horses with infectious nor aseptic ulcerative keratitis. These data support that CXL can be performed in the standing horse, but further studies are required to compare CXL to conventional medical treatment in equine keratitis and to optimize the CXL protocol in this species. PMID:23803176

  19. Feruloylated arabinoxylans are oxidatively cross-linked by extracellular maize peroxidase but not by horseradish peroxidase.

    PubMed

    Burr, Sally J; Fry, Stephen C

    2009-09-01

    Covalent cross-linking of soluble extracellular arabinoxylans in living maize cultures, which models the cross-linking of wall-bound arabinoxylans, is due to oxidation of feruloyl esters to oligoferuloyl esters and ethers. The oxidizing system responsible could be H2O2/peroxidase, O2/laccase, or reactive oxygen species acting non-enzymically. To distinguish these possibilities, we studied arabinoxylan cross-linking in vivo and in vitro. In living cultures, exogenous, soluble, extracellular, feruloylated [pentosyl-3H]arabinoxylans underwent cross-linking, beginning abruptly 8 d after sub-culture. Cross-linking was suppressed by iodide, an H2O2 scavenger, indicating dependence on endogenous H2O2. However, exogenous H2O2 did not cause precocious cross-linking, despite the constant presence of endogenous peroxidases, suggesting that younger cultures contained natural cross-linking inhibitors. Dialysed culture-filtrates cross-linked [3H]arabinoxylans in vitro only if H2O2 was also added, indicating a peroxidase requirement. This cross-linking was highly ionic-strength-dependent. The peroxidases responsible were heat-labile, although relatively heat-stable peroxidases (assayed on o-dianisidine) were also present. Surprisingly, added horseradish peroxidase, even after heat-denaturation, blocked the arabinoxylan-cross-linking action of maize peroxidases, suggesting that the horseradish protein was a competing substrate for [3H]arabinoxylan coupling. In conclusion, we show for the first time that cross-linking of extracellular arabinoxylan in living maize cultures is an action of apoplastic peroxidases, some of whose unusual properties we report.

  20. J-integral fracture toughness and tearing modulus measurement of radiation cross-linked UHMWPE.

    PubMed

    Gomoll, A; Wanich, T; Bellare, A

    2002-11-01

    Radiation and chemical cross-linking of medical grade ultrahigh molecular weight polyethylene (UHMWPE) has recently been utilized in an effort to improve wear performance of total joint replacement components. However, reductions in mechanical properties with cross-linking are cause for concern regarding the use of cross-linked UHMWPE for high-stress applications such as in total knee replacement prostheses. In this study, the fracture behavior of radiation cross-linked UHMWPE was compared to that of uncross-linked UHMWPE. The Rice and Sorensen model that utilizes mechanical parameters obtained from uniaxial tensile and compact tension tests was used to calculate the steady state J-integral fracture toughness, Jss, for radiation cross-linked UHMWPE. Jss decreased monotonically with increase in radiation dose. UHMWPE exhibited tough, ductile tearing behavior with stable crack growth when it was cross-linked using a gamma radiation dose of 0-50 kGy. However, in cross-linked UHMWPE irradiated to a dose of 100 and 200 kGy, unstable fracture occurred spontaneously upon attaining the initial crack driving force, J1c. This indicates that a high degree of cross-linking is less desirable for high-stress applications in orthopaedic implants. However, a substantial increase in J1c, even at a low degree of cross-linking, suggests that a low degree of cross-linking may be beneficial for resistance to delamination and catastrophic failure, both of which require an initiation step for the fracture to propagate in the material. This mechanical test should, however, be considered along with fatigue tests and joint simulator testing before determination of an appropriate amount of cross-linking for total joint replacement prostheses that experience high stresses.

  1. Collagen Cross-Linking Using Riboflavin and Ultraviolet-A for Corneal Thinning Disorders

    PubMed Central

    Pron, G; Ieraci, L; Kaulback, K

    2011-01-01

    , watching television or reading, difficult to perform. Keratoconus is the most common form of corneal thinning disorder and involves a noninflammatory chronic disease process of progressive corneal thinning. Although the specific cause for the biomechanical alterations in the corneal stroma is unknown, there is a growing body of evidence suggesting that genetic factors may play an important role. Keratoconus is a rare disease (< 0.05% of the population) and is unique among chronic eye diseases because it has an early onset, with a median age of 25 years. Disease management for this condition follows a step-wise approach depending on disease severity. Contact lenses are the primary treatment of choice when there is irregular astigmatism associated with the disease. Patients are referred for corneal transplants as a last option when they can no longer tolerate contact lenses or when lenses no longer provide adequate vision. Keratoconus is one of the leading indications for corneal transplants and has been so for the last 3 decades. Despite the high success rate of corneal transplants (up to 20 years) there are reasons to defer it as long as possible. Patients with keratoconus are generally young and a longer-term graft survival of at least 30 or 40 years may be necessary. The surgery itself involves lengthy time off work and postsurgery, while potential complications include long-term steroid use, secondary cataracts, and glaucoma. After a corneal transplant, keratoconus may recur resulting in a need for subsequent interventions. Residual refractive errors and astigmatism can remain challenges after transplantation, and high refractive surgery and regraft rates in KC patients have been reported. Visual rehabilitation or recovery of visual acuity after transplant may be slow and/or unsatisfactory to patients. Description of Technology/Therapy Corneal cross-linking involves the use of riboflavin (vitamin B2) and ultraviolet-A (UVA) radiation. A UVA irradiation device known as

  2. Acute Corneal Hydrops 3 Years after Intra-corneal Ring Segments and Corneal Collagen Cross-linking

    PubMed Central

    Antonios, Rafic; Dirani, Ali; Fadlallah, Ali; Chelala, Elias; Hamadeh, Adib; Jarade, Elias

    2016-01-01

    This case report describes a 15-year-old male with allergic conjunctivitis and keratoconus, who underwent uneventful intra-corneal ring segment (ICRS) implantation and corneal collagen cross-linking (CXL) in the right eye. During the follow-up periods, the patient was noted to have several episodes of allergic conjunctivitis that were treated accordingly. At the 2 years postoperatively, he presented with another episode of allergic conjunctivitis and progression of keratoconus was suspected on topography. However, the patient was lost to follow-up, until he presented with acute hydrops at 3 years postoperatively. There are no reported cases of acute corneal hydrops in cross-linked corneas. We suspect the young age, allergic conjunctivitis and eye rubbing may be a risk factors associated with possible progression of keratoconus after CXL. Prolonged follow-up and aggressive control of the allergy might be necessary in similar cases. PMID:26957859

  3. Chromatin structure analysis of single gene molecules by psoralen cross-linking and electron microscopy.

    PubMed

    Brown, Christopher R; Eskin, Julian A; Hamperl, Stephan; Griesenbeck, Joachim; Jurica, Melissa S; Boeger, Hinrich

    2015-01-01

    Nucleosomes occupy a central role in regulating eukaryotic gene expression by blocking access of transcription factors to their target sites on chromosomal DNA. Analysis of chromatin structure and function has mostly been performed by probing DNA accessibility with endonucleases. Such experiments average over large numbers of molecules of the same gene, and more recently, over entire genomes. However, both digestion and averaging erase the structural variation between molecules indicative of dynamic behavior, which must be reconstructed for any theory of regulation. Solution of this problem requires the structural analysis of single gene molecules. In this chapter, we describe a method by which single gene molecules are purified from the yeast Saccharomyces cerevisiae and cross-linked with psoralen, allowing the determination of nucleosome configurations by transmission electron microscopy. We also provide custom analysis software that semi-automates the analysis of micrograph data. This single-gene technique enables detailed examination of chromatin structure at any genomic locus in yeast.

  4. Actin stress in cell reprogramming

    PubMed Central

    Guo, Jun; Wang, Yuexiu; Sachs, Frederick; Meng, Fanjie

    2014-01-01

    Cell mechanics plays a role in stem cell reprogramming and differentiation. To understand this process better, we created a genetically encoded optical probe, named actin–cpstFRET–actin (AcpA), to report forces in actin in living cells in real time. We showed that stemness was associated with increased force in actin. We reprogrammed HEK-293 cells into stem-like cells using no transcription factors but simply by softening the substrate. However, Madin-Darby canine kidney (MDCK) cell reprogramming required, in addition to a soft substrate, Harvey rat sarcoma viral oncogene homolog expression. Replating the stem-like cells on glass led to redifferentiation and reduced force in actin. The actin force probe was a FRET sensor, called cpstFRET (circularly permuted stretch sensitive FRET), flanked by g-actin subunits. The labeled actin expressed efficiently in HEK, MDCK, 3T3, and bovine aortic endothelial cells and in multiple stable cell lines created from those cells. The viability of the cell lines demonstrated that labeled actin did not significantly affect cell physiology. The labeled actin distribution was similar to that observed with GFP-tagged actin. We also examined the stress in the actin cross-linker actinin. Actinin force was not always correlated with actin force, emphasizing the need for addressing protein specificity when discussing forces. Because actin is a primary structural protein in animal cells, understanding its force distribution is central to understanding animal cell physiology and the many linked reactions such as stress-induced gene expression. This new probe permits measuring actin forces in a wide range of experiments on preparations ranging from isolated proteins to transgenic animals. PMID:25422450

  5. The role of cyclase-associated protein in regulating actin filament dynamics - more than a monomer-sequestration factor.

    PubMed

    Ono, Shoichiro

    2013-08-01

    Dynamic reorganization of the actin cytoskeleton is fundamental to a number of cell biological events. A variety of actin-regulatory proteins modulate polymerization and depolymerization of actin and contribute to actin cytoskeletal reorganization. Cyclase-associated protein (CAP) is a conserved actin-monomer-binding protein that has been studied for over 20 years. Early studies have shown that CAP sequesters actin monomers; recent studies, however, have revealed more active roles of CAP in actin filament dynamics. CAP enhances the recharging of actin monomers with ATP antagonistically to ADF/cofilin, and also promotes the severing of actin filaments in cooperation with ADF/cofilin. Self-oligomerization and binding to other proteins regulate activities and localization of CAP. CAP has crucial roles in cell signaling, development, vesicle trafficking, cell migration and muscle sarcomere assembly. This Commentary discusses the recent advances in our understanding of the functions of CAP and its implications as an important regulator of actin cytoskeletal dynamics, which are involved in various cellular activities.

  6. Self-assembly made durable: water-repellent materials formed by cross-linking fullerene derivatives.

    PubMed

    Wang, Jiaobing; Shen, Yanfei; Kessel, Stefanie; Fernandes, Paulo; Yoshida, Kaname; Yagai, Shiki; Kurth, Dirk G; Möhwald, Helmuth; Nakanishi, Takashi

    2009-01-01

    Fullerene flakes: A diacetylene-functionalized fullerene derivative self-organizes into flakelike microparticles (see picture). Both the diacetylene and C(60) moieties can be effectively cross-linked, which leads to supramolecular materials with remarkable resistivity to solvent, heat, and mechanical stress. Moreover, the surface of the cross-linked flakelike objects is highly durable and water-repellent.

  7. Chemistry and Physical Properties of Melt Processed- and Solution- Cross Linked Corn Zein

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Corn zein was cross linked with the glutaraldehyde (GDA) using glacial acetic acid (HAc) as catalyst. The objectives are to enhance the mechanical properties of poured films and to compare them with compression molded tensile bars from melt processed zein. Chemistry of the cross linking reaction w...

  8. PREPARATION OF NOVEL METALLIC AND BIMETALLIC CROSS-LINKED POLY (VINYL ALCOHOL) NANOCOMPOSITES UNDER MICROWAVE IRRADIATION

    EPA Science Inventory

    A facile method utilizing microwave irradiation is described that accomplishes the cross-linking reaction of PVA with metallic and bimetallic systems. Nanocomposites of PVA-cross-linked metallic systems such as Pt, Cu, and In and bimetallic systems such as Pt-In, Ag-Pt, Pt-Fe, Cu...

  9. NOVEL METALLIC AND BIMETALLIC CROSS-LINKED POLY (VINYL ALCOHOL) NANOCOMPOSITES PREPARED UNDER MICROWAVE IRRADIATION

    EPA Science Inventory

    A facile microwave irradiation approach that results in a cross-linking reaction of poly (vinyl alcohol) (PVA) with metallic and bimetallic systems is described. Nanocomposites of PVA cross-linked metallic systems such as Pt, Cu, and In and bimetallic systems such as Pt-In, Ag-P...

  10. 21 CFR 177.2710 - Styrene-divinylbenzene resins, cross-linked.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ....2710 Section 177.2710 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN... for Use Only as Components of Articles Intended for Repeated Use § 177.2710 Styrene-divinylbenzene resins, cross-linked. Styrene-divinylbenzene cross-linked copolymer resins may be safely used as...

  11. 21 CFR 177.2710 - Styrene-divinylbenzene resins, cross-linked.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ....2710 Section 177.2710 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN... for Use Only as Components of Articles Intended for Repeated Use § 177.2710 Styrene-divinylbenzene resins, cross-linked. Styrene-divinylbenzene cross-linked copolymer resins may be safely used as...

  12. 21 CFR 177.2710 - Styrene-divinylbenzene resins, cross-linked.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ....2710 Section 177.2710 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN... for Use Only as Components of Articles Intended for Repeated Use § 177.2710 Styrene-divinylbenzene resins, cross-linked. Styrene-divinylbenzene cross-linked copolymer resins may be safely used as...

  13. Preparation of size tunable giant vesicles from cross-linked dextran(ethylene glycol) hydrogels.

    PubMed

    López Mora, Néstor; Hansen, Jesper S; Gao, Yue; Ronald, Andrew A; Kieltyka, Roxanne; Malmstadt, Noah; Kros, Alexander

    2014-02-25

    We present a novel chemically cross-linked dextran-poly(ethylene glycol) hydrogel substrate for the preparation of dense vesicle suspensions under physiological ionic strength conditions. These vesicles can be easily diluted for individual study. Modulating the degree of cross-linking within the hydrogel network results in tuning of the vesicle size distribution.

  14. Evaluation of cross-linking methods for electrospun gelatin on cell growth and viability.

    PubMed

    Sisson, Kristin; Zhang, Chu; Farach-Carson, Mary C; Chase, D Bruce; Rabolt, John F

    2009-07-13

    The creation of a tissue engineering scaffold via electrospinning that has minimal toxicity and uses a solvent system composed of solvents with low toxicity and different cross-linking agents was investigated. First, a solvent system of acetic acid/ethyl acetate/water (50:30:20) with gelatin as a solute was evaluated. The optimum system for electrospinning a scaffold with the desired properties resulted from a gelatin concentration of 10 wt %. Several different methods were used to cross-link the electrospun gelatin fibers, including vapor-phase glutaraldehyde, aqueous phase genipin, and glyceraldehyde, as well as reactive oxygen species from a plasma cleaner. Because glutaraldehyde at high concentrations has been shown to be toxic, we explored other cross-linking methods. Using reactive oxygen species from a plasma cleaner is an easy alternative; however, the degradation reaction dominated the cross-linking reaction and the scaffolds degraded after only a few hours in aqueous medium at 37 °C. Glyceraldehyde and genipin were established as good options for cross-linking agents because of the low toxicity of these cross-linkers and the resistance to dissolution of the cross-linked fibers in cell culture medium at 37 °C. MG63 osteoblastic cells were grown on each of the cross-linked scaffolds. A proliferation assay showed that the cells proliferated as well or better on the cross-linked scaffolds than on traditional two-dimensional polystyrene culture plates.

  15. Probing structural elements in RNA using engineered disulfide cross-links.

    PubMed Central

    Maglott, E J; Glick, G D

    1998-01-01

    Three analogs of unmodified yeast tRNAPhe, each possessing a single disulfide cross-link, have been designed and synthesized. One cross-link is between G1 and C72 in the amino acid acceptor stem, a second cross-link is in the central D region of yeast tRNAPhe between C11 and C25 and the third cross-link bridges U16 and C60 at the D loop/T loop interface. Air oxidation to form the cross-links is quantitative and analysis of the cross-linked products by native and denaturing PAGE, RNase T1 mapping, Pb(II) cleavage, UV cross-linking and thermal denaturation demonstrates that the disulfide bridges do not alter folding of the modified tRNAs relative to the parent sequence. The finding that cross-link formation between thiol-derivatized residues correlates with the position of these groups in the crystal structure of native yeast tRNAPhe and that the modifications do not significantly perturb native structure suggests that this methodology should be applicable to the study of RNA structure, conformational dynamics and folding pathways. PMID:9469841

  16. Persulfate initiated ultra-low cross-linked poly(N-isopropylacrylamide) microgels possess an unusual inverted cross-linking structure.

    PubMed

    Virtanen, O L J; Mourran, A; Pinard, P T; Richtering, W

    2016-05-07

    Cross-linking density and distribution are decisive for the mechanical and other properties of stimuli-sensitive poly(N-isopropylacrylamide) microgels. Here we investigate the structure of ultra-low cross-linked microgels by static light scattering and scanning force microscopy, and show that they have an inverted cross-linking structure with respect to conventional microgels, contrary to what has been assumed previously. The conventional microgels have the largest polymer volume fraction in the core from where the particle density decays radially outwards, whereas ultra-low cross-linked particles have the highest polymer volume fraction close to the surface. On a solid substrate these particles form buckled shapes at high surface coverage, as shown by scanning force micrographs. The special structure of ultra-low cross-linked microgels is attributed to cross-linking of the particle surface, which is exposed to hydrogen abstraction by radicals generated from persulfate initiators during and after polymerization. The particle core, which is less accessible to the diffusion of radicals, has consequently a lower polymer volume fraction in the swollen state. By systematic variation of the cross-linker concentration it is shown that the cross-linking contribution from peroxide under typical synthesis conditions is weaker than that from the use of 1 mol% N,N'-methylenebisacrylamide. Soft deformable hydrogel particles are of interest because they emulate biological tissues, and understanding the underlying synthesis principle enables tailoring the microgel structure for biomimetic applications. Deformability of microgels is usually controlled by the amount of added cross-linker; here we however highlight an alternative approach through structural softness.

  17. A genome-wide analysis reveals that the Drosophila transcription factor Lola promotes axon growth in part by suppressing expression of the actin nucleation factor Spire

    PubMed Central

    2011-01-01

    Background The phylogenetically conserved transcription factor Lola is essential for many aspects of axon growth and guidance, synapse formation and neural circuit development in Drosophila. To date it has been difficult, however, to obtain an overall view of Lola functions and mechanisms. Results We use expression microarrays to identify the lola-dependent transcriptome in the Drosophila embryo. We find that lola regulates the expression of a large selection of genes that are known to affect each of several lola-dependent developmental processes. Among other loci, we find lola to be a negative regulator of spire, an actin nucleation factor that has been studied for its essential role in oogenesis. We show that spire is expressed in the nervous system and is required for a known lola-dependent axon guidance decision, growth of ISNb motor axons. We further show that reducing spire gene dosage suppresses this aspect of the lola phenotype, verifying that derepression of spire is an important contributor to the axon stalling phenotype of embryonic motor axons in lola mutants. Conclusions These data shed new light on the molecular mechanisms of many lola-dependent processes, and also identify several developmental processes not previously linked to lola that are apt to be regulated by this transcription factor. These data further demonstrate that excessive expression of the actin nucleation factor Spire is as deleterious for axon growth in vivo as is the loss of Spire, thus highlighting the need for a balance in the elementary steps of actin dynamics to achieve effective neuronal morphogenesis. PMID:22129300

  18. Investigation of anisotropic thermal transport in cross-linked polymers

    NASA Astrophysics Data System (ADS)

    Simavilla, David Nieto

    Thermal transport in lightly cross-linked polyisoprene and polybutadine subjected to uniaxial elongation is investigated experimentally. We employ two experimental techniques to assess the effect that deformation has on this class of materials. The first technique, which is based on Forced Rayleigh Scattering (FRS), allows us to measure the two independent components of the thermal diffusivity tensor as a function of deformation. These measurements along with independent measurements of the tensile stress and birefringence are used to evaluate the stress-thermal and stress-optic rules. The stress-thermal rule is found to be valid for the entire range of elongations applied. In contrast, the stress-optic rule fails for moderate to large stretch ratios. This suggests that the degree of anisotropy in thermal conductivity depends on both orientation and tension in polymer chain segments. The second technique, which is based on infrared thermography (IRT), allows us to measure anisotropy in thermal conductivity and strain induced changes in heat capacity. We validate this method measurements of anisotropic thermal conductivity by comparing them with those obtained using FRS. We find excellent agreement between the two techniques. Uncertainty in the infrared thermography method measurements is estimated to be about 2-5 %. The accuracy of the method and its potential application to non-transparent materials makes it a good alternative to extend current research on anisotropic thermal transport in polymeric materials. A second IRT application allows us to investigate the dependence of heat capacity on deformation. We find that heat capacity increases with stretch ratio in polyisoprene specimens under uniaxial extension. The deviation from the equilibrium value of heat capacity is consistent with an independent set of experiments comparing anisotropy in thermal diffusivity and conductivity employing FRS and IRT techniques. We identify finite extensibility and strain

  19. Chemically cross-linked silk fibroin hydrogel with enhanced elastic properties, biodegradability, and biocompatibility

    PubMed Central

    Kim, Min Hee; Park, Won Ho

    2016-01-01

    In this study, the synthesis of silk fibroin (SF) hydrogel via chemical cross-linking reactions of SF due to gamma-ray (γ-ray) irradiation was investigated, as were the resultant hydrogel’s properties. Two different hydrogels were investigated: physically cross-linked SF hydrogel and chemically cross-linked SF hydrogel irradiated at different doses of γ-rays. The effects of the irradiation dose and SF concentration on the hydrogelation of SF were examined. The chemically cross-linked SF hydrogel was compared with the physically cross-linked one with regard to secondary structure and gel strength. Furthermore, the swelling behavior, crystallinity, and biodegradation of the SF hydrogels were characterized. To assay cell proliferation, the cell viability of human mesenchymal stem cells on the lyophilized SF hydrogel scaffolds was evaluated, and no significant cytotoxicity against human mesenchymal stem cells was observed. PMID:27382283

  20. Modified dextran cross-linked electrospun gelatin nanofibres for biomedical applications.

    PubMed

    Jalaja, K; Kumar, P R Anil; Dey, Tuli; Kundu, Subhas C; James, Nirmala R

    2014-12-19

    Electrospun gelatin nanofibres attract attention of bioengineering arena because of its excellent biocompatibility and structural resemblance with native extracellular matrix. In this study, we have developed gelatin nanofibres using an innovative cross-linking approach to minimize cytotoxic effects. Gelatin was dissolved in water:acetic acid (8:2, v/v) solution and electrospun to form nanofibres with diameter in the range of 156 ± 30 nm. The nanofibres were cross-linked with a modified polysaccharide, namely, dextran aldehyde (DA). Cross-linking with DA could be achieved without compromising the fibrous architecture. DA cross-linked gelatin nanofibres maintained the fibrous morphology in aqueous medium. These mats exhibit improved mechanical properties and gradual degradation behaviour. The nanofibres were evaluated for cytotoxicity, cell adhesion, viability, morphology and proliferation using L-929 fibroblast cells. The results confirmed that DA cross-linked mats were non cytotoxic towards L-929 cells with good cell adhesion, spreading and proliferation.

  1. Robust Gold Nanoparticle Sheets by Ligand Cross-Linking at the Air-Water Interface.

    PubMed

    Kosif, Irem; Kratz, Katrina; You, Siheng Sean; Bera, Mrinal K; Kim, Kyungil; Leahy, Brian; Emrick, Todd; Lee, Ka Yee C; Lin, Binhua

    2017-02-28

    We report the results of cross-linking of two-dimensional gold nanoparticle (Au-NP) assemblies at the air-water interface in situ. We introduce an aqueous soluble ruthenium benzylidene catalyst into the water subphase to generate a robust, elastic two-dimensional network of nanoparticles containing cyclic olefins in their ligand framework. The most striking feature of the cross-linked Au-NP assemblies is that the extended connectivity of the nanoparticles enables the film to preserve much of its integrity under compression and expansion, features that are absent in its non-cross-linked counterparts. The cross-linking process appears to "stitch" the nanoparticle crystalline domains together, allowing the cross-linked monolayers to behave like a piece of fabric under lateral compression.

  2. Collagen cross-linking of skin in patients with amyotrophic lateral sclerosis

    NASA Technical Reports Server (NTRS)

    Ono, S.; Yamauchi, M.

    1992-01-01

    Collagen cross-links of skin tissue (left upper arm) from 11 patients with amyotrophic lateral sclerosis (ALS) and 9 age-matched control subjects were quantified. It was found that patients with ALS had a significant reduction in the content of an age-related, stable cross-link, histidinohydroxylysinonorleucine, that was negatively correlated with the duration of illness. The contents of sodium borohydride-reducible labile cross-links, dehydro-hydroxylysinonorleucine and dehydro-histidinohydroxymerodesmosine, were significantly increased and were positively associated with the duration of illness (r = 0.703, p less than 0.05 and r = 0.684, p less than 0.05, respectively). The results clearly indicate that during the course of ALS, the cross-linking pathway of skin collagen runs counter to its normal aging, resulting in a "rejuvenation" phenomenon of skin collagen. Thus, cross-linking of skin collagen is affected in ALS.

  3. Assessment of protein function following cross-linking by alpha-dicarbonyls.

    PubMed

    Miller, Antonia G; Gerrard, Juliet A

    2005-06-01

    Protein cross-linking via the Maillard reaction with alpha-dicarbonyl compounds has been the subject of intense scrutiny in the literature. We report here a study of the impact of this cross-linking on enzyme function. Protein function following glycation was examined by treating ribonuclease A with methylglyoxal, glyoxal, and diacetyl, which cross-linked the enzyme and impaired its activity. The effects of two reported Maillard reaction inhibitors, aminoguanidine and 3,5-dimethylpyrazole-1-carboxamidine, on the cross-linking reaction were assessed, with a parallel measurement of the effect on enzyme activity. The results demonstrate that preventing protein cross-linking does not necessarily preserve enzyme activity. These results cast doubt on the likely efficacy of some purported antiaging compounds in vivo.

  4. Modified gum arabic cross-linked gelatin scaffold for biomedical applications.

    PubMed

    Sarika, P R; Cinthya, Kuriakose; Jayakrishnan, A; Anilkumar, P R; James, Nirmala Rachel

    2014-10-01

    The present work deals with development of modified gum arabic cross-linked gelatin scaffold for cell culture. A new biocompatible scaffold was developed by cross-linking gelatin (Gel) with gum arabic, a polysaccharide. Gum arabic was subjected to periodate oxidation to obtain gum arabic aldehyde (GAA). GAA was reacted with gelatin under appropriate pH to prepare the cross-linked hydrogel. Cross-linking occurred due to Schiff's base reaction between aldehyde groups of oxidized gum arabic and amino groups of gelatin. The scaffold prepared from the hydrogel was characterized by swelling properties, degree of cross-linking, in vitro degradation and scanning electron microscopy (SEM). Cytocompatibility evaluation using L-929 and HepG2 cells confirmed non-cytotoxic and non-adherent nature of the scaffold. These properties are essential for generating multicellular spheroids and hence the scaffold is proposed to be a suitable candidate for spheroid cell culture.

  5. Chemically cross-linked silk fibroin hydrogel with enhanced elastic properties, biodegradability, and biocompatibility.

    PubMed

    Kim, Min Hee; Park, Won Ho

    2016-01-01

    In this study, the synthesis of silk fibroin (SF) hydrogel via chemical cross-linking reactions of SF due to gamma-ray (γ-ray) irradiation was investigated, as were the resultant hydrogel's properties. Two different hydrogels were investigated: physically cross-linked SF hydrogel and chemically cross-linked SF hydrogel irradiated at different doses of γ-rays. The effects of the irradiation dose and SF concentration on the hydrogelation of SF were examined. The chemically cross-linked SF hydrogel was compared with the physically cross-linked one with regard to secondary structure and gel strength. Furthermore, the swelling behavior, crystallinity, and biodegradation of the SF hydrogels were characterized. To assay cell proliferation, the cell viability of human mesenchymal stem cells on the lyophilized SF hydrogel scaffolds was evaluated, and no significant cytotoxicity against human mesenchymal stem cells was observed.

  6. A Study into the Collision-induced Dissociation (CID) Behavior of Cross-Linked Peptides.

    PubMed

    Giese, Sven H; Fischer, Lutz; Rappsilber, Juri

    2016-03-01

    Cross-linking/mass spectrometry resolves protein-protein interactions or protein folds by help of distance constraints. Cross-linkers with specific properties such as isotope-labeled or collision-induced dissociation (CID)-cleavable cross-linkers are in frequent use to simplify the identification of cross-linked peptides. Here, we analyzed the mass spectrometric behavior of 910 unique cross-linked peptides in high-resolution MS1 and MS2 from published data and validate the observation by a ninefold larger set from currently unpublished data to explore if detailed understanding of their fragmentation behavior would allow computational delivery of information that otherwise would be obtained via isotope labels or CID cleavage of cross-linkers. Isotope-labeled cross-linkers reveal cross-linked and linear fragments in fragmentation spectra. We show that fragment mass and charge alone provide this information, alleviating the need for isotope-labeling for this purpose. Isotope-labeled cross-linkers also indicate cross-linker-containing, albeit not specifically cross-linked, peptides in MS1. We observed that acquisition can be guided to better than twofold enrich cross-linked peptides with minimal losses based on peptide mass and charge alone. By help of CID-cleavable cross-linkers, individual spectra with only linear fragments can be recorded for each peptide in a cross-link. We show that cross-linked fragments of ordinary cross-linked peptides can be linearized computationally and that a simplified subspectrum can be extracted that is enriched in information on one of the two linked peptides. This allows identifying candidates for this peptide in a simplified database search as we propose in a search strategy here. We conclude that the specific behavior of cross-linked peptides in mass spectrometers can be exploited to relax the requirements on cross-linkers.

  7. Carbodiimide cross-linking of amniotic membranes in the presence of amino acid bridges.

    PubMed

    Lai, Jui-Yang

    2015-06-01

    The purpose of this study was to investigate the carbodiimide cross-linking of amniotic membrane (AM) in the presence of amino acid bridges. The biological tissues were treated with glycine, lysine, or glutamic acid and chemically cross-linked to examine the role of amino acid types in collagenous biomaterial processing. Results of zeta potential measurements showed that the use of uncharged, positively and negatively charged amino acids dictates the charge state of membrane surface. Tensile strength and water content measurements demonstrated that the addition of lysine molecules to the cross-linking system can increase the cross-linking efficiency and dehydration degree while the introduction of glutamic acid in the AM samples decreases the number of cross-links per unit mass of chemically modified tissue collagen. The differences in the cross-linking density further determined the thermal and biological stability by differential scanning calorimetry and in vitro degradation tests. As demonstrated in matrix permeability studies, the improved formation of covalent cross-linkages imposed by lysine facilitated construction of stronger cross-linking structures. In contrast, the added glycine molecules were insufficient to enhance the resistances of the proteinaceous matrices to thermal denaturation and enzymatic degradation. The cytocompatibility of these biological tissue membranes was evaluated by using human corneal epithelial cell cultures. Results of cell viability, metabolic activity, and pro-inflammatory gene expression level showed that the AM materials cross-linked with carbodiimide in the presence of different types of amino acids are well tolerated without evidence of detrimental effect on cell growth. In addition, the amino acid treated and carbodiimide cross-linked AM implants had good biocompatibility in the anterior chamber of the rabbit eye model. Our findings suggest that amino acid type is a very important engineering parameter to mediate

  8. Upregulation of human immunodeficiency virus (HIV) replication by CD4 cross-linking in peripheral blood mononuclear cells of HIV-infected adults.

    PubMed Central

    Than, S; Oyaizu, N; Tetali, S; Romano, J; Kaplan, M; Pahwa, S

    1997-01-01

    This study was conducted with peripheral blood mononuclear cells from 67 human immunodeficiency virus (HIV)-infected adults. It supports the hypothesis that cross-linking of CD4 molecules by HIV gp120 can result in HIV upregulation and spread of infection. Underlying mechanisms include activation of latent infection by factors in addition to, or other than, tumor necrosis factor alpha. PMID:9223523

  9. Upregulation of human immunodeficiency virus (HIV) replication by CD4 cross-linking in peripheral blood mononuclear cells of HIV-infected adults.

    PubMed

    Than, S; Oyaizu, N; Tetali, S; Romano, J; Kaplan, M; Pahwa, S

    1997-08-01

    This study was conducted with peripheral blood mononuclear cells from 67 human immunodeficiency virus (HIV)-infected adults. It supports the hypothesis that cross-linking of CD4 molecules by HIV gp120 can result in HIV upregulation and spread of infection. Underlying mechanisms include activation of latent infection by factors in addition to, or other than, tumor necrosis factor alpha.

  10. Cortactin involvement in the keratinocyte growth factor and fibroblast growth factor 10 promotion of migration and cortical actin assembly in human keratinocytes

    SciTech Connect

    Ceccarelli, Simona; Cardinali, Giorgia; Aspite, Nicaela; Picardo, Mauro; Marchese, Cinzia; Torrisi, Maria Rosaria; Mancini, Patrizia . E-mail: patrizia.mancini@uniroma1.it

    2007-05-15

    Keratinocyte growth factor (KGF/FGF7) and fibroblast growth factor 10 (FGF10/KGF2) regulate keratinocyte proliferation and differentiation by binding to the tyrosine kinase KGF receptor (KGFR). KGF induces keratinocyte motility and cytoskeletal rearrangement, whereas a direct role of FGF10 on keratinocyte migration is not clearly established. Here we analyzed the motogenic activity of FGF10 and KGF on human keratinocytes. Migration assays and immunofluorescence of actin cytoskeleton revealed that FGF10 is less efficient than KGF in promoting migration and exerts a delayed effect in inducing lamellipodia and ruffles formation. Both growth factors promoted phosphorylation and subsequent membrane translocation of cortactin, an F-actin binding protein involved in cell migration; however, FGF10-induced cortactin phosphorylation was reduced, more transient and delayed with respect to that promoted by KGF. Cortactin phosphorylation induced by both growth factors was Src-dependent, while its membrane translocation and cell migration were blocked by either Src and PI3K inhibitors, suggesting that both pathways are involved in KGF- and FGF10-dependent motility. Furthermore, siRNA-mediated downregulation of cortactin inhibited KGF- and FGF10-induced migration. These results indicate that cortactin is involved in keratinocyte migration promoted by both KGF and FGF10.

  11. Cholera toxin treatment of vascular smooth muscle cells decreases smooth muscle α-actin content and abolishes the platelet-derived growth factor-BB-stimulated DNA synthesis

    PubMed Central

    Sachinidis, Agapios; Seul, Claudia; Gouni-Berthold, Ioanna; Seewald, Stefan; Ko, Yon; Vetter, Hans; Fingerle, Jürgen; Hoppe, Jürgen

    2000-01-01

    The second messenger cyclic AMP regulates diverse biological processes such as cell morphology and cell growth. We examined the role of the second messenger cyclic AMP on rat aortic vascular smooth muscle cell (VSMC) morphology and the intracellular transduction pathway mediated by platelet-derived growth factor β-receptor (PDGF-Rβ). The effect of PDGF-BB on VSMCs growth was assessed by [3H]-thymidine incorporation. Tyrosine phosphorylation of PDGF-Rβ, PLC-γ1, ERK1 and ERK2, p125FAK and paxillin as well as Sm α-actin was examined by the chemiluminescence Western blotting method. Actin mRNA level was quantitated by Northern blotting. Visualization of Sm α-actin filaments, paxillin and PDGF-Rβ was performed by immunfluorescence microscopy. Cholera toxin (CTX; 10 nM) treatment lead to a large and sustained increase in the cyclic AMP concentration after 2 h which correlated with change of VSMC morphology including complete disruption of the Sm α-actin filament array and loss of focal adhesions. Treatment of VSMCs with CTX did not influence tyrosine phosphorylation of p125FAK and paxillin but decreased the content of a Sm α-actin protein. Maximal decrease of 70% was observed after 24 h of treatment. CTX also caused a 90% decrease of the actin mRNA level. CTX treatment completely abolished PDGF-BB stimulated DNA-synthesis although PDGF-Rβ level and subcellular distribution and translocation was not altered. Furthermore CTX attenuated the PDGF-BB-induced tyrosine phosphorylation of the PDGF-Rβ, PI 3′-K, PLC-γ1 and ERK1/2 indicating an action of cyclic AMP on PDGF-β receptor. We conclude that although cyclic AMP attenuates the PDGF-Rβ mediated intracellular transduction pathway, an intact actin filament may be required for the PDGF-BB-induced DNA synthesis in VSMCs. PMID:10928958

  12. In vitro and in vivo biocompatibility of dextran dialdehyde cross-linked gelatin hydrogel films.

    PubMed

    Draye, J P; Delaey, B; Van de Voorde, A; Van Den Bulcke, A; De Reu, B; Schacht, E

    1998-09-01

    The biosafety of a new hydrogel wound dressing material consisting of dextran dialdehyde cross-linked gelatin was evaluated (i) in vitro in cultures of dermal fibroblasts, epidermal keratinocytes, and endothelial cells, three cell types which play a major role in the process of cutaneous wound healing, and (ii) in vivo by subcutaneous implantation studies in mice. The cytotoxicities of this hydrogel, two semi-occlusive polyurethane dressings (Tegaderm and OpSite), and a hydrocolloid dressing (DuoDERM) were compared by measuring cell survival with the tetrazolium salt reduction (MTT) assay after incubations of the wound dressing samples for up to 6 d, in the presence of--but not in direct contact with--the cells. In vitro, the degree of cytotoxicity of the new hydrogel was greater in keratinocyte cultures than in fibroblast and endothelial cell cultures, and increased upon longer incubation time. In keratinocyte cultures, the semi-occlusive polyurethane dressings, the hydrocolloid, and the hydrogel dressings induced low, high and acceptable degrees of cytotoxicity, respectively. The toxicity of the isolated hydrogel components was assessed in Balb MK keratinocyte cultures. In these cells, epidermal growth-factor-stimulated thymidine incorporation into DNA was higher in the presence of gelatin. By contrast, concentrations of dextran dialdehyde as low as 0.002% were found to significantly decrease thymidine incorporation (P < 0.01). Subcutaneous implantation studies in mice showed that in vivo the hydrogel was biocompatible since the foreign body reaction seen around the implanted hydrogel samples was moderate and became minimal upon increasing implantation time. These results indicate that dextran dialdehyde cross-linked gelatin hydrogels have an appropriate biocompatibility.

  13. Structure of the ribosome post-recycling complex probed by chemical cross-linking and mass spectrometry

    PubMed Central

    Kiosze-Becker, Kristin; Ori, Alessandro; Gerovac, Milan; Heuer, André; Nürenberg-Goloub, Elina; Rashid, Umar Jan; Becker, Thomas; Beckmann, Roland; Beck, Martin; Tampé, Robert

    2016-01-01

    Ribosome recycling orchestrated by the ATP binding cassette (ABC) protein ABCE1 can be considered as the final—or the first—step within the cyclic process of protein synthesis, connecting translation termination and mRNA surveillance with re-initiation. An ATP-dependent tweezer-like motion of the nucleotide-binding domains in ABCE1 transfers mechanical energy to the ribosome and tears the ribosome subunits apart. The post-recycling complex (PRC) then re-initiates mRNA translation. Here, we probed the so far unknown architecture of the 1-MDa PRC (40S/30S·ABCE1) by chemical cross-linking and mass spectrometry (XL-MS). Our study reveals ABCE1 bound to the translational factor-binding (GTPase) site with multiple cross-link contacts of the helix–loop–helix motif to the S24e ribosomal protein. Cross-linking of the FeS cluster domain to the ribosomal protein S12 substantiates an extreme lever-arm movement of the FeS cluster domain during ribosome recycling. We were thus able to reconstitute and structurally analyse a key complex in the translational cycle, resembling the link between translation initiation and ribosome recycling. PMID:27824037

  14. Methicillin-Resistant Staphylococcus aureus Ocular Infection after Corneal Cross-Linking for Keratoconus: Potential Association with Atopic Dermatitis

    PubMed Central

    Fasciani, Romina; Agresta, Antonio; Caristia, Alice; Mosca, Luigi; Scupola, Andrea; Caporossi, Aldo

    2015-01-01

    Purpose. To report the risk of methicillin-resistant Staphylococcus aureus (MRSA) ocular infection after UVA-riboflavin corneal collagen cross-linking in a patient with atopic dermatitis. Methods. A 22-year-old man, with bilateral evolutive keratoconus and atopic dermatitis, underwent UVA-riboflavin corneal cross-linking and presented with rapidly progressive corneal abscesses and cyclitis in the treated eye five days after surgery. The patient was admitted to the hospital and treated with broad-spectrum antimicrobic therapy. Results. The patient had positive cultures for MRSA, exhibiting a strong resistance to antibiotics. Antibiotic therapy was modified and targeted accordingly. The intravitreal reaction is extinguished, but severe damage of ocular structures was unavoidable. Conclusion. Riboflavin/UVA corneal cross-linking is considered a safe procedure and is extremely effective in halting keratoconus' progression. However, this procedure is not devoid of infectious complications, due to known risk factors and/or poor patients' hygiene compliance in the postoperative period. Atopic dermatitis is a common disease among patients with keratoconus and Staphylococcus aureus colonization is commonly found in patients with atopic dermatitis. Therefore, comorbidity with atopic dermatitis should be thoroughly assessed through clinical history before surgery. A clinical evaluation within three days after surgery and the imposition of strict personal hygiene rules are strongly recommended. PMID:25866692

  15. Determination of protein conformation by isotopically labelled cross-linking and dedicated software

    NASA Astrophysics Data System (ADS)

    Nielsen, Tina; Thaysen-Andersen, Morten; Larsen, Nanna; Jørgensen, Flemming S.; Houen, Gunnar; Højrup, Peter

    2007-12-01

    Chemical cross-linking in conjunction with mass spectrometry (MS) can be used for sensitive and rapid investigation of the three-dimensional structure of proteins at low resolution. However, the resulting data are very complex, and on the bioinformatic side, there still exists an urgent need for improving computer software for (semi-) automated cross-linking data analysis. In this study, we have developed dedicated software for rapid and confident identification and validation of cross-linked species using an isotopic labelled cross-linker approach in combination with MS. Deuterated (+4 Da) and non-deuterated (+0 Da) bis(sulfosuccinimidyl)suberate, BS3, was used as homobifunctional cross-linker to tag the cross-linked regions. Peptides generated from proteolysis were separated using high performance liquid chromatography, and peptide mass fingerprinting was obtained for the individual fractions using matrix-assisted laser-desorption ionisation time-of-flight (MALDI TOF) MS. The resulting peptide mass lists were combined and transferred to the program, ProteinXXX, which generated the theoretical mass values of all combinations of cross-linked peptides and dead-end cross-links and compared this to the obtained mass lists. In addition, screening for 4 Da-separated signals aided the identification of potential cross-linked species. Sequence information of these candidates was then obtained using MALDI TOF TOF. The cross-linked peptides could then be validated based on the match of the fragmentation pattern and the theoretical values produced by ProteinXXX. This semi-automated interpretation provided a high analysis speed of cross-linking data, with efficient and confident identification of cross-linked species. Four experiments using different conditions showed a high degree of reproducibility as only 1 and 2 cross-links out of 36 identified was not observed in two experiments. The method was tested using human placenta calreticulin (CRT). Based on the identified cross-links

  16. Formation and Destabilization of Actin Filaments with Tetramethylrhodamine-Modified Actin

    PubMed Central

    Kudryashov, Dmitry S.; Phillips, Martin; Reisler, Emil

    2004-01-01

    Actin labeling at Cys374 with tethramethylrhodamine derivatives (TMR-actin) has been widely used for direct observation of the in vitro filaments growth, branching, and treadmilling, as well as for the in vivo visualization of actin cytoskeleton. The advantage of TMR-actin is that it does not lock actin in filaments (as rhodamine-phalloidin does), possibly allowing for its use in investigating the dynamic assembly behavior of actin polymers. Although it is established that TMR-actin alone is polymerization incompetent, the impact of its copolymerization with unlabeled actin on filament structure and dynamics has not been tested yet. In this study, we show that TMR-actin perturbs the filaments structure when copolymerized with unlabeled actin; the resulting filaments are more fragile and shorter than the control filaments. Due to the increased severing of copolymer filaments, TMR-actin accelerates the polymerization of unlabeled actin in solution also at mole ratios lower than those used in most fluorescence microscopy experiments. The destabilizing and severing effect of TMR-actin is countered by filament stabilizing factors, phalloidin, S1, and tropomyosin. These results point to an analogy between the effects of TMR-actin and severing proteins on F-actin, and imply that TMR-actin may be inappropriate for investigations of actin filaments dynamics. PMID:15298916

  17. Novel antimicrobial superporous cross-linked chitosan/pyromellitimide benzoyl thiourea hydrogels.

    PubMed

    Mohamed, Nadia A; Abd El-Ghany, Nahed A; Fahmy, Mona M

    2016-01-01

    In this work, chitosan (CS) was cross-linked with different amounts of pyromellitimide benzoyl thiourea moieties. The structure of the cross-linked CS was confirmed by elemental analyses, FTIR and (1)H- NMR spectroscopy. The cross-linking process proceeds via reacting of the amino groups of CS with the isothiocyanate groups of the N,N'-bis [4-(isothiocyanate carbonyl)phenyl] pyromellitimide cross-linker. The amount of the cross-linker was varied with respect to CS to produce four new pyromellitimide benzoyl thiourea cross-linked CS (PIBTU-CS) hydrogels designated as PIBTU-CS-1, PIBTU-CS-2, PIBTU-CS-3, and PIBTU-CS-4 of increasing cross-linking degree percent of 11, 22, 44 and 88%, respectively. The scanning electron microscopy observation indicates the extremely porous structure of the hydrogels. XRD results showed that the crystallinity of CS was decreased upon cross-linking. The four hydrogels exhibit a higher antibacterial activity on Bacillus subtilis and Streptococcus pneumoniae as Gram positive bacteria and against Escherichia coli as Gram negative bacteria and higher antifungal activity on Aspergillus fumigatus, Syncephalastrum racemosum and Geotricum candidum than that of the parent CS as shown from their higher inhibition zone diameters and their lower MIC values. The swell ability of the hydrogel as well as their antimicrobial activity increased with increasing cross-linking density.

  18. Probing structures of large protein complexes using zero-length cross-linking.

    PubMed

    Rivera-Santiago, Roland F; Sriswasdi, Sira; Harper, Sandra L; Speicher, David W

    2015-11-01

    Structural mass spectrometry (MS) is a field with growing applicability for addressing complex biophysical questions regarding proteins and protein complexes. One of the major structural MS approaches involves the use of chemical cross-linking coupled with MS analysis (CX-MS) to identify proximal sites within macromolecules. Identified cross-linked sites can be used to probe novel protein-protein interactions or the derived distance constraints can be used to verify and refine molecular models. This review focuses on recent advances of "zero-length" cross-linking. Zero-length cross-linking reagents do not add any atoms to the cross-linked species due to the lack of a spacer arm. This provides a major advantage in the form of providing more precise distance constraints as the cross-linkable groups must be within salt bridge distances in order to react. However, identification of cross-linked peptides using these reagents presents unique challenges. We discuss recent efforts by our group to minimize these challenges by using multiple cycles of LC-MS/MS analysis and software specifically developed and optimized for identification of zero-length cross-linked peptides. Representative data utilizing our current protocol are presented and discussed.

  19. The use of highly cross-linked polyethylene in total knee arthroplasty.

    PubMed

    Lachiewicz, Paul F; Geyer, Mark R

    2011-03-01

    Polyethylene wear, with resultant particle-induced osteolysis, is a cause of late failure of total knee arthroplasty. The causes of both wear and osteolysis are multifactorial; still, improvements in the polyethylene liner have been investigated. Available highly cross-linked polyethylene tibial liners and patellar prostheses differ greatly in the amount and method of irradiation, thermal treatments, and sterilization techniques they undergo. Several varieties of highly cross-linked polyethylene reduce the gravimetric and volumetric wear of tibial liners in knee simulator studies. However, reduced fracture toughness and the generation of smaller and possibly more reactive particles also have been reported with some varieties of polyethylene. Clinical studies of the use of highly cross-linked polyethylene in total knee arthroplasty are limited. Two nonrandomized trials of highly cross-linked polyethylene in total knee arthroplasty have reported a nonsignificant decrease in radiolucent lines at 2 and 5 years, respectively. The risks of using highly cross-linked polyethylene include fracture of the liner or of a posterior-stabilized tibial post, liner dislodgement or locking mechanism disruption, and possibly more osteolysis. Highly cross-linked polyethylene tibial liners may be considered for younger, more active patients. However, until additional clinical results are available, a cautious approach is warranted to the widespread use of highly cross-linked polyethylene in total knee arthroplasty.

  20. The 5'-GNC site for DNA interstrand cross-linking is conserved for diepoxybutane stereoisomers.

    PubMed

    Millard, Julie T; Hanly, Trevor C; Murphy, Kris; Tretyakova, Natalia

    2006-01-01

    The bifunctional alkylating agent 1,2,3,4-diepoxybutane forms interstrand DNA-DNA cross-links between the N7 positions of deoxyguanosine residues on opposite strands of the duplex. For racemic diepoxybutane, these cross-links predominate within 5'-GNC/3'CNG sequences, where N is any nucleotide. We used denaturing polyacrylamide gel electrophoresis (dPAGE) to examine the role of stereochemistry in the cross-linking reaction, subjecting a restriction fragment to cross-linking with S,S-DEB, R,R-DEB, or meso-DEB. DNA cross-links generated by each isomer were isolated by dPAGE, and the sites of cross-linking were identified by sequencing gel analysis of DNA fragments generated by hot piperidine cleavage. We found that the 5'-GNC consensus sequence of racemic DEB is conserved, but the efficiencies of cross-linking vary, with S,S- > R,R- > meso-DEB. These results help explain the observed differences between the biological activities of DEB stereoisomers.

  1. Phased psoralen cross-links do not bend the DNA double helix

    SciTech Connect

    Haran, T.E.; Crothers, D.M.

    1988-09-06

    Although the chemical reaction of psoralens with nucleic acids is well understood, the structure of psoralen-DNA cross-linked products is still not clear. Model building studies based on the crystal structure of the psoralen-thymine monoadduct suggest that each cross-link bends the DNA double helix by 46.5/sup 0/. Here the authors use gel electrophoresis to test the validity of the current models. They have synthesized a series of DNA fragments (21-24 base pairs in length), each containing one unique T-A site for 4'-(hydroxymethyl)-4,5'8-trimethylpsoralen (HMT) cross-linking. Because of an estimated 28/sup 0/ unwinding of the helix by HMT, one expects that the 22-bp cross-linked fragment will be repeated nearly in phase with the average helical screw when multimerized. In that sequence ligation will maximally amplify any deformation to the double helix. They find that the ligated multimers of cross-linked DNA migrate close to the multimers of non-cross-linked DNA on polyacrylamide gels. These observations place an upper limit of 10/sup 0/ on DNA bending induced by psoralen cross-linking and indicate unwinding by about 1 bp, as well as stiffening of the double helix. These properties are not unexpected for classical intercalators.

  2. Lysyl hydroxylase 2 induces a collagen cross-link switch in tumor stroma

    PubMed Central

    Chen, Yulong; Terajima, Masahiko; Yang, Yanan; Sun, Li; Ahn, Young-Ho; Pankova, Daniela; Puperi, Daniel S.; Watanabe, Takeshi; Kim, Min P.; Blackmon, Shanda H.; Rodriguez, Jaime; Liu, Hui; Behrens, Carmen; Wistuba, Ignacio I.; Minelli, Rosalba; Scott, Kenneth L.; Sanchez-Adams, Johannah; Guilak, Farshid; Pati, Debananda; Thilaganathan, Nishan; Burns, Alan R.; Creighton, Chad J.; Martinez, Elisabeth D.; Zal, Tomasz; Grande-Allen, K. Jane; Yamauchi, Mitsuo; Kurie, Jonathan M.

    2015-01-01

    Epithelial tumor metastasis is preceded by an accumulation of collagen cross-links that heighten stromal stiffness and stimulate the invasive properties of tumor cells. However, the biochemical nature of collagen cross-links in cancer is still unclear. Here, we postulated that epithelial tumorigenesis is accompanied by changes in the biochemical type of collagen cross-links. Utilizing resected human lung cancer tissues and a p21CIP1/WAF1-deficient, K-rasG12D-expressing murine metastatic lung cancer model, we showed that, relative to normal lung tissues, tumor stroma contains higher levels of hydroxylysine aldehyde–derived collagen cross-links (HLCCs) and lower levels of lysine aldehyde–derived cross-links (LCCs), which are the predominant types of collagen cross-links in skeletal tissues and soft tissues, respectively. Gain- and loss-of-function studies in tumor cells showed that lysyl hydroxylase 2 (LH2), which hydroxylates telopeptidyl lysine residues on collagen, shifted the tumor stroma toward a high-HLCC, low-LCC state, increased tumor stiffness, and enhanced tumor cell invasion and metastasis. Together, our data indicate that LH2 enhances the metastatic properties of tumor cells and functions as a regulatory switch that controls the relative abundance of biochemically distinct types of collagen cross-links in the tumor stroma. PMID:25664850

  3. Synthesis and Characterization of Cross-linked Polymer Electrolyte Membranes for Supercapacitor

    NASA Astrophysics Data System (ADS)

    Rosi, Memoria; Ekaputra, Muhamad Prama; Abdullah, Mikrajuddin; Khairurrijal

    2010-10-01

    Cross-linked polyvinyl alcohol (PVA) electrolyte membranes have been synthesized by using a solution casting method. In this study, PVA was blended with oxidative cross-linked agent (zinc acetate) and nano-sized silica as filler to stabilize PVA matrix and enhance conductivity. The cross-linked membranes were immersed into lithium hydroxide (LiOH) aqueous solution to increase their ionic conductivity. Two techniques were used to characterize the resulted membranes including Fourier transform infra red (FTIR) and AC impedance spectroscopies. The results showed that absorption peaks of C-O-C group and Si-O-Si are presence in the FTIR spectra attributed to the cross-linking process. Impedance spectra indicated that the contribution of ionic dopant (LiOH) to enhance conductivity is insignificant. The highest conductivity of the studied cross-linked PVA membrane is 1.34×10-3 S cm-1 corresponding to 5% LiOH dopant concentration of cross-linked PVA-zinc acetate-nano silica membrane. The present study also suggested that the solution casting is appropriate for cross-linked membrane synthesis.

  4. The Synergy of Double Cross-linking Agents on the Properties of Styrene Butadiene Rubber Foams

    PubMed Central

    Shao, Liang; Ji, Zhan-You; Ma, Jian-Zhong; Xue, Chao-Hua; Ma, Zhong-Lei; Zhang, Jing

    2016-01-01

    Sulfur (S) cross-linking styrene butadiene rubber (SBR) foams show high shrinkage due to the cure reversion, leading to reduced yield and increased processing cost. In this paper, double cross-linking system by S and dicumyl peroxide (DCP) was used to decrease the shrinkage of SBR foams. Most importantly, the synergy of double cross-linking agents was reported for the first time to our knowledge. The cell size and its distribution of SBR foams were investigated by FESEM images, which show the effect of DCP content on the cell structure of the SBR foams. The relationships between shrinkage and crystalline of SBR foams were analyzed by the synergy of double cross-linking agents, which were demonstrated by FTIR, Raman spectra, XRD, DSC and TGA. When the DCP content was 0.6 phr, the SBR foams exhibit excellent physical and mechanical properties such as low density (0.223 g/cm3), reduced shrinkage (2.25%) and compression set (10.96%), as well as elevated elongation at break (1.78 × 103%) and tear strength (54.63 N/mm). The results show that these properties are related to the double cross-linking system of SBR foams. Moreover, the double cross-linking SBR foams present high electromagnetic interference (EMI) shielding properties compared with the S cross-linking SBR foams. PMID:27841307

  5. Chemistry and physical properties of melt-processed and solution-cross-linked corn zein.

    PubMed

    Sessa, David J; Mohamed, Abdellatif; Byars, Jeffrey A

    2008-08-27

    Corn zein was cross-linked with glutaraldehyde (GDA) using glacial acetic acid (HAc) as catalyst. The objectives are to evaluate the swelling characteristics of GDA cross-linked zein gels in water, ethanol, and their combinations. Similar formulations, upon solvent evaporation, form films. The mechanical properties of the films are compared to compression molded tensile bars from GDA melt-processed zein as a second objective. Chemistry of the cross-linking reaction was based on the aldehyde binding characteristics defined by use of fluorescence spectroscopy; sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) to demonstrate the cross-linking reaction; FTIR to observe absorption differences of the cross-linked product; differential scanning calorimetry, dynamic mechanical analysis and thermogravimetric analysis to assess thermal properties; and the use of Instron Universal Testing Machine to evaluate mechanical properties. A reaction mechanism for acid catalyzed GDA cross-linking of zein is proposed. Thermal and mechanical properties of tensile bars cut from either film or formed by compression molding were similar, where both showed increased tensile strengths, ductility and stiffness when compared with unmodified controls. Samples that were reacted with 8% GDA by weight based on weight of zein from either process retained their integrity when tensile bars from each were subjected to boiling water for 10 min or soaking in either water or HAc for 24 h. The melt-processed, cross-linked zein is a more environmentally friendly method that would eliminate the need for HAc recovery.

  6. Composition of cross-linked 125I-follitropin-receptor complexes

    SciTech Connect

    Shin, J.; Ji, T.H.

    1985-10-15

    Both of the alpha and beta subunits of intact human follitropin (FSH) were radioiodinated with SVI-sodium iodide and chloramine-T and could be resolved on sodium dodecyl sulfate-polyacrylamide gels. Radioiodinated FSH was affinity-cross-linked with a cleavable (nondisulfide) homobifunctional reagent to its membrane receptor on the porcine granulosa cell surface as well as to a Triton X-100-solubilized form of the receptor. Cross-linked samples revealed three additional bands of slower electrophoretic mobility, corresponding to 65, 83, and 117 kDa, in addition to the hormone bands. The hormone alpha beta dimer band corresponded to 43 kDa. Formation of the three bands requires the SVI-hormone to bind specifically to the receptor with subsequent cross-linking. Binding was prevented by an excess of the native hormone but not by other hormones. A monofunctional analog of the cross-linking reagent failed to produce the three bands. Reagent concentration-dependent cross-linking revealed that their formation was sequential; smaller complexes formed first and then larger ones. When gels of cross-linked complexes were treated to cleave covalent cross-links and then electrophoresed in a second dimension, 18-, 22-, and 34-kDa components were released, in addition to the alpha and beta subunits of the hormone.

  7. Electrospun gelatin nanofibers: a facile cross-linking approach using oxidized sucrose.

    PubMed

    Jalaja, K; James, Nirmala R

    2015-02-01

    Gelatin nanofibers were fabricated via electrospinning with minimal toxicity from solvents and cross-linking agents. Electrospinning was carried out using a solvent system based on water and acetic acid (8:2, v/v). Acetic acid concentration was kept as minimum as possible to reduce the toxic effects. Electrospun gelatin nanofibers were cross-linked with oxidized sucrose. Sucrose was oxidized by periodate oxidation to introduce aldehyde functionality. Cross-linking with oxidized sucrose could be achieved without compromising the nanofibrous architecture. Cross-linked gelatin nanofibers maintained the fibrous morphology even after keeping in contact with aqueous medium. The morphology of the cross-linked nanofibrous mats was examined by scanning electron microscopy (SEM). Oxidized sucrose cross-linked gelatin nanofibers exhibited improved thermal and mechanical properties. The nanofibrous mats were evaluated for cytotoxicity and cell viability using L-929 fibroblast cells. The results confirmed that oxidized sucrose cross-linked gelatin nanofibers were non-cytotoxic towards L-929 cells with good cell viability.

  8. Probing structures of large protein complexes using zero-length cross-linking

    PubMed Central

    Rivera-Santiago, Roland F.; Sriswasdi, Sira; Harper, Sandra L.; Speicher, David W.

    2015-01-01

    Structural mass spectrometry (MS) is a field with growing applicability for addressing complex biophysical questions regarding proteins and protein complexes. One of the major structural MS approaches involves the use of chemical cross-linking coupled with MS analysis (CX-MS) to identify proximal sites within macromolecules. Identified cross-linked sites can be used to probe novel protein–protein interactions or the derived distance constraints can be used to verify and refine molecular models. This review focuses on recent advances of “zero-length” cross-linking. Zero-length cross-linking reagents do not add any atoms to the cross-linked species due to the lack of a spacer arm. This provides a major advantage in the form of providing more precise distance constraints as the cross-linkable groups must be within salt bridge distances in order to react. However, identification of cross-linked peptides using these reagents presents unique challenges. We discuss recent efforts by our group to minimize these challenges by using multiple cycles of LC–MS/MS analysis and software specifically developed and optimized for identification of zero-length cross-linked peptides. Representative data utilizing our current protocol are presented and discussed. PMID:25937394

  9. The Synergy of Double Cross-linking Agents on the Properties of Styrene Butadiene Rubber Foams

    NASA Astrophysics Data System (ADS)

    Shao, Liang; Ji, Zhan-You; Ma, Jian-Zhong; Xue, Chao-Hua; Ma, Zhong-Lei; Zhang, Jing

    2016-11-01

    Sulfur (S) cross-linking styrene butadiene rubber (SBR) foams show high shrinkage due to the cure reversion, leading to reduced yield and increased processing cost. In this paper, double cross-linking system by S and dicumyl peroxide (DCP) was used to decrease the shrinkage of SBR foams. Most importantly, the synergy of double cross-linking agents was reported for the first time to our knowledge. The cell size and its distribution of SBR foams were investigated by FESEM images, which show the effect of DCP content on the cell structure of the SBR foams. The relationships between shrinkage and crystalline of SBR foams were analyzed by the synergy of double cross-linking agents, which were demonstrated by FTIR, Raman spectra, XRD, DSC and TGA. When the DCP content was 0.6 phr, the SBR foams exhibit excellent physical and mechanical properties such as low density (0.223 g/cm3), reduced shrinkage (2.25%) and compression set (10.96%), as well as elevated elongation at break (1.78 × 103%) and tear strength (54.63 N/mm). The results show that these properties are related to the double cross-linking system of SBR foams. Moreover, the double cross-linking SBR foams present high electromagnetic interference (EMI) shielding properties compared with the S cross-linking SBR foams.

  10. Cross-linking of microtubules by microtubule-associated proteins (MAPs) from the brine shrimp, Artemia.

    PubMed

    Campbell, E J; MacKinlay, S A; MacRae, T H

    1989-05-01

    Microtubules induced with taxol to assemble in cell-free extracts of the brine shrimp, Artemia, are cross-linked by microtubule-associated proteins (MAPs). When the MAPs, extracted from taxol-stabilized microtubules with 1 M-NaCl are co-assembled with purified Artemia or mammalian neural tubulin, reconstitution of cross-linking between microtubules occurs. The most prominent non-tubulin protein associated with reconstituted cross-linked microtubules has a molecular weight of 49,000 but we cannot yet exclude the possibility that other proteins may be responsible for the cross-linking. Cross-linkers are separated by varying distances while cross-linked microtubules, prepared under different conditions, are 6.9-7.7 nm apart. Cross-linking of microtubules by MAPs occurs whether MAPs are added to assembling tubulin or to microtubules, and it is not disrupted by ATP. The MAPs are heat-sensitive and do not stabilize microtubules to cold. Immunological characterization of Artemia MAPs on Western blots indicates that Artemia lack MAP 1, MAP 2 and tau. Our results clearly demonstrate that Artemia contain novel MAPs with the ability to cross-link microtubules from phylogenetically disparate organisms in an ATP-independent manner.

  11. Actinic reticuloid

    SciTech Connect

    Marx, J.L.; Vale, M.; Dermer, P.; Ragaz, A.; Michaelides, P.; Gladstein, A.H.

    1982-09-01

    A 58-year-old man has his condition diagnosed as actinic reticuloid on the basis of clinical and histologic findings and phototesting data. He had clinical features resembling mycosis fungoides in light-exposed areas. Histologic findings disclosed a bandlike infiltrate with atypical mononuclear cells in the dermis and scattered atypical cells in the epidermis. Electron microscopy disclosed mononuclear cells with bizarre, convoluted nuclei, resembling cerebriform cells of Lutzner. Phototesting disclosed a diminished minimal erythemal threshold to UV-B and UV-A. Microscopic changes resembling actinic reticuloid were reproduced in this patient 24 and 72 hours after exposure to 15 minimal erythemal doses of UV-B.

  12. Novel chitosan-based films cross-linked by genipin with improved physical properties.

    PubMed

    Jin, J; Song, M; Hourston, D J

    2004-01-01

    Novel cross-linked chitosan-based films were prepared using the solution casting technique. A naturally occurring and nontoxic cross-linking agent, genipin, was used to form the chitosan and chitosan/poly(ethylene oxide) (PEO) blend networks, where two types of PEO were used, one with a molecular weight of 20 000 g/mol (HPEO) and the other of 600 g/mol (LPEO). Genipin is used in traditional Chinese medicine and extracted from gardenia fruit. Importantly, it overcomes the problem of physiological toxicity inherent in the use of some common synthetic chemicals as cross-linking agents. The mechanical properties and the stability in water of cross-linked and un-crosslinked chitosan and chitosan/PEO blend films were investigated. It was shown that, compared to the transparent yellow, un-cross-linked chitosan/PEO blend films, the genipin-cross-linked chitosan-based film, blue in color, was more elastic, was more stable, and had better mechanical properties. Genipin-cross-linking produced chitosan networks that were insoluble in acidic and alkaline solutions but were able to swell in these aqueous media. The swelling characteristics of the films exhibit sensitivity to the environmental pH and temperature. The surface properties of the films were also examined by contact angle measurements using water and mixtures of water/ethanol. The results showed that, with the one exception of cross-linked pure chitosan in 100% water, the cross-linked chitosan and chitosan/PEO blends were more hydrophobic than un-crosslinked ones.

  13. Maturation of Collagen Ketoimine Cross-links by an Alternative Mechanism to Pyridinoline Formation in Cartilage*

    PubMed Central

    Eyre, David R.; Weis, Mary Ann; Wu, Jiann-Jiu

    2010-01-01

    The tensile strength of fibrillar collagens depends on stable intermolecular cross-links formed through the lysyl oxidase mechanism. Such cross-links based on hydroxylysine aldehydes are particularly important in cartilage, bone, and other skeletal tissues. In adult cartilages, the mature cross-linking structures are trivalent pyridinolines, which form spontaneously from the initial divalent ketoimines. We examined whether this was the complete story or whether other ketoimine maturation products also form, as the latter are known to disappear almost completely from mature tissues. Denatured, insoluble, bovine articular cartilage collagen was digested with trypsin, and cross-linked peptides were isolated by copper chelation chromatography, which selects for their histidine-containing sequence motifs. The results showed that in addition to the naturally fluorescent pyridinoline peptides, a second set of cross-linked peptides was recoverable at a high yield from mature articular cartilage. Sequencing and mass spectral analysis identified their origin from the same molecular sites as the initial ketoimine cross-links, but the latter peptides did not fluoresce and were nonreducible with NaBH4. On the basis of their mass spectra, they were identical to their precursor ketoimine cross-linked peptides, but the cross-linking residue had an M+188 adduct. Considering the properties of an analogous adduct of identical added mass on a glycated lysine-containing peptide from type II collagen, we predicted that similar dihydroxyimidazolidine structures would form from their ketoimine groups by spontaneous oxidation and free arginine addition. We proposed the trivial name arginoline for the ketoimine cross-link derivative. Mature bovine articular cartilage contains about equimolar amounts of arginoline and hydroxylysyl pyridinoline based on peptide yields. PMID:20363745

  14. Photo-cross-linking of amniotic membranes for limbal epithelial cell cultivation.

    PubMed

    Lai, Jui-Yang

    2014-12-01

    In the present study, we developed photo-cross-linked amniotic membrane (AM) as a limbal stem cell niche. After ultraviolet (UV) irradiation for varying time periods, the biological tissues were studied by determinations of cross-linking structure, degradability, and nutrient permeation ability. Our results showed that the number of cross-links per unit mass of AM significantly increased with increasing illumination time from 5 to 50 min. However, the cross-link formation was inhibited by longer irradiation time (i.e., 150 min), probably due to the scission of tissue collagen chains through irradiation. The biological stability and matrix permeability of photo-cross-linked AM materials strongly depended on their cross-linking densities affected by the UV irradiation. In vitro biocompatibility studies including cell viability and pro-inflammatory gene expression analyses demonstrated that, irrespective of the irradiation time employed, the physically cross-linked biological tissues exhibited negligible cytotoxicity and similar interleukin-6 (IL-6) mRNA levels. The data clearly indicate that these AM matrices do not cause potential harm to the corneal epithelial cells. After the growth of limbal epithelial cells (LECs) on AM substrates, Western blot analyses were conducted to examine the expression of ABCG2. It was found that the ability of UV-irradiated AM to maintain the undifferentiated precursor cell phenotype was significantly enhanced with increasing extent of photo-cross-linking. In summary, the UV irradiation time may have a profound influence on the fabrication of photo-cross-linked AM matrices for LEC cultivation.

  15. Chemistry of collagen cross-links: glucose-mediated covalent cross-linking of type-IV collagen in lens capsules.

    PubMed Central

    Bailey, A J; Sims, T J; Avery, N C; Miles, C A

    1993-01-01

    The incubation of lens capsules with glucose in vitro resulted in changes in the mechanical and thermal properties of type-IV collagen consistent with increased cross-linking. Differential scanning calorimetry (d.s.c.) of fresh lens capsules showed two major peaks at melting temperatures Tm 1 and Tm 2 at approx. 54 degrees C and 90 degrees C, which can be attributed to the denaturation of the triple helix and 7S domains respectively. Glycosylation of lens capsules in vitro for 24 weeks caused an increase in Tm 1 from 54 degrees C to 61 degrees C, while non-glycosylated, control incubated capsules increased to a Tm 1 of 57 degrees C. The higher temperature required to denature the type-IV collagen after incubation in vitro suggested increased intermolecular cross-linking. Glycosylated lens capsules were more brittle than fresh samples, breaking at a maximum strain of 36.8 +/- 1.8% compared with 75.6 +/- 6.3% for the fresh samples. The stress at maximum strain (or 'strength') was dramatically reduced from 12.0 to 4.7 N.mm.mg-1 after glycosylation in vitro. The increased constraints within the system leading to loss of strength and increased brittleness suggested not only the presence of more cross-links but a difference in the location of these cross-links compared with the natural lysyl-aldehyde-derived cross-links. The chemical nature of the fluorescent glucose-derived cross-link following glycosylation was determined as pentosidine, at a concentration of 1 pentosidine molecule per 600 collagen molecules after 24 weeks incubation. Pentosidine was also determined in the lens capsules obtained from uncontrolled diabetics at a level of about 1 per 100 collagen molecules. The concentration of these pentosidine cross-links is far too small to account for the observed changes in the thermal and mechanical properties following incubation in vitro, clearly indicating that another as yet undefined, but apparently more important cross-linking mechanism mediated by glucose is

  16. Demonstration of prominent actin filaments in the root columella

    NASA Technical Reports Server (NTRS)

    Collings, D. A.; Zsuppan, G.; Allen, N. S.; Blancaflor, E. B.; Brown, C. S. (Principal Investigator)

    2001-01-01

    The distribution of actin filaments within the gravity-sensing columella cells of plant roots remains poorly understood, with studies over numerous years providing inconsistent descriptions of actin organization in these cells. This uncertainty in actin organization, and thus in actin's role in graviperception and gravisignaling, has led us to investigate actin arrangements in the columella cells of Zea mays L., Medicago truncatula Gaertn., Linum usitatissiilium L. and Nicotianla benthamiana Domin. Actin organization was examined using a combination of optimized immunofluorescence techniques, and an improved fluorochrome-conjugated phalloidin labeling method reliant on 3-maleimidobenzoyl-N-hydroxy-succinimide ester (MBS) cross-linking combined with glycerol permeabilization. Confocal microscopy of root sections labeled with anti-actin antibodies revealed patterns suggestive of actin throughout the columella region. These patterns included short and fragmented actin bundles, fluorescent rings around amyloplasts and intense fluorescence originating from the nucleus. Additionally, confocal microscopy of MBS-stabilized and Alexa Fluor-phalloidin-labeled root sections revealed a previously undetected state of actin organization in the columella. Discrete actin structures surrounded the amyloplasts and prominent actin cables radiated from the nuclear surface toward the cell periphery. Furthermore, the cortex of the columella cells contained fine actin bundles (or single filaments) that had a predominant transverse orientation. We also used confocal microscopy of plant roots expressing endoplasmic reticulum (ER)-targeted green fluorescent protein to demonstrate rapid ER movements within the columella cells, suggesting that the imaged actin network is functional. The successful identification of discrete actin structures in the root columella cells forms the perception and signaling.

  17. Kinetically controlled patterning of highly cross-linked phosphonium photopolymers using simple anion exchange.

    PubMed

    Guterman, Ryan; Gillies, Elizabeth R; Ragogna, Paul J

    2015-05-12

    A phosphonium salt possessing three methacrylate groups has been incorporated into a photopolymeric system to generate highly cross-linked polyelectrolyte networks. Emergent chemical and physical properties in the polymers were observed and attributed to the tandem increase in cross-link density and ion-content upon incorporation of the self-cross-linking cation. Anion-exchange with bis(trifluoromethylsulfonyl)imide or dodecylbenzenesulfonate resulted in significant differences in wettability and ion-exchange behavior. The passivating effects of dodecylbenzenesulfonate were utilized to selectively pattern fluorescein dye into the polymer network, highlighting a new patterning procedure using ionic-bond forming reactions.

  18. Chances and pitfalls of chemical cross-linking with amine-reactive N-hydroxysuccinimide esters.

    PubMed

    Kalkhof, Stefan; Sinz, Andrea

    2008-09-01

    In this report we summarize our experiences with the reaction products of N-hydroxysuccinimide (NHS) esters, which are widely used for chemical cross-linking of lysine residues in proteins. We describe the products, which should be scrutinized during data analysis using customized software when NHS esters are employed for chemical cross-linking. Reaction products of NHS esters were observed not only with lysines, but also with serines, tyrosines, and threonines. This report is intended to be a practical guide for those working in the field of chemical cross-linking and mass spectrometry.

  19. Chitosan-cross-linked osmium polymer composites as an efficient platform for electrochemical biosensors.

    PubMed

    Jirimali, Harishchandra Digambar; Nagarale, Rajaram Krishna; Lee, Jong Myung; Saravanakumar, Durai; Shin, Woonsup

    2013-07-22

    A new family of chitosan-cross-linked osmium polymer composites was prepared and its electrochemical properties were examined. The composites were prepared by quaternization of the poly(4-vinylpyridine) osmium bipyridyl polymer (PVP-Os) which was then cross-linked with chitosan, yielding PVP-Os/chitosan. Films made of the composites showed improved mass and electron transport owing to the porous and hydrophilic structure which is derived from the cross-links between the Os polymer and chitosan. The rate for glucose oxidation was enhanced four times when glucose oxidase (GOx) was immobilized on PVP-Os/chitosan compared immobilization on PVP-Os.

  20. Enzymatically cross-linked hyperbranched polyglycerol hydrogels as scaffolds for living cells.

    PubMed

    Wu, Changzhu; Strehmel, Christine; Achazi, Katharina; Chiappisi, Leonardo; Dernedde, Jens; Lensen, Marga C; Gradzielski, Michael; Ansorge-Schumacher, Marion B; Haag, Rainer

    2014-11-10

    Although several strategies are now available to enzymatically cross-link linear polymers to hydrogels for biomedical use, little progress has been reported on the use of dendritic polymers for the same purpose. Herein, we demonstrate that horseradish peroxidase (HRP) successfully catalyzes the oxidative cross-linking of a hyperbranched polyglycerol (hPG) functionalized with phenol groups to hydrogels. The tunable cross-linking results in adjustable hydrogel properties. Because the obtained materials are cytocompatible, they have great potential for encapsulating living cells for regenerative therapy. The gel formation can be triggered by glucose and controlled well under various environmental conditions.

  1. Double-helical nucleic acids with cross-linked strands: synthesis and applications in molecular biology

    NASA Astrophysics Data System (ADS)

    Antsypovitch, Sergei I.; Oretskaya, Tat'yana S.

    1998-03-01

    Data on the methods employed for cross-linking of DNA strands and for the synthesis of oligonucleotide duplexes with cross-links between strands are summarised. Existing methods are systematised; their advantages and drawbacks are discussed. The examples of applications of DNA duplexes with covalently cross-linked chains for the study of protein-nucleic acid recognition and mechanisms of action of nucleic acid-binding proteins for gaining information about the spatial structure of nucleic acids, and for the solution of other problems of molecular biology are given. The bibliography includes 131 references.

  2. Effect of cross-linking with calcium ions on the physical properties of alginate films.

    PubMed

    Russo, R; Malinconico, M; Santagata, G

    2007-10-01

    Three films of sodium alginate, with different amounts of guluronic fraction, were investigated with different techniques. On increasing the fraction of guluronic units the chain-to-chain interaction was promoted. The three samples were ionically cross-linked with calcium ions by soaking the films in a solution of calcium chloride. The introduction of the cross-linking points caused an appreciable change in the physical properties, and the results were discussed in terms of different composition of the materials and in terms of the increased free volume during the cross-linking process.

  3. The interaction of the chaperonin tailless complex polypeptide 1 (TCP1) ring complex (TRiC) with ribosome-bound nascent chains examined using photo-cross-linking.

    PubMed

    McCallum, C D; Do, H; Johnson, A E; Frydman, J

    2000-05-01

    The eukaryotic chaperonin tailless complex polypeptide 1 (TCP1) ring complex (TRiC) (also called chaperonin containing TCP1 [CCT]) is a hetero-oligomeric complex that facilitates the proper folding of many cellular proteins. To better understand the manner in which TRiC interacts with newly translated polypeptides, we examined its association with nascent chains using a photo-cross-linking approach. To this end, a series of ribosome-bound nascent chains of defined lengths was prepared using truncated mRNAs. Photoactivatable probes were incorporated into these (35)S- labeled nascent chains during translation. Upon photolysis, TRiC was cross-linked to ribosome-bound polypeptides exposing at least 50-90 amino acids outside the ribosomal exit channel, indicating that the chaperonin associates with much shorter nascent chains than indicated by previous studies. Cross-links were observed for nascent chains of the cytosolic proteins actin, luciferase, and enolase, but not to ribosome-bound preprolactin. The pattern of cross-links became more complex as the nascent chain increased in length. These results suggest a chain length-dependent increase in the number of TRiC subunits involved in the interaction that is consistent with the idea that the substrate participates in subunit-specific contacts with the chaperonin. Both ribosome isolation by centrifugation through sucrose cushions and immunoprecipitation with anti-puromycin antibodies demonstrated that the photoadducts form on ribosome-bound polypeptides. Our results indicate that TRiC/CCT associates with the translating polypeptide shortly after it emerges from the ribosome and suggest a close association between the chaperonin and the translational apparatus.

  4. Differential binding of tropomyosin isoforms to actin modified with m-maleimidobenzoyl-N-hydroxysuccinimide ester and fluorescein-5-isothiocyanate.

    PubMed

    Skórzewski, Radosław; Robaszkiewicz, Katarzyna; Jarzebińska, Justyna; Suder, Piotr; Silberring, Jerzy; Moraczewska, Joanna

    2009-11-01

    Differential interactions of tropomyosin (TM) isoforms with actin can be important for determination of the thin filament functions. A mechanism of tropomyosin binding to actin was studied by comparing interactions of five alphaTM isoforms with actin modified with m-maleimidobenzoyl-N-hydroxysuccinimide ester (MBS) and with fluorescein-5-isothiocyanate (FITC). MBS attachment sites were revealed with mass spectrometry methods. We found that the predominant actin fraction was cross-linked by MBS within subdomain 3. A smaller fraction of the modified actin was cross-linked within subdomain 2 and between subdomains 2 and 1. Moreover, investigated actins carried single labels in subdomains 1, 2, and 3. Such extensive modification caused a large decrease in actin affinity for skeletal and smooth muscle tropomyosins, nonmuscle TM2, and chimeric TM1b9a. In contrast, binding of nonmuscle isoform TM5a was less affected. Isoform's affinity for actin modified in subdomain 2 by binding of FITC to Lys61 was intermediate between the affinity for native actin and MBS-modified actin except for TM5a, which bound to FITC-actin with similar affinity as to actin modified with MBS. The analysis of binding curves according to the McGhee-von Hippel model revealed that binding to an isolated site, as well as cooperativity of binding to a contiguous site, was affected by both actin modifications in a TM isoform-specific manner.

  5. Use of a fusion protein between GFP and an actin-binding domain to visualize transient filamentous-actin structures.

    PubMed

    Pang, K M; Lee, E; Knecht, D A

    1998-03-26

    Many important processes in eukaryotic cells involve changes in the quantity, location and the organization of actin filaments [1] [2] [3]. We have been able to visualize these changes in live cells using a fusion protein (GFP-ABD) comprising the green fluorescent protein (GFP) of Aequorea victoria and the 25 kDa highly conserved actin-binding domain (ABD) from the amino terminus of the actin cross-linking protein ABP-120 [4]. In live cells of the soil amoeba Dictyostelium that were expressing GFP-ABD, the three-dimensional architecture of the actin cortex was clearly visualized. The pattern of GFP-ABD fluorescence in these cells coincided with that of rhodamine-phalloidin, indicating that GFP-ABD specifically binds filamentous (F) actin. On the ventral surface of non-polarized vegetative cells, a broad ring of F actin periodically assembled and contracted, whereas in polarized cells there were transient punctate F-actin structures; cells cycled between the polarized and non-polarized morphologies. During the formation of pseudopods, an increase in fluorescence intensity coincided with the initial outward deformation of the membrane. This is consistent with the models of pseudopod extension that predict an increase in the local density of actin filaments. In conclusion, GFP-ABD specifically binds F actin and allows the visualization of F-actin dynamics and cellular behavior simultaneously.

  6. Botulinum toxin type A targets RhoB to inhibit lysophosphatidic acid-stimulated actin reorganization and acetylcholine release in nerve growth factor-treated PC12 cells.

    PubMed

    Ishida, Hiroshi; Zhang, Xieping; Erickson, Kelly; Ray, Prabhati

    2004-09-01

    Botulinum toxin type A (BoNT/A) produced by Clostridium botulinum inhibits Ca2+-dependent acetylcholine (ACh) release (neuroexocytosis) at peripheral neuromuscular junctions, sometimes causing neuromuscular paralysis. This inhibitory effect is attributed to its metalloprotease activity to cleave the 25-kDa synaptosomal-associated protein, which is essential for the exocytotic machinery. However, deletion of this protein does not result in a complete block of neuroexocytosis, suggesting that botulinum-mediated inhibition may occur via another mechanism. Rho GTPases, a class of small GTP-binding proteins (G proteins), control actin cytoskeletal organization, thereby regulating a variety of cellular functions in various cells, including neuronal cells. We have shown that the G protein activator lysophosphatidic acid (LPA) triggered actin reorganization followed by Ca2+-dependent ACh release in nerve growth factor-treated PC12 cells and that BoNT/A blocked both events through degradation of RhoB by the proteasome. Overexpression of wild-type RhoB caused actin reorganization and enhanced the release of ACh by LPA to overcome toxin's inhibitory effect on actin reorganization/exocytosis stimulated by LPA, whereas overexpression of a dominant negative RhoB inhibited ACh release, regardless of LPA and/or toxin treatment. Finally, a knockdown of the RhoB gene via sequence-specific, post-transcriptional gene silencing reduced RhoB expression in PC12 cells, resulting in total inhibition of both actin reorganization and ACh release induced by LPA. We conclude that the RhoB signaling pathway regulates ACh release via actin cytoskeletal reorganization and that botulinum toxin inhibits neuroexocytosis by targeting RhoB pathway.

  7. Cross-linking proteins with bimetallic tetracarboxylate compounds of transition metals

    DOEpatents

    Kostic, Nenad M.; Chen, Jian

    1991-03-05

    Stable cross-linked complexes of transition-metal tetracarboxylates and proteins are formed. The preferred transition-metal is rhodium. The protein may be collagen or an enzyme such as a proteolytic enzyme.

  8. In Vivo Oxidative Stability Changes of Highly Cross-Linked Polyethylene Bearings: An Ex Vivo Investigation.

    PubMed

    Rowell, Shannon L; Reyes, Christopher R; Malchau, Henrik; Muratoglu, Orhun K

    2015-10-01

    The development of highly cross-linked UHMWPEs focused on stabilizing radiation-induced free radicals as the sole precursor to oxidative degradation. However, secondary in vivo oxidation mechanisms have been discovered. After a preliminary post-operative analysis, we subjected highly cross-linked retrievals with 1-4 years in vivo durations and never-implanted controls to accelerated aging to predict the extent to which their oxidative stability was compromised in vivo. Lipid absorption, oxidation, and hydroperoxides were measured using infrared spectroscopy. Gravimetric swelling was used to measure cross-link density. After aging, all retrievals, except vitamin E-stabilized components, regardless of initial lipid levels or oxidation, showed significant oxidative degradation, demonstrated by subsurface oxidative peaks, increased hydroperoxides and decreased cross-link density, compared to their post-operative material properties and never-implanted counterparts, confirming oxidative stability changes.

  9. Effect of cross-link density on carbon dioxide separation in polydimtheylsiloxane-norbornene membranes

    SciTech Connect

    Hong, Tao; Niu, Zhenbin; Hu, Xunxiang; Gmernicki, Kevin R.; Cheng, Shiwang; Fan, Fei; Johnson, Joseph C.; Hong, Eunice K.; Mahurin, Shannon Mark; Jiang, De-en; Long, Brian K.; Mays, Jimmy; Sokolov, Alexei P.; Saito, Tomonori

    2015-10-01

    Here, the development of high-performance materials for carbon dioxide separation and capture will significantly contribute to a solution for climate change. Herein, (bicycloheptenyl)ethylterminated polydimethylsiloxane (PDMSPNB) membranes with varied cross-link densities were synthesized via ring-opening metathesis polymerization. The developed polymer membranes show higher permeability and better selectivity than those of conventional cross-linked PDMS membrane. The achieved performance (CO2 permeability ≈ 6800 Barrer; CO2/N2 selectivity ≈ 14) is very promising for practical applications. The key to achieving this high performance is the use of an in situ cross-linking method for difunctional PDMS macromonomers, which provides lightly cross-linked membranes. By combining positron annihilation lifetime spectroscopy, broadband dielectric spectroscopy, and gas solubility measurements, key parameters necessary for achieving excellent performance have been elucidated.

  10. Effect of cross-link density on carbon dioxide separation in polydimethylsiloxane-norbornene membranes

    SciTech Connect

    Hong, Tao; Niu, Zhenbin; Hu, Xunxiang; Gmernicki, Kevin; Cheng, Shiwang; Fan, Fei; Johnson, J. Casey; Hong, Eunice; Mahurin, Shannon; Jiang, De -en; Long, Brian; Mays, Jimmy; Sokolov, Alexei; Saito, Tomonori

    2015-01-01

    The development of high performance materials for CO2 separation and capture will significantly contribute to a solution for climate change. In this work, (bicycloheptenyl) ethyl terminated polydimethylsiloxane (PDMSPNB) membranes with varied cross-link densities were synthesized via ring-opening metathesis polymerization. The developed polymer membranes show higher permeability and better selectivity than those of conventional cross-linked PDMS membrane. The achieved performance (CO2 permeability ~ 6800 Barrer and CO2/N2 selectivity ~ 14) is very promising for practical applications. The key to achieving this high performance is the use of an in-situ cross-linking method of the difunctional PDMS macromonomers, which provides lightly cross-linked membranes. By combining positron annihilation lifetime spectroscopy, broadband dielectric spectroscopy and gas solubility measurements, we have elucidated the key parameters necessary for achieving their excellent performance.

  11. Collagen type IX from human cartilage: a structural profile of intermolecular cross-linking sites.

    PubMed Central

    Diab, M; Wu, J J; Eyre, D R

    1996-01-01

    Type IX collagen, a quantitatively minor collagenous component of cartilage, is known to be associated with and covalently cross-linked to type II collagen fibrils in chick and bovine cartilage. Type IX collagen molecules have also been shown to form covalent cross-links with each other in bovine cartilage. In the present study we demonstrate by structural analysis and location of cross-linking sites that, in human cartilage, type IX collagen is covalently cross-linked to type II collagen and to other molecules of type IX collagen. We also present evidence that, if the proteoglycan form of type IX collagen is present in human cartilage, it can only be a minor component of the matrix, similar to findings with bovine cartilage. PMID:8660302

  12. Effect of cross-link density on carbon dioxide separation in polydimtheylsiloxane-norbornene membranes

    DOE PAGES

    Hong, Tao; Niu, Zhenbin; Hu, Xunxiang; ...

    2015-10-01

    Here, the development of high-performance materials for carbon dioxide separation and capture will significantly contribute to a solution for climate change. Herein, (bicycloheptenyl)ethylterminated polydimethylsiloxane (PDMSPNB) membranes with varied cross-link densities were synthesized via ring-opening metathesis polymerization. The developed polymer membranes show higher permeability and better selectivity than those of conventional cross-linked PDMS membrane. The achieved performance (CO2 permeability ≈ 6800 Barrer; CO2/N2 selectivity ≈ 14) is very promising for practical applications. The key to achieving this high performance is the use of an in situ cross-linking method for difunctional PDMS macromonomers, which provides lightly cross-linked membranes. By combining positron annihilationmore » lifetime spectroscopy, broadband dielectric spectroscopy, and gas solubility measurements, key parameters necessary for achieving excellent performance have been elucidated.« less

  13. Effect of cross-link density on carbon dioxide separation in polydimethylsiloxane-norbornene membranes

    DOE PAGES

    Hong, Tao; Niu, Zhenbin; Hu, Xunxiang; ...

    2015-01-01

    The development of high performance materials for CO2 separation and capture will significantly contribute to a solution for climate change. In this work, (bicycloheptenyl) ethyl terminated polydimethylsiloxane (PDMSPNB) membranes with varied cross-link densities were synthesized via ring-opening metathesis polymerization. The developed polymer membranes show higher permeability and better selectivity than those of conventional cross-linked PDMS membrane. The achieved performance (CO2 permeability ~ 6800 Barrer and CO2/N2 selectivity ~ 14) is very promising for practical applications. The key to achieving this high performance is the use of an in-situ cross-linking method of the difunctional PDMS macromonomers, which provides lightly cross-linked membranes.more » By combining positron annihilation lifetime spectroscopy, broadband dielectric spectroscopy and gas solubility measurements, we have elucidated the key parameters necessary for achieving their excellent performance.« less

  14. Plasticizer migration from cross-linked flexible PVC. 1. Effects on tribology

    NASA Astrophysics Data System (ADS)

    Pannico, M.; Persico, P.; Ambrogi, V.; Carfagna, C.

    2010-06-01

    Utilization of soft PVC is restricted by plasticizer migration that can affect material properties, as well as its toxicity. Modifying the chemical structure of PVC is one of the most effective tool to reduce the diffusion of plasticizer. In this work, a soft cross-linked PVC was obtained using a difunctional amine, namely isophoron diamine (IPDA) as the cross-linking agent. The gel content (wt %) was evaluated by weighting the insoluble portion obtained through solvent extraction technique. Thermogravimetric analysis (TGA) revealed that cross-linking reactions promote thermal degradation phenomena in the polymer matrix. Tribological properties of soft uncross-linked, cross-linked and rigid PVC were determined. Soft formulations were held in contact for 32 days with rigid PVC sheets. Plasticizer migration towards the interface causes an increase of dynamic friction compared to that of the reference rigid PVC.

  15. Cross-linking proteins with bimetallic tetracarboxylate compounds of transition metals

    DOEpatents

    Kostic, N.M.; Chen, J.

    1991-03-05

    Stable cross-linked complexes of transition-metal tetracarboxylates and proteins are formed. The preferred transition-metal is rhodium. The protein may be collagen or an enzyme such as a proteolytic enzyme. No Drawings

  16. Inverted bulk-heterojunction solar cell with cross-linked hole-blocking layer

    PubMed Central

    Udum, Yasemin; Denk, Patrick; Adam, Getachew; Apaydin, Dogukan H.; Nevosad, Andreas; Teichert, Christian; S. White, Matthew.; S. Sariciftci, Niyazi.; Scharber, Markus C.

    2014-01-01

    We have developed a hole-blocking layer for bulk-heterojunction solar cells based on cross-linked polyethylenimine (PEI). We tested five different ether-based cross-linkers and found that all of them give comparable solar cell efficiencies. The initial idea that a cross-linked layer is more solvent resistant compared to a pristine PEI layer could not be confirmed. With and without cross-linking, the PEI layer sticks very well to the surface of the indium–tin–oxide electrode and cannot be removed by solvents used to process PEI or common organic semiconductors. The cross-linked PEI hole-blocking layer functions for multiple donor–acceptor blends. We found that using cross-linkers improves the reproducibility of the device fabrication process. PMID:24817837

  17. A Review of Collagen Cross-Linking in Cornea and Sclera

    PubMed Central

    Zhang, Xiao; Tao, Xiang-chen; Zhang, Jian; Li, Zhi-wei; Xu, Yan-yun; Wang, Yu-meng; Zhang, Chun-xiao; Mu, Guo-ying

    2015-01-01

    Riboflavin/UVA cross-linking is a technique introduced in the past decades for the treatment of keratoconus, keratectasia, and infectious keratitis. Its efficacy and safety have been investigated with clinical and laboratory studies since its first clinical application by Wollensak for the treatment of keratoconus. Although its complications are encountered during clinical practice, such as infection inducing risk, minimal invasion merits a further investigation on its future application in clinical practice. Recently, collagen cross-linking in sclera shows a promising prospect. In present study, we summarized the representative studies describing the clinical and laboratory application of collagen cross-linking published in past decades and provided our opinion on the positive and negative results of cross-linking in the treatment of ophthalmic disorders. PMID:25922758

  18. Ionically cross-linked hyaluronic acid: wetting, lubrication, and viscoelasticity of a modified adhesion barrier gel

    PubMed Central

    Vorvolakos, Katherine; Isayeva, Irada S; Luu, Hoan-My Do; Patwardhan, Dinesh V; Pollack, Steven K

    2011-01-01

    Hyaluronic acid (HA), in linear or cross-linked form, is a common component of cosmetics, personal care products, combination medical products, and medical devices. In all cases, the ability of the HA solution or gel to wet surfaces and/or disrupt and lubricate interfaces is a limiting feature of its mechanism of action. We synthesized ferric ion–cross-linked networks of HA based on an adhesion barrier, varied the degree of cross-linking, and performed wetting goniometry, viscometry, and dynamic mechanical analysis. As cross-linking increases, so do contact angle, viscosity, storage modulus, and loss modulus; thus, wetting and lubrication are compromised. These findings have implications in medical device materials, such as adhesion barriers and mucosal drug delivery vehicles. PMID:22915924

  19. Effect of Cross-Link Density on Carbon Dioxide Separation in Polydimethylsiloxane-Norbornene Membranes.

    PubMed

    Hong, Tao; Niu, Zhenbin; Hu, Xunxiang; Gmernicki, Kevin; Cheng, Shiwang; Fan, Fei; Johnson, J Casey; Hong, Eunice; Mahurin, Shannon; Jiang, De-en; Long, Brian; Mays, Jimmy; Sokolov, Alexei; Saito, Tomonori

    2015-11-01

    The development of high-performance materials for carbon dioxide separation and capture will significantly contribute to a solution for climate change. Herein, (bicycloheptenyl)ethyl-terminated polydimethylsiloxane (PDMSPNB) membranes with varied cross-link densities were synthesized via ring-opening metathesis polymerization. The developed polymer membranes show higher permeability and better selectivity than those of conventional cross-linked PDMS membrane. The achieved performance (CO2 permeability≈6800 Barrer; CO2 /N2 selectivity≈14) is very promising for practical applications. The key to achieving this high performance is the use of an in situ cross-linking method for difunctional PDMS macromonomers, which provides lightly cross-linked membranes. By combining positron annihilation lifetime spectroscopy, broadband dielectric spectroscopy, and gas solubility measurements, key parameters necessary for achieving excellent performance have been elucidated.

  20. Streamlined discovery of cross-linked chromatin complexes and associated histone modifications by mass spectrometry.

    PubMed

    Zee, Barry M; Alekseyenko, Artyom A; McElroy, Kyle A; Kuroda, Mitzi I

    2016-02-16

    Posttranslational modifications (PTMs) are key contributors to chromatin function. The ability to comprehensively link specific histone PTMs with specific chromatin factors would be an important advance in understanding the functions and genomic targeting mechanisms of those factors. We recently introduced a cross-linked affinity technique, BioTAP-XL, to identify chromatin-bound protein interactions that can be difficult to capture with native affinity techniques. However, BioTAP-XL was not strictly compatible with similarly comprehensive analyses of associated histone PTMs. Here we advance BioTAP-XL by demonstrating the ability to quantify histone PTMs linked to specific chromatin factors in parallel with the ability to identify nonhistone binding partners. Furthermore we demonstrate that the initially published quantity of starting material can be scaled down orders of magnitude without loss in proteomic sensitivity. We also integrate hydrophilic interaction chromatography to mitigate detergent carryover and improve liquid chromatography-mass spectrometric performance. In summary, we greatly extend the practicality of BioTAP-XL to enable comprehensive identification of protein complexes and their local chromatin environment.

  1. DNA-protein cross-links produced by various chemicals in cultured human lymphoma cells.

    PubMed

    Costa, M; Zhitkovich, A; Harris, M; Paustenbach, D; Gargas, M

    1997-04-11

    Chemicals such as cis-platinum, formaldehyde, chromate, copper, and certain arsenic compounds have been shown to produce DNA-protein cross-links in human in vitro cell systems at high doses, such as those in the cytotoxic range. Thus far there have only been a limited number of other chemicals evaluated for their ability to produce cross-links. The purpose of the work described here was to evaluate whether select industrial chemicals can form DNA-protein cross-links in human cells in vitro. We evaluated acetaldehyde, acrolein, diepoxybutane, paraformaldehyde, 2-furaldehyde, propionaldehyde, chloroacetaldehyde, sodium arsenite, and a deodorant tablet [Mega Blue; hazardous component listed as tris(hydroxymethyl)nitromethane]. Short- and long-term cytotoxicity was evaluated and used to select appropriate doses for in vitro testing. DNA-protein cross-linking was evaluated at no fewer than three doses and two cell lysate washing temperatures (45 and 65 degrees C) in Epstein-Barr virus (EBV) human Burkitt's lymphoma cells. The two washing temperatures were used to assess the heat stability of the DNA-protein cross-link, 2-Furaldehyde, acetaldehyde, and propionaldehyde produced statistically significant increases in DNA-protein cross-links at washing temperatures of 45 degrees C, but not 65 degrees C, and at or above concentrations of 5, 17.5, and 75 mM, respectively. Acrolein, diepoxybutane, paraformaldehyde, and Mega Blue produced statistically significant increases in DNA-protein cross-links washed at 45 and 65 degrees C at or above concentrations of 0.15 mM, 12.5 mM, 0.003%, and 0.1%, respectively. Sodium arsenite and chloroacetaldehyde did not produce significantly increased DNA-protein cross-links at either temperature nor at any dose tested. Excluding paraformaldehyde and 2-furaldehyde treatments, significant increases in DNA-protein cross-links were observed only at doses that resulted in complete cell death within 4 d following dosing. This work demonstrates that

  2. Intra-molecular cross-linking of acidic residues for protein structure studies.

    SciTech Connect

    Kruppa, Gary Hermann; Young, Malin M.; Novak, Petr; Schoeniger, Joseph S.

    2005-03-01

    Intra-molecular cross-linking has been suggested as a method of obtaining distance constraints that would be useful in developing structural models of proteins. Recent work published on intra-molecular cross-linking for protein structural studies has employed commercially available primary amine selective reagents that can cross-link lysine residues to other lysine residues or the amino terminus. Previous work using these cross-linkers has shown that for several proteins of known structure, the number of cross-links that can be obtained experimentally may be small compared to what would be expected from the known structure, due to the relative reactivity, distribution, and solvent accessibility of the lysines in the protein sequence. To overcome these limitations we have investigated the use of cross-linking reagents that can react with other reactive sidechains in proteins. We used 1-Ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) to activate the carboxylic acid containing residues, aspartic acid (D), glutamic acid (E), and the carboxy terminus (O), for cross-linking reactions. Once activated, the DEO sidechains can react to form 'zero-length' cross-links with nearby primary amine containing resides, lysines (K) and the amino terminus (X), via the formation of a new amide bond. We also show that the EDC-activated DEO sidechains can be cross-linked to each other using dihydrazides, two hydrazide moieties connected by an alkyl cross-linker ann of variable length. Using these reagents, we have found three new 'zero-length' cross-links in ubiquitin consistent with its known structure (M1-E16, M1-E18, and K63-E64). Using the dihydrazide cross-linkers, we have identified 2 new cross-links (D21-D32 and E24-D32) unambiguously. Using a library of dihydrazide cross-linkers with varying arm length, we have shown that there is a minimum arm length required for the DEO-DEO cross-links of 5.8 angstroms. These results show that additional structural information

  3. Computational exploration of polymer nanocomposite mechanical property modification via cross-linking topology.

    PubMed

    Lacevic, Naida; Gee, Richard H; Saab, Andrew; Maxwell, Robert

    2008-09-28

    Molecular dynamics simulations have been performed in order to study the effects of nanoscale filler cross-linking topologies and loading levels on the mechanical properties of a model elastomeric nanocomposite. The model system considered here is constructed from octafunctional polyhedral oligomeric silsesquioxane (POSS) dispersed in a poly(dimethylsiloxane) (PDMS) matrix. Shear moduli, G, have been computed for pure and for filled and unfilled PDMS as a function of cross-linking density, POSS fill loading level, and polymer network topology. The results reported here show that G increases as the cross-linking (covalent bonds formed between the POSS and the PDMS network) density increases. Further, G is found to have a strong dependence on cross-linking topology. The increase in shear modulus, G, for POSS filled PDMS is significantly higher than that for unfilled PDMS cross-linked with standard molecular species, suggesting an enhanced reinforcement mechanism for POSS. In contrast, in blended systems (POSS/PDMS mixture with no cross-linking) G was not observed to significantly increase with POSS loading. Finally, we find intriguing differences in the structural arrangement of bond strains between the cross-linked and the blended systems. In the unfilled PDMS the distribution of highly strained bonds appears to be random, while in the POSS filled system, the strained bonds form a netlike distribution that spans the network. Such a distribution may form a structural network "holding" the composite together and resulting in increases in G compared to an unfilled, cross-linked system. These results are of importance for engineering of new POSS-based multifunctional materials with tailor-made mechanical properties.

  4. 2-(2-methoxyethoxy)ethyl methacrylate hydrogels with gradient of cross-link density

    NASA Astrophysics Data System (ADS)

    Kadlubowski, Slawomir; Matusiak, Malgorzata; Adamus, Agnieszka; Olejniczak, Magdalena N.; Kozanecki, Marcin

    2016-01-01

    Electron beam irradiation of 2-(2-methoxyethoxy)ethyl methacrylate and ethylene glycol dimethacrylate mixtures leads to the formation of cross-linked structures that exhibit a gradient of cross-link density, as demonstrated by gel fraction, swelling and Differential Scanning Calorimetry analysis. The reason for observed phase separation is formation of the high molecular weight clusters and its precipitation before gelation dose. This effect can be controlled/influenced by absorbed dose and cross-linker concentration.

  5. A novel fibre-ensemble level constitutive model for exogenous cross-linked collagenous tissues

    PubMed Central

    Sacks, Michael S.; Wognum, Silvia

    2016-01-01

    Exogenous cross-linking of soft collagenous tissues is a common method for biomaterial development and medical therapies. To enable improved applications through computational methods, physically realistic constitutive models are required. Yet, despite decades of research, development and clinical use, no such model exists. In this study, we develop the first rigorous full structural model (i.e. explicitly incorporating various features of the collagen fibre architecture) for exogenously cross-linked soft tissues. This was made possible, in-part, with the use of native to cross-linked matched experimental datasets and an extension to the collagenous structural constitutive model so that the uncross-linked collagen fibre responses could be mapped to the cross-linked configuration. This allowed us to separate the effects of cross-linking from kinematic changes induced in the cross-linking process, which in turn allowed the non-fibrous tissue matrix component and the interaction effects to be identified. It was determined that the matrix could be modelled as an isotropic material using a modified Yeoh model. The most novel findings of this study were that: (i) the effective collagen fibre modulus was unaffected by cross-linking and (ii) fibre-ensemble interactions played a large role in stress development, often dominating the total tissue response (depending on the stress component and loading path considered). An important utility of the present model is its ability to separate the effects of exogenous cross-linking on the fibres from changes due to the matrix. Applications of this approach include the utilization in the design of novel chemical treatments to produce specific mechanical responses and the study of fatigue damage in bioprosthetic heart valve biomaterials. PMID:26855761

  6. Computational exploration of polymer nanocomposite mechanical property modification via cross-linking topology

    SciTech Connect

    Lacevic, N; Gee, R; Saab, A; Maxwell, R

    2008-04-24

    Molecular dynamics simulations have been performed in order to study the effects of nanoscale filler cross-linking topologies and loading levels on the mechanical properties of a model elastomeric nanocomposite. The model system considered here is constructed from octa-functional polyhedral oligomeric silsesquioxane (POSS) dispersed in a poly(dimethylsiloxane) (PDMS) matrix. Shear moduli, G, have been computed for pure and for filled and unfilled PDMS as a function of cross-linking density, POSS fill loading level, and polymer network topology. The results reported here show that G increases as the cross-linking (covalent bonds formed between the POSS and the PDMS network) density increases. Further, G is found to have a strong dependence on cross-linking topology. The increase in shear modulus, G, for POSS filled PDMS is significantly higher than that for unfilled PDMS cross-linked with standard molecular species, suggesting an enhanced reinforcement mechanism for POSS. In contrast, in blended systems (POSS/PDMS mixture with no cross-linking) G was not observed to significantly increase with POSS loading. Finally, we find intriguing differences in the structural arrangement of bond strains between the cross-linked and the blended systems. In the unfilled PDMS the distribution of highly strained bonds appears to be random, while in the POSS filled system, the strained bonds form a net-like distribution that spans the network. Such a distribution may form a structural network 'holding' the composite together and resulting in increases in G compared to an unfilled, cross-linked system. These results are of importance for engineering of new POSS-based multifunctional materials with tailor-made mechanical properties.

  7. A novel fibre-ensemble level constitutive model for exogenous cross-linked collagenous tissues.

    PubMed

    Sacks, Michael S; Zhang, Will; Wognum, Silvia

    2016-02-06

    Exogenous cross-linking of soft collagenous tissues is a common method for biomaterial development and medical therapies. To enable improved applications through computational methods, physically realistic constitutive models are required. Yet, despite decades of research, development and clinical use, no such model exists. In this study, we develop the first rigorous full structural model (i.e. explicitly incorporating various features of the collagen fibre architecture) for exogenously cross-linked soft tissues. This was made possible, in-part, with the use of native to cross-linked matched experimental datasets and an extension to the collagenous structural constitutive model so that the uncross-linked collagen fibre responses could be mapped to the cross-linked configuration. This allowed us to separate the effects of cross-linking from kinematic changes induced in the cross-linking process, which in turn allowed the non-fibrous tissue matrix component and the interaction effects to be identified. It was determined that the matrix could be modelled as an isotropic material using a modified Yeoh model. The most novel findings of this study were that: (i) the effective collagen fibre modulus was unaffected by cross-linking and (ii) fibre-ensemble interactions played a large role in stress development, often dominating the total tissue response (depending on the stress component and loading path considered). An important utility of the present model is its ability to separate the effects of exogenous cross-linking on the fibres from changes due to the matrix. Applications of this approach include the utilization in the design of novel chemical treatments to produce specific mechanical responses and the study of fatigue damage in bioprosthetic heart valve biomaterials.

  8. Collagen and elastin cross-linking is altered during aberrant late lung development associated with hyperoxia.

    PubMed

    Mižíková, Ivana; Ruiz-Camp, Jordi; Steenbock, Heiko; Madurga, Alicia; Vadász, István; Herold, Susanne; Mayer, Konstantin; Seeger, Werner; Brinckmann, Jürgen; Morty, Rory E

    2015-06-01

    Maturation of the lung extracellular matrix (ECM) plays an important role in the formation of alveolar gas exchange units. A key step in ECM maturation is cross-linking of collagen and elastin, which imparts stability and functionality to the ECM. During aberrant late lung development in bronchopulmonary dysplasia (BPD) patients and animal models of BPD, alveolarization is blocked, and the function of ECM cross-linking enzymes is deregulated, suggesting that perturbed ECM cross-linking may impact alveolarization. In a hyperoxia (85% O2)-based mouse model of BPD, blunted alveolarization was accompanied by alterations to lung collagen and elastin levels and cross-linking. Total collagen levels were increased (by 63%). The abundance of dihydroxylysinonorleucine collagen cross-links and the dihydroxylysinonorleucine-to-hydroxylysinonorleucine ratio were increased by 11 and 18%, respectively, suggestive of a profibrotic state. In contrast, insoluble elastin levels and the abundance of the elastin cross-links desmosine and isodesmosine in insoluble elastin were decreased by 35, 30, and 21%, respectively. The lung collagen-to-elastin ratio was threefold increased. Treatment of hyperoxia-exposed newborn mice with the lysyl oxidase inhibitor β-aminopropionitrile partially restored normal collagen levels, normalized the dihydroxylysinonorleucine-to-hydroxylysinonorleucine ratio, partially normalized desmosine and isodesmosine cross-links in insoluble elastin, and partially restored elastin foci structure in the developing septa. However, β-aminopropionitrile administration concomitant with hyperoxia exposure did not improve alveolarization, evident from unchanged alveolar surface area and alveoli number, and worsened septal thickening (increased by 12%). These data demonstrate that collagen and elastin cross-linking are perturbed during the arrested alveolarization of developing mouse lungs exposed to hyperoxia.

  9. Thickness-dependent glass transition temperature and charge mobility in cross-linked polyfluorene thin films

    NASA Astrophysics Data System (ADS)

    Liu, Dan; Qin, Hui; Zhang, Jinghui; Wang, Tao

    2016-11-01

    We report thickness-dependent glass transition temperature (Tg) and charge mobility in cross-linked thin films made of conjugated polymer poly(9,9-dioctylfluorene-co-N -(4-butylphenyl)diphenylamine) (TFB). Monotonic Tg depressions with reducing film thickness in thermally and UV cross-linked TFB thin films supported on Si-SiOx substrates are observed through ellipsometry measurements, suggesting that a surface mobile layer with enhanced chain dynamics still exists in cross-linked TFB thin films, even with a high cross-linking percentage. Data fitting using a three-layer model shows that the Tg in the interface, bulk and surface layer both increases with increasing cross-linking, while the thickness of the interface and surface layer increases and reduces, respectively. Cross-linking of TFB thin film generates traps that hinder charge transport and consequently reduce charge mobility. The charge mobility converges in thick (>140 nm) and thin (<40 nm) TFB films but shows strong thickness dependence in between, reducing from 4.0 ×10-4c m2/V s in a 180-nm film to 0.1 ×10-4c m2/V s in a 20-nm thin film.

  10. Ruthenium-catalyzed photo cross-linking of fibrin-based engineered tissue

    PubMed Central

    Bjork, Jason W.; Johnson, Sandra L.; Tranquillo, Robert T.

    2012-01-01

    Most cross-linking methods utilize chemistry or physical processes that are detrimental to cells and tissue development. Those that are not as harmful often do not provide a level of strength that ultimately meets the required application. The purpose of this work was to investigate the use of a ruthenium-sodium persulfate cross-linking system to form dityrosine in fibrin-based engineered tissue. By utilizing the tyrosine residues inherent to fibrin and cell-deposited proteins, at least 3-fold mechanical strength increases and 10-fold stiffness increases were achieved after cross-linking. This strengthening and stiffening effect was found to increase with culture duration prior to cross-linking such that physiologically relevant properties were obtained. Fibrin was not required for this effect as demonstrated by testing with collagen-based engineered tissue. Cross-linked tissues were implanted subcutaneously and shown to have minimal inflammation after 30 days, similar to non-cross-linked controls. Overall, the method employed is rapid, non-toxic, minimally inflammatory, and is capable of increasing strength and stiffness of engineered tissues to physiological levels. PMID:21196047

  11. Triple shape memory effects of cross-linked polyethylene/polypropylene blends with cocontinuous architecture.

    PubMed

    Zhao, Jun; Chen, Min; Wang, Xiaoyan; Zhao, Xiaodong; Wang, Zhenwen; Dang, Zhi-Min; Ma, Lan; Hu, Guo-Hua; Chen, Fenghua

    2013-06-26

    In this paper, the triple shape memory effects (SMEs) observed in chemically cross-linked polyethylene (PE)/polypropylene (PP) blends with cocontinuous architecture are systematically investigated. The cocontinuous window of typical immiscible PE/PP blends is the volume fraction of PE (v(PE)) of ca. 30-70 vol %. This architecture can be stabilized by chemical cross-linking. Different initiators, 2,5-dimethyl-2,5-di(tert-butylperoxy)-hexane (DHBP), dicumylperoxide (DCP) coupled with divinylbenzene (DVB) (DCP-DVB), and their mixture (DHBP/DCP-DVB), are used for the cross-linking. According to the differential scanning calorimetry (DSC) measurements and gel fraction calculations, DHBP produces the best cross-linking and DCP-DVB the worst, and the mixture, DHBP/DCP-DVB, is in between. The chemical cross-linking causes lower melting temperature (Tm) and smaller melting enthalpy (ΔHm). The prepared triple shape memory polymers (SMPs) by cocontinuous immiscible PE/PP blends with v(PE) of 50 vol % show pronounced triple SMEs in the dynamic mechanical thermal analysis (DMTA) and visual observation. This new strategy of chemically cross-linked immiscible blends with cocontinuous architecture can be used to design and prepare new SMPs with triple SMEs.

  12. Exogenous collagen cross-linking recovers tendon functional integrity in an experimental model of partial tear.

    PubMed

    Fessel, Gion; Wernli, Jeremy; Li, Yufei; Gerber, Christian; Snedeker, Jess G

    2012-06-01

    We investigated the hypothesis that exogenous collagen cross-linking can augment intact regions of tendon to mitigate mechanical propagation of partial tears. We first screened the low toxicity collagen cross-linkers genipin, methylglyoxal and ultra-violet (UV) light for their ability to augment tendon stiffness and failure load in rat tail tendon fascicles (RTTF). We then investigated cross-linking effects in load bearing equine superficial digital flexor tendons (SDFT). Data indicated that all three cross-linking agents augmented RTTF mechanical properties but reduced native viscoelasticity. In contrast to effects observed in fascicles, methylglyoxal treatment of SDFT detrimentally affected tendon mechanical integrity, and in the case of UV did not alter tendon mechanics. As in the RTTF experiments, genipin cross-linking of SDFT resulted in increased stiffness, higher failure loads and reduced viscoelasticity. Based on this result we assessed the efficacy of genipin in arresting tendon tear propagation in cyclic loading to failure. Genipin cross-linking secondary to a mid-substance biopsy-punch significantly reduced tissue strains, increased elastic modulus and increased resistance to fatigue failure. We conclude that genipin cross-linking of injured tendons holds potential for arresting tendon tear progression, and that implications of the treatment on matrix remodeling in living tendons should now be investigated.

  13. Fibromodulin Interacts with Collagen Cross-linking Sites and Activates Lysyl Oxidase*

    PubMed Central

    Bihan, Dominique; Bonna, Arkadiusz; Rubin, Kristofer; Farndale, Richard W.

    2016-01-01

    The hallmark of fibrotic disorders is a highly cross-linked and dense collagen matrix, a property driven by the oxidative action of lysyl oxidase. Other fibrosis-associated proteins also contribute to the final collagen matrix properties, one of which is fibromodulin. Its interactions with collagen affect collagen cross-linking, packing, and fibril diameter. We investigated the possibility that a specific relationship exists between fibromodulin and lysyl oxidase, potentially imparting a specific collagen matrix phenotype. We mapped the fibromodulin-collagen interaction sites using the collagen II and III Toolkit peptide libraries. Fibromodulin interacted with the peptides containing the known collagen cross-linking sites and the MMP-1 cleavage site in collagens I and II. Interestingly, the interaction sites are closely aligned within the quarter-staggered collagen fibril, suggesting a multivalent interaction between fibromodulin and several collagen helices. Furthermore, we detected an interaction between fibromodulin and lysyl oxidase (a major collagen cross-linking enzyme) and mapped the interaction site to 12 N-terminal amino acids on fibromodulin. This interaction also increases the activity of lysyl oxidase. Together, the data suggest a fibromodulin-modulated collagen cross-linking mechanism where fibromodulin binds to a specific part of the collagen domain and also forms a complex with lysyl oxidase, targeting the enzyme toward specific cross-linking sites. PMID:26893379

  14. Dynamic OCT measurements of corneal biomechanical properties after UV cross-linking in the rabbit

    NASA Astrophysics Data System (ADS)

    Twa, Michael D.; Li, Jiasong; Manapuram, Ravi K.; Menodiado, Floredes M.; Singh, Manmohan; Aglyamov, Salavat; Emelianov, Stanislav; Larin, Kirill V.

    2013-03-01

    Structural properties of the cornea determine the shape and optical quality of the eye. Keratoconus, a structural degeneration of the cornea, is often treated with UV-induced collagen cross-linking to increase tissue resistance to further deformation and degeneration. Optimal treatments would be customized to the individual and consider preexisting structural properties as well as the effects induced by treatment and this requires the capability to noninvasively measure tissue properties. The purpose of this study is to use novel methods of optical elastography to study the effects of UV-induced corneal collagen cross-linking in the rabbit eye. Low-amplitude (<1μm) elastic flexural waves were generated using focused air-pulse stimulation. Elastic wave propagation was measured over a 10x10mm area using Phase Stabilized Swept Source Optical Coherence Elastography (PhS-SSOCE) with a sensitivity of ~ 10 nm. Wave amplitude and velocity were computed and compared in tissues before and after UV cross-linking. Wave amplitude was decreased by the cross-linking treatment, while wave velocity was greater in cross-linked tissue than it was in the untreated cornea. Decreased wave amplitude and increased wave velocity after cross-linking is consistent with increased tissue stiffness. This was confirmed by conventional mechanical tension testing. These results demonstrate that the combination of the PhS-SSOCE and focused air pulse stimulation is capable of measuring low amplitude tissue motion and quantifying corneal stiffness.

  15. Fibromodulin Interacts with Collagen Cross-linking Sites and Activates Lysyl Oxidase.

    PubMed

    Kalamajski, Sebastian; Bihan, Dominique; Bonna, Arkadiusz; Rubin, Kristofer; Farndale, Richard W

    2016-04-08

    The hallmark of fibrotic disorders is a highly cross-linked and dense collagen matrix, a property driven by the oxidative action of lysyl oxidase. Other fibrosis-associated proteins also contribute to the final collagen matrix properties, one of which is fibromodulin. Its interactions with collagen affect collagen cross-linking, packing, and fibril diameter. We investigated the possibility that a specific relationship exists between fibromodulin and lysyl oxidase, potentially imparting a specific collagen matrix phenotype. We mapped the fibromodulin-collagen interaction sites using the collagen II and III Toolkit peptide libraries. Fibromodulin interacted with the peptides containing the known collagen cross-linking sites and the MMP-1 cleavage site in collagens I and II. Interestingly, the interaction sites are closely aligned within the quarter-staggered collagen fibril, suggesting a multivalent interaction between fibromodulin and several collagen helices. Furthermore, we detected an interaction between fibromodulin and lysyl oxidase (a major collagen cross-linking enzyme) and mapped the interaction site to 12 N-terminal amino acids on fibromodulin. This interaction also increases the activity of lysyl oxidase. Together, the data suggest a fibromodulin-modulated collagen cross-linking mechanism where fibromodulin binds to a specific part of the collagen domain and also forms a complex with lysyl oxidase, targeting the enzyme toward specific cross-linking sites.

  16. Cross-linking proteins by laccase-catalyzed oxidation: importance relative to other modifications.

    PubMed

    Steffensen, Charlotte L; Andersen, Mogens L; Degn, Peter E; Nielsen, Jacob H

    2008-12-24

    Laccase-catalyzed oxidation was able to induce intermolecular cross-links in beta-lactoglobulin, and ferulic acid-mediated laccase-catalyzed oxidation was able to induce intermolecular cross-links in alpha-casein, whereas transglutaminase cross-linked only alpha-casein. In addition, different patterns of laccase-induced oxidative modifications were detected, including dityrosine formation, formation of fluorescent tryptophan oxidation products, and carbonyls derived from histidine, tryptophan, and methionine. Laccase-catalyzed oxidation as well as transglutaminase induced only minor changes in surface tension of the proteins, and the changes could not be correlated to protein cross-linking. The presence of ferulic acid was found to influence the effect of laccase, allowing laccase to form irreducible intermolecular cross-links in beta-lactoglobulin and resulting in proteins exercising higher surface tensions due to cross-linking as well as other oxidative modifications. The outcome of using ferulic acid-mediated laccase-catalyzed oxidation to modify the functional properties of proteinaceous food components or other biosystems is expected to be highly dependent on the protein composition, resulting in different changes of the functional properties.

  17. xComb: a cross-linked peptide database approach to protein-protein interaction analysis.

    PubMed

    Panchaud, Alexandre; Singh, Pragya; Shaffer, Scott A; Goodlett, David R

    2010-05-07

    We developed an informatic method to identify tandem mass spectra composed of chemically cross-linked peptides from those of linear peptides and to assign sequence to each of the two unique peptide sequences. For a given set of proteins the key software tool, xComb, combs through all theoretically feasible cross-linked peptides to create a database consisting of a subset of all combinations represented as peptide FASTA files. The xComb library of select theoretical cross-linked peptides may then be used as a database that is examined by a standard proteomic search engine to match tandem mass spectral data sets to identify cross-linked peptides. The database search may be conducted against as many as 50 proteins with a number of common proteomic search engines, e.g. Phenyx, Sequest, OMSSA, Mascot and X!Tandem. By searching against a peptide library of linearized, cross-linked peptides, rather than a linearized protein library, search times are decreased and the process is decoupled from any specific search engine. A further benefit of decoupling from the search engine is that protein cross-linking studies may be conducted with readily available informatics tools for which scoring routines already exist within the proteomic community.

  18. Cross-linking of a polyomavirus attachment protein to its mouse kidney cell receptor

    SciTech Connect

    Griffith, G.R.; Consigli, R.A.

    1986-06-01

    The authors used photoaffinity cross-linking with the heterobifunctional cross-linker N-hydroxysuccinimidyl 4-azidobenzoate (HSAB) to covalently link polyomavirus to a mouse kidney cell surface component. The virus-HSAB combination was adsorbed to the cells and then cross-linked and isolated in monopinocytotic vesicles from the cells after endocytosis. The cross-linked product was identified on sodium dodecyl sulfate-polyacrylamide gels by the presence of a new band carrying /sup 125/I-labeled virion protein with a higher molecular mass than the normal virion protein bands. A single new band, with an apparent molecular mass of 120 kilodaltons (120 kDa), was identified by this procedure. This band was formed only in the presence of the HSAB cross-linker when virions were bound to the cells. The band also copurified with cross-linked virions when virion-containing vesicles were treated with detergent to remove the cell membrane. Antibody treatments that blocked up to 100% of virus binding and internalization also blocked cross-linking, as measured by the formation of the 120-kDa band. The 120-kDa band was characterized by preparation of antibody against the excised band from the gel. These data have demonstrated that molecules of possible biological significance in the binding of polyomavirus to mouse kidney cells have been cross-linked and that cell surface molecules have been identified that may be characterized further for possible receptor function in polyomavirus attachment.

  19. Cross-linking of DNA through HMGA1 suggests a DNA scaffold

    PubMed Central

    Vogel, Benjamin; Löschberger, Anna; Sauer, Markus; Hock, Robert

    2011-01-01

    Binding of proteins to DNA is usually considered 1D with one protein bound to one DNA molecule. In principle, proteins with multiple DNA binding domains could also bind to and thereby cross-link different DNA molecules. We have investigated this possibility using high-mobility group A1 (HMGA1) proteins, which are architectural elements of chromatin and are involved in the regulation of multiple DNA-dependent processes. Using direct stochastic optical reconstruction microscopy (dSTORM), we could show that overexpression of HMGA1a-eGFP in Cos-7 cells leads to chromatin aggregation. To investigate if HMGA1a is directly responsible for this chromatin compaction we developed a DNA cross-linking assay. We were able to show for the first time that HMGA1a can cross-link DNA directly. Detailed analysis using point mutated proteins revealed a novel DNA cross-linking domain. Electron microscopy indicates that HMGA1 proteins are able to create DNA loops and supercoils in linearized DNA confirming the cross-linking ability of HMGA1a. This capacity has profound implications for the spatial organization of DNA in the cell nucleus and suggests cross-linking activities for additional nuclear proteins. PMID:21596776

  20. Bifunctional Electrophiles Cross-Link Thioredoxins with Redox Relay Partners in Cells

    PubMed Central

    Naticchia, Matthew R.; Brown, Haley A.; Garcia, Francisco J.; Lamade, Andrew M.; Justice, Samantha L.; Herrin, Rachelle P.; Morano, Kevin A.; West, James D.

    2013-01-01

    Thioredoxin protects cells against oxidative damage by reducing disulfide bonds in improperly oxidized proteins. Previously, we found that the baker's yeast cytosolic thioredoxin Trx2 undergoes cross-linking to form several protein-protein complexes in cells treated with the bifunctional electrophile divinyl sulfone (DVSF). Here, we report that the peroxiredoxin Tsa1 and the thioredoxin reductase Trr1, both of which function in a redox relay network with thioredoxin, become cross-linked in complexes with Trx2 upon DVSF treatment. Treatment of yeast with other bifunctional electrophiles, including diethyl acetylenedicarboxylate (DAD), mechlorethamine (HN2), and 1,2,3,4-diepoxybutane (DEB), resulted in the formation of similar cross-linked complexes. Cross-linking of Trx2 and Tsa1 to other proteins by DVSF and DAD is dependent on modification of the active site Cys residues within these proteins. In addition, the human cytosolic thioredoxin, cytosolic thioredoxin reductase, and peroxiredoxin 2 form cross-linked complexes to other proteins in the presence of DVSF, although each protein shows different susceptibilities to modification by DAD, HN2, and DEB. Taken together, our results indicate that bifunctional electrophiles potentially disrupt redox homeostasis in yeast and human cells by forming cross-linked complexes between thioredoxins and their redox partners. PMID:23414292

  1. Antifouling coatings based on covalently cross-linked agarose film via thermal azide-alkyne cycloaddition.

    PubMed

    Xu, Li Qun; Pranantyo, Dicky; Neoh, Koon-Gee; Kang, En-Tang; Teo, Serena Lay-Ming; Fu, Guo Dong

    2016-05-01

    Coatings based on thin films of agarose-poly(ethylene glycol) (Agr-PEG) cross-linked systems are developed as environmentally-friendly and fouling-resistant marine coatings. The Agr-PEG cross-linked systems were prepared via thermal azide-alkyne cycloaddition (AAC) using azido-functionalized Agr (AgrAz) and activated alkynyl-containing poly(2-propiolamidoethyl methacrylate-co-poly(ethylene glycol)methyl ether methacrylate) P(PEMA-co-PEGMEMA) random copolymers as the precursors. The Agr-PEG cross-linked systems were further deposited onto a SS surface, pre-functionalized with an alkynyl-containing biomimetic anchor, dopamine propiolamide, to form a thin film after thermal treatment. The thin film-coated SS surfaces can effectively reduce the adhesion of marine algae and the settlement of barnacle cyprids. Upon covalent cross-linking, the covalently cross-linked Agr-PEG films coated SS surfaces exhibit good stability in flowing artificial seawater, and enhanced resistance to the settlement of barnacle cyprids, in comparison to that of the surfaces coated with physically cross-linked AgrAz films.

  2. Cross-linked chitosan improves the mechanical properties of calcium phosphate-chitosan cement.

    PubMed

    Aryaei, Ashkan; Liu, Jason; Jayatissa, Ahalapitiya H; Jayasuriya, A Champa

    2015-09-01

    Calcium phosphate (CaP) cements are highly applicable and valuable materials for filling bone defects by minimally invasive procedures. The chitosan (CS) biopolymer is also considered as one of the promising biomaterial candidates in bone tissue engineering. In the present study, some key features of CaP-CS were significantly improved by developing a novel CaP-CS composite. For this purpose, CS was the first cross-linked with tripolyphosphate (TPP) and then mixed with CaP matrix. A group of CaP-CS samples without cross-linking was also prepared. Samples were fabricated and tested based on the known standards. Additionally, the effect of different powder (P) to liquid (L) ratios was also investigated. Both cross-linked and uncross-linked CaP-CS samples showed excellent washout resistance. The most significant effects were observed on Young's modulus and compressive strength in wet condition as well as surface hardness. In dry conditions, the Young's modulus of cross-linked samples was slightly improved. Based on the presented results, cross-linking does not have a significant effect on porosity. As expected, by increasing the P/L ratio of a sample, ductility and injectability were decreased. However, in the most cases, mechanical properties were enhanced. The results have shown that cross-linking can improve the mechanical properties of CaP-CS and hence it can be used for bone tissue engineering applications.

  3. MARCKS actin-binding capacity mediates actin filament assembly during mitosis in human hepatic stellate cells.

    PubMed

    Rombouts, Krista; Mello, Tommaso; Liotta, Francesco; Galli, Andrea; Caligiuri, Alessandra; Annunziato, Francesco; Pinzani, Massimo

    2012-08-15

    Cross-linking between the actin cytoskeleton and plasma membrane actin-binding proteins is a key interaction responsible for the mechanical properties of the mitotic cell. Little is known about the identity, the localization, and the function of actin filament-binding proteins during mitosis in human hepatic stellate cells (hHSC). The aim of the present study was to identify and analyze the cross talk between actin and myristoylated alanine-rich kinase C substrate (MARCKS), an important PKC substrate and actin filament-binding protein, during mitosis in primary hHSC. Confocal analysis and chromosomal fraction analysis of mitotic hHSC demonstrated that phosphorylated (P)-MARCKS displays distinct phase-dependent localizations, accumulates at the perichromosomal layer, and is a centrosomal protein belonging to the chromosomal cytosolic fraction. Aurora B kinase (AUBK), an important mitotic regulator, β-actin, and P-MARCKS concentrate at the cytokinetic midbody during cleavage furrow formation. This localization is critical since MARCKS-depletion in hHSC is characterized by a significant loss in cytosolic actin filaments and cortical β-actin that induces cell cycle inhibition and dislocation of AUBK. A depletion of AUBK in hHSC affects cell cycle, resulting in multinucleation. Quantitative live cell imaging demonstrates that the actin filament-binding capacity of MARCKS is key to regulate mitosis since the cell cycle inhibitory effect in MARCKS-depleted cells caused abnormal cell morphology and an aberrant cytokinesis, resulting in a significant increase in cell cycle time. These findings implicate that MARCKS, an important PKC substrate, is essential for proper cytokinesis and that MARCKS and its partner actin are key mitotic regulators during cell cycle in hHSC.

  4. Actin restricts FcεRI diffusion and facilitates antigen-induced receptor immobilisation

    PubMed Central

    Andrews, Nicholas L.; Lidke, Keith A.; Pfeiffer, Janet R.; Burns, Alan R.; Wilson, Bridget S.; Oliver, Janet M.; Lidke, Diane S.

    2010-01-01

    The actin cytoskeleton has been implicated in restricting diffusion of plasma membrane components. Here, simultaneous observations of quantum dot-labelled FcεRI motion and GFP-tagged actin dynamics provide direct evidence that actin filament bundles define micron-sized domains that confine mobile receptors. Dynamic reorganisation of actin structures occurs over seconds, making the location and dimensions of actin-defined domains time dependent. Multiple FcεRI often maintain extended close proximity without detectable correlated motion, suggesting that they are co-confined within membrane domains. FcεRI signalling is activated by cross-linking with multivalent antigen. We show that receptors become immobilised within seconds of cross-linking. Disruption of the actin cytoskeleton results in delayed immobilisation kinetics and increased diffusion of cross-linked clusters. These results implicate actin in membrane partitioning that not only restricts diffusion of membrane proteins, but also dynamically influences their long-range mobility, sequestration, and response to ligand binding. PMID:18641640

  5. Elucidation for Electro-chemical Degradation of Cross-linked Polyethylene Insulated Power Cable

    NASA Astrophysics Data System (ADS)

    Miyashita, Yoshitsugu

    The water treeing phenomenon has been recognized as an ultimate difficulty for long-term service life of cross-linked polyethylene (XLPE) insulated power cables. Elucidation of substantial mechanistic pathway of water treeing is quite important to suppress water treeing. Many factors which affect water treeing have been found, such as electrical stress, existence of water, temperature and mechanical stress, and somewhat complicated synergestic interaction between them has also been pointed out. These factors make water treeing very difficult to be understood. The author studied the role of water in this phenomenon. To obtain certain analogical evidence for water treeing, a series of experiments was conducted by employing, as substituents of water, several organic solvents which possess relatively high dielectric constant and dissimilar electrochemical behavior, respectively. Consequently, it was found that a new type of treeing in XLPE matrix could be generated by certain organic solvent without water, and such treeing became easier to incept and propagate when the solvent contained particular electrolyte. Further, it was also indicated that cation was more effective than anion on such solvent treeing. Water tree inception and propagation mechanism was discussed from these experimental results.

  6. A single charge in the actin binding domain of fascin can independently tune the linear and non-linear response of an actin bundle network.

    PubMed

    Maier, M; Müller, K W; Heussinger, C; Köhler, S; Wall, W A; Bausch, A R; Lieleg, O

    2015-05-01

    Actin binding proteins (ABPs) not only set the structure of actin filament assemblies but also mediate the frequency-dependent viscoelastic moduli of cross-linked and bundled actin networks. Point mutations in the actin binding domain of those ABPs can tune the association and dissociation dynamics of the actin/ABP bond and thus modulate the network mechanics both in the linear and non-linear response regime. We here demonstrate how the exchange of a single charged amino acid in the actin binding domain of the ABP fascin triggers such a modulation of the network rheology. Whereas the overall structure of the bundle networks is conserved, the transition point from strain-hardening to strain-weakening sensitively depends on the cross-linker off-rate and the applied shear rate. Our experimental results are consistent both with numerical simulations of a cross-linked bundle network and a theoretical description of the bundle network mechanics which is based on non-affine bending deformations and force-dependent cross-link dynamics.

  7. Fascin regulates nuclear actin during Drosophila oogenesis

    PubMed Central

    Kelpsch, Daniel J.; Groen, Christopher M.; Fagan, Tiffany N.; Sudhir, Sweta; Tootle, Tina L.

    2016-01-01

    Drosophila oogenesis provides a developmental system with which to study nuclear actin. During Stages 5–9, nuclear actin levels are high in the oocyte and exhibit variation within the nurse cells. Cofilin and Profilin, which regulate the nuclear import and export of actin, also localize to the nuclei. Expression of GFP-tagged Actin results in nuclear actin rod formation. These findings indicate that nuclear actin must be tightly regulated during oogenesis. One factor mediating this regulation is Fascin. Overexpression of Fascin enhances nuclear GFP-Actin rod formation, and Fascin colocalizes with the rods. Loss of Fascin reduces, whereas overexpression of Fascin increases, the frequency of nurse cells with high levels of nuclear actin, but neither alters the overall nuclear level of actin within the ovary. These data suggest that Fascin regulates the ability of specific cells to accumulate nuclear actin. Evidence indicates that Fascin positively regulates nuclear actin through Cofilin. Loss of Fascin results in decreased nuclear Cofilin. In addition, Fascin and Cofilin genetically interact, as double heterozygotes exhibit a reduction in the number of nurse cells with high nuclear actin levels. These findings are likely applicable beyond Drosophila follicle development, as the localization and functions of Fascin and the mechanisms regulating nuclear actin are widely conserved. PMID:27535426

  8. Helical buckling of actin inside filopodia generates traction.

    PubMed

    Leijnse, Natascha; Oddershede, Lene B; Bendix, Poul M

    2015-01-06

    Cells can interact with their surroundings via filopodia, which are membrane protrusions that extend beyond the cell body. Filopodia are essential during dynamic cellular processes like motility, invasion, and cell-cell communication. Filopodia contain cross-linked actin filaments, attached to the surrounding cell membrane via protein linkers such as integrins. These actin filaments are thought to play a pivotal role in force transduction, bending, and rotation. We investigated whether, and how, actin within filopodia is responsible for filopodia dynamics by conducting simultaneous force spectroscopy and confocal imaging of F-actin in membrane protrusions. The actin shaft was observed to periodically undergo helical coiling and rotational motion, which occurred simultaneously with retrograde movement of actin inside the filopodium. The cells were found to retract beads attached to the filopodial tip, and retraction was found to correlate with rotation and coiling of the actin shaft. These results suggest a previously unidentified mechanism by which a cell can use rotation of the filopodial actin shaft to induce coiling and hence axial shortening of the filopodial actin bundle.

  9. Helical buckling of actin inside filopodia generates traction

    PubMed Central

    Leijnse, Natascha; Oddershede, Lene B.; Bendix, Poul M.

    2015-01-01

    Cells can interact with their surroundings via filopodia, which are membrane protrusions that extend beyond the cell body. Filopodia are essential during dynamic cellular processes like motility, invasion, and cell–cell communication. Filopodia contain cross-linked actin filaments, attached to the surrounding cell membrane via protein linkers such as integrins. These actin filaments are thought to play a pivotal role in force transduction, bending, and rotation. We investigated whether, and how, actin within filopodia is responsible for filopodia dynamics by conducting simultaneous force spectroscopy and confocal imaging of F-actin in membrane protrusions. The actin shaft was observed to periodically undergo helical coiling and rotational motion, which occurred simultaneously with retrograde movement of actin inside the filopodium. The cells were found to retract beads attached to the filopodial tip, and retraction was found to correlate with rotation and coiling of the actin shaft. These results suggest a previously unidentified mechanism by which a cell can use rotation of the filopodial actin shaft to induce coiling and hence axial shortening of the filopodial actin bundle. PMID:25535347

  10. Chemical cross-linking of bovine retinal transducin and cGMP phosphodiesterase.

    PubMed

    Hingorani, V N; Tobias, D T; Henderson, J T; Ho, Y K

    1988-05-15

    The bifunctional reagents para-phenyldimaleimide and maleimidobenzoyl-N-hydroxysuccinimide ester were used to chemically cross-link the subunits of the transducin and cGMP phosphodiesterase (PDE) complexes of bovine rod photoreceptor cells. The cross-linked products were identified by Western immunoblotting using antisera against purified subunits of transducin (T alpha and T beta gamma) and PDE. Oligomeric cross-linked products of transducin subunits as large as (T alpha beta gamma)3 were observed in the latent form of transducin with bound GDP. In addition to the expected T alpha beta and T beta gamma cross-linked products, a (T alpha gamma)2 structure was detected. The close proximity of T alpha and T gamma suggests that T gamma may play a role in conferring the specificity of the interaction between T alpha and rhodopsin. Most of the oligomeric cross-linked structures between T alpha and T beta gamma were diminished in the activated form of transducin, with guanosine 5'-(beta, gamma-imidotriphosphate) (Gpp(NH)p) bound. However, cross-linking between T beta and T gamma was not altered. These results suggest that transducin exists as an oligomer in solution which dissociates upon the binding of Gpp(NH)p. To identify the possible interacting domains between the T alpha, T beta, and T gamma subunits, the cross-linked products were subjected to limited tryptic proteolysis. Several cross-linked tryptic peptides of transducin subunits were found and include the cross-linked products of the N terminus 15-kDa fragment of T beta and the C terminus 5-kDa fragment of T alpha, T gamma and the 12-kDa fragment of T alpha, T gamma and the 15-kDa as well as the 23-kDa fragments of T beta, and an intra-T alpha cross-linked product of the 2- and 21-kDa fragments. These results have allowed the construction of a topographical model for the transducin subunits. The organization of the subunits of PDE (P alpha, P beta, and P gamma) was also studied. The formation of the high

  11. Solid lipid nanoparticles coated with cross-linked polymeric double layer for oral delivery of curcumin.

    PubMed

    Wang, Taoran; Ma, Xiaoyu; Lei, Yu; Luo, Yangchao

    2016-12-01

    Solid lipid nanoparticles (SLNs) are regarded as promising carriers to improve the safety and effectiveness of delivery for drugs and nutrients, however, the clinic applications for oral administration are limited by their poor stability in gastrointestinal conditions. In this study, surface modification was explored to confer new physicochemical properties to SLNs and thus achieve enhanced functionalities. Novel SLNs with biopolymeric double layer (DL) coating using two natural biopolymers, i.e. caseinate (NaCas) and pectin, were prepared to encapsulate and deliver curcumin, a lipophilic bioactive compound studied as a model drug/nutrient. The DL coating was chemically cross-linked by creating covalent bonds between NaCas and pectin, using two different cross-linkers, i.e. glutaraldehyde (GA) and 1-Ethyl-3-(3-dimethylaminopropyl) carbodiimide/N-Hydroxysuccinimide (EDC/NHS). Prior to cross-linking, the mean particle size, polydispersity index and zeta potential of DL-SLNs were 300-330nm, 0.25-0.30, -45-40mV, respectively. It was found that cross-linking with GA had a more prominent effect on particle size and polydispersity index than EDC/NHS. The cross-linking process significantly improved physicochemical properties of DL-SLNs, resulting in higher encapsulation efficiency and loading capacity, better stability and slower release profile in simulated gastrointestinal conditions. Particularly, an optimal zero-order release kinetic was observed for EDC/NHS crosslinked DL-SLNs. The electron microscopy revealed that both cross-linked DL-SLNs exhibited spherical shape with homogeneous size and smooth surface. Encapsulation of curcumin in SLNs dramatically enhanced its antioxidant activity in aqueous condition. The cross-linking process further helped spray drying of SLNs by forming homogenous powder particles. These results indicated that coating with cross-linked polymers could significantly improve the physicochemical properties of SLNs and expand their potentials as

  12. Biodegradation of differently cross-linked collagen membranes: an experimental study in the rat.

    PubMed

    Rothamel, Daniel; Schwarz, Frank; Sager, Martin; Herten, Monika; Sculean, Anton; Becker, Jürgen

    2005-06-01

    The aim of the present study was to compare the biodegradation of differently cross-linked collagen membranes in rats. Five commercially available and three experimental membranes (VN) were included: (1) BioGide (BG) (non-cross-linked porcine type I and III collagens), (2) BioMend (BM), (3) BioMendExtend (BME) (glutaraldehyde cross-linked bovine type I collagen), (4) Ossix (OS) (enzymatic-cross-linked bovine type I collagen), (5) TutoDent (TD) (non-cross-linked bovine type I collagen, and (6-8) VN(1-3) (chemical cross-linked porcine type I and III collagens). Specimens were randomly allocated in unconnected subcutaneous pouches separated surgically on the back of 40 wistar rats, which were divided into five groups (2, 4, 8, 16, and 24 weeks), including eight animals each. After 2, 4, 8, 16, and 24 weeks of healing, the rats were sacrificed and explanted specimens were prepared for histologic and histometric analysis. The following parameters were evaluated: biodegradation over time, vascularization, tissue integration, and foreign body reaction. Highest vascularization and tissue integration was noted for BG followed by BM, BME, and VN(1); TD, VN(2), and VN(3) showed prolongated, while OS exhibited no vascularization. Subsequently, biodegradation of BG, BM, BME and VN(1) was faster than TD, VN(2), and VN(3). OS showed only a minute amount of superficial biodegradation 24 weeks following implantation. Biodegradation of TD, BM, BME, VN(2), and VN(3) was associated with the presence of inflammatory cells. Within the limits of the present study, it was concluded that cross-linking of bovine and porcine-derived collagen types I and III was associated with (i) prolonged biodegradation, (ii) decreased tissue integration and vascularization, and (iii) in case of TD, BM, BME, VN(2), and VN(3) foreign body reactions.

  13. Conformational Transitions of the Cross-linking Domains of Elastin during Self-assembly*

    PubMed Central

    Reichheld, Sean E.; Muiznieks, Lisa D.; Stahl, Richard; Simonetti, Karen; Sharpe, Simon; Keeley, Fred W.

    2014-01-01

    Elastin is the intrinsically disordered polymeric protein imparting the exceptional properties of extension and elastic recoil to the extracellular matrix of most vertebrates. The monomeric precursor of elastin, tropoelastin, as well as polypeptides containing smaller subsets of the tropoelastin sequence, can self-assemble through a colloidal phase separation process called coacervation. Present understanding suggests that self-assembly is promoted by association of hydrophobic domains contained within the tropoelastin sequence, whereas polymerization is achieved by covalent joining of lysine side chains within distinct alanine-rich, α-helical cross-linking domains. In this study, model elastin polypeptides were used to determine the structure of cross-linking domains during the assembly process and the effect of sequence alterations in these domains on assembly and structure. CD temperature melts indicated that partial α-helical structure in cross-linking domains at lower temperatures was absent at physiological temperature. Solid-state NMR demonstrated that β-strand structure of the cross-linking domains dominated in the coacervate state, although α-helix was predominant after subsequent cross-linking of lysine side chains with genipin. Mutation of lysine residues to hydrophobic amino acids, tyrosine or alanine, leads to increased propensity for β-structure and the formation of amyloid-like fibrils, characterized by thioflavin-T binding and transmission electron microscopy. These findings indicate that cross-linking domains are structurally labile during assembly, adapting to changes in their environment and aggregated state. Furthermore, the sequence of cross-linking domains has a dramatic effect on self-assembly properties of elastin-like polypeptides, and the presence of lysine residues in these domains may serve to prevent inappropriate ordered aggregation. PMID:24550393

  14. Click Cross-Linking-Improved Waterborne Polymers for Environment-Friendly Coatings and Adhesives.

    PubMed

    Hu, Jianqing; Peng, Kaimei; Guo, Jinshan; Shan, Dingying; Kim, Gloria B; Li, Qiyao; Gerhard, Ethan; Zhu, Liang; Tu, Weiping; Lv, Weizhong; Hickner, Michael A; Yang, Jian

    2016-07-13

    Waterborne polymers, including waterborne polyurethanes (WPU), polyester dispersions (PED), and polyacrylate emulsions (PAE), are employed as environmentally friendly water-based coatings and adhesives. An efficient, fast, stable, and safe cross-linking strategy is always desirable to impart waterborne polymers with improved mechanical properties and water/solvent/thermal and abrasion resistance. For the first time, click chemistry was introduced into waterborne polymer systems as a cross-linking strategy. Click cross-linking rendered waterborne polymer films with significantly improved tensile strength, hardness, adhesion strength, and water/solvent resistance compared to traditional waterborne polymer films. For example, click cross-linked WPU (WPU-click) has dramatically improved the mechanical strength (tensile strength increased from 0.43 to 6.47 MPa, and Young's modulus increased from 3 to 40 MPa), hardness (increased from 59 to 73.1 MPa), and water resistance (water absorption percentage dropped from 200% to less than 20%); click cross-linked PED (PED-click) film also possessed more than 3 times higher tensile strength (∼28 MPa) than that of normal PED (∼8 MPa). The adhesion strength of click cross-linked PAE (PAE-click) to polypropylene (PP) was also improved (from 3 to 5.5 MPa). In addition, extra click groups can be preserved after click cross-linking for further functionalization of the waterborne polymeric coatings/adhesives. In this work, we have demonstrated that click modification could serve as a convenient and powerful approach to significantly improve the performance of a variety of traditional coatings and adhesives.

  15. In situ forming chitosan hydrogels prepared via ionic/covalent co-cross-linking.

    PubMed

    Moura, M José; Faneca, H; Lima, M Pedroso; Gil, M Helena; Figueiredo, M Margarida

    2011-09-12

    In situ forming chitosan hydrogels have been prepared via coupled ionic and covalent cross-linking. Thus, different amounts of genipin (0.05, 0.10, 0.15, and 0.20% (w/w)), used as a chemical cross-linker, were added to a solution of chitosan that was previously neutralized with a glycerol-phosphate complex (ionic cross-linker). In this way, it was possible to overcome the pH barrier of the chitosan solution, to preserve its thermosensitive character, and to enhance the extent of cross-linking in the matrix simultaneously. To investigate the contributions of the ionic cross-linking and the chemical cross-linking, separately, we prepared the hydrogels without the addition of either genipin or the glycerol-phosphate complex. The addition of genipin to the neutralized solution disturbs the ionic cross-linking process and the chemical cross-linking becomes the dominant process. Moreover, the genipin concentration was used to modulate the network structure and performance. The more promising formulations were fully characterized, in a hydrated state, with respect to any equilibrium swelling, the development of internal structure, the occurrence of in vitro degradability and cytotoxicity, and the creation of in vivo injectability. Each of the hydrogel systems exhibited a notably high equilibrium water content, arising from the fact that their internal structure (examined by conventional SEM, and environmental SEM) was highly porous with interconnecting pores. The porosity and the pore size distribution were quantified by mercury intrusion porosimetry. Although all gels became degraded in the presence of lysozyme, their degradation rate greatly depended on the genipin load. Through in vitro viability tests, the hydrogel-based formulations were shown to be nontoxic. The in vivo injection of a co-cross-linking formulation revealed that the gel was rapidly formed and localized at the injection site, remaining in position for at least 1 week.

  16. Cross-linking of wheat gluten proteins during production of hard pretzels.

    PubMed

    Rombouts, Ine; Lagrain, Bert; Brijs, Kristof; Delcour, Jan A

    2012-06-01

    The impact of the hot alkaline dip, prior to pretzel-baking, on the types and levels of cross-links between wheat proteins was studied. Protein extractability of pretzel dough in sodium dodecyl sulfate containing buffer decreased during alkaline dipping [45 s, 1.0% (w/v) NaOH, 90°C], and even more during baking (3 min at 250°C) and drying (10 min at 135°C). Reducing agent increased the extractability partly, indicating that both reducible (disulfide, SS) and non-reducible (non-SS) protein cross-links had been formed. The decrease in cystine levels suggested β-elimination of cystine releasing Cys and dehydroalanine (DHA). Subsequent reaction of DHA with Lys and Cys, induced the unusual and potentially cross-linking amino acids lysinoalanine (LAL) and lanthionine (LAN), respectively, in alkaline dipped dough (7 μmol LAN/g protein) and in the end product (9 μmol LAL and 50 μmol LAN/g protein). The baking/drying step increased sample redness, decreased Lys levels more than expected based on LAL formation (57 μmol/g protein), and induced a loss of reducing sugars (99 μmol/g protein), which suggested the potential contribution of Maillard-derived cross-links to the observed extractability loss. However, levels of Maillard products which possibly cross-link proteins, are small compared to DHA-derived cross-links. Higher dipping temperatures, longer dipping times, and higher NaOH concentrations increased protein extractability losses and redness, as well as LAL and LAN levels in the end product. No indications for Maillard-derived cross-links or LAL in pretzel dough immediately after dipping were found, even when severe dipping conditions were used.

  17. Preparation and characterization of resistant starch type IV nanoparticles through ultrasonication and miniemulsion cross-linking.

    PubMed

    Ding, Yongbo; Zheng, Jiong; Xia, Xuejuan; Ren, Tingyuan; Kan, Jianquan

    2016-05-05

    This study aimed to assess the properties of resistant starch type IV (chemically modified starch, RS4) prepared from a new and convenient synthesis route by using ultrasonication combined with water-in-oil miniemulsion cross-linking technique. A three-factor Box-Behnken design and optimization was used to minimize particle size through the developed RS4 nanoparticles. The predicted minimized Z-Avel (576.1nm) under the optimum conditions of the process variables (ultrasonic power, 214.57W; sonication time, 114.73min; and oil/water ratio, 10.54:1) was very close to the experimental value (651.0nm) determined in a batch experiment. After preparing the RS4 nanoparticles, morphological, physical, chemical, and functional properties were assessed. Results revealed that RS4 nanoparticle size reached about 600nm. Scanning electron microscopy images showed that ultrasonication induced notches and grooves on the surface. Under polarized light, the polarized cross was impaired. X-ray diffraction results revealed that the crystalline structure was disrupted. Smaller or no endotherms were exhibited in DSC analysis. In the FTIR graph, new peaks at 1532.91 and 1451.50cm(-1) were observed, and pasting properties were reduced. Amylose content, solubility, and SP increased, but RS content decreased. Anti-digestibility remained after ultrasonication. The prepared RS4 nanoparticles could be extensively used in biomedical applications and in the development of new medical materials.

  18. Optimization of β-cyclodextrin cross-linked polymer for monitoring of quercetin

    NASA Astrophysics Data System (ADS)

    Zhu, Xiashi; Ping, Wenhui

    2014-11-01

    A novel method for the separation/analysis of quercetin was described, which was based on the investigation of the inclusion interactions of β-cyclodextrin cross-linked polymer (β-CDCP) with quercetin (Qu) and the adsorption behavior of Qu on β-CDCP. The inclusion interaction of β-CDCP with Qu was studied through FTIR, TGA and 13C NMR. Under the optimum conditions, the preconcentration factor of the proposed method was approximately 8.8, the β-CDCP could be used repeatedly for 30 times and offered better recovery. The linear range, limit of detection (LOD) and the relative standard deviation (RSD) was found to be 0.10-12.0 μg mL-1, 4.6 ng mL-1 and 3.10% (n = 3, c = 2.0 μg mL-1) respectively. This technique had been successfully applied to the determination of Qu in real samples.

  19. Biomimetic hydration lubrication with various polyelectrolyte layers on cross-linked polyethylene orthopedic bearing materials.

    PubMed

    Kyomoto, Masayuki; Moro, Toru; Saiga, Kenichi; Hashimoto, Masami; Ito, Hideya; Kawaguchi, Hiroshi; Takatori, Yoshio; Ishihara, Kazuhiko

    2012-06-01

    Natural joints rely on fluid thin-film lubrication by the hydrated polyelectrolyte layer of cartilage. However, current artificial joints with polyethylene (PE) surfaces have considerably less efficient lubrication and thus much greater wear, leading to osteolysis and aseptic loosening. This is considered a common factor limiting prosthetic longevity in total hip arthroplasty (THA). However, such wear could be mitigated by surface modification to mimic the role of cartilage. Here we report the development of nanometer-scale hydrophilic layers with varying charge (nonionic, cationic, anionic, or zwitterionic) on cross-linked PE (CLPE) surfaces, which could fully mimic the hydrophilicity and lubricity of the natural joint surface. We present evidence to support two lubrication mechanisms: the primary mechanism is due to the high level of hydration in the grafted layer, where water molecules act as very efficient lubricants; and the secondary mechanism is repulsion of protein molecules and positively charged inorganic ions by the grafted polyelectrolyte layer. Thus, such nanometer-scaled hydrophilic polymers or polyelectrolyte layers on the CLPE surface of acetabular cup bearings could confer high durability to THA prosthetics.

  20. Superamphiphobic surfaces constructed by cross-linked hollow SiO2 spheres

    NASA Astrophysics Data System (ADS)

    Cui, Weihao; Wang, Tao; Yan, Aili; Wang, Sheng

    2017-04-01

    By using stringed carbon spheres as template material, a series of hierarchical 3D cross-linked SiO2 coated carbon spheres and hollow SiO2 spheres were fabricated, and spray-coated on glass slides, followed by the fluorination treatment with per-fluorotrichlorosilane. The surface characterization and surface wettability data indicated that hollow SiO2 spheres spray-coated surfaces showed better superhydrophobicity and superoleophobcity properties than the corresponding solid C@SiO2 coated surface. This study further demonstrated that superamphiphobicity depends on two critical factors, namely a cavity- and spot-rich hierarchical structure and the size and shape of overhangs. Moreover, the optimal conditions for the preparation of hollow SiO2 coated glass slide were identified after a systematic investigation of various concentrations of the carbon precursor and tetraethylorthosilicate. It was found that when 0.10 g carbon spheres prepared from 1.0 M carbon precursor were used as the template and 20 mg/mL tetraethylorthosilicate was used as silica precursor, the hollow SiO2 coated glass slide exhibited the best superamphiphobic performance, with the highest contact angles and lowest sliding angles for various liquids, such as water, olive oil, n-hexadecane and n-dodecane.

  1. Novel enzymatically cross-linked hyaluronan hydrogels support the formation of 3D neuronal networks.

    PubMed

    Broguiere, Nicolas; Isenmann, Luca; Zenobi-Wong, Marcy

    2016-08-01

    Hyaluronan (HA) is an essential component of the central nervous system's extracellular matrix and its high molecular weight (MW) form has anti-inflammatory and anti-fibrotic properties relevant for regenerative medicine. Here, we introduce a new hydrogel based on high MW HA which is cross-linked using the transglutaminase (TG) activity of the activated blood coagulation factor XIII (FXIIIa). These HA-TG gels have significant advantages for neural tissue engineering compared to previous HA gels. Due to their chemical inertness in the absence of FXIIIa, the material can be stored long-term, is stable in solution, and shows no cytotoxicity. The gelation is completely cell-friendly due to the specificity of the enzyme and the gelation rate can be tuned from seconds to hours at physiological pH and independently of stiffness. The gels are injectable, and attach covalently to fibrinogen and fibrin, two common bioactive components in in vitro tissue engineering, as well as proteins present in vivo, allowing the gels to covalently bind to brain or spinal cord defects. These optimal chemical and bioactive properties of HA-TG gels enabled the formation of 3D neuronal cultures of unprecedented performance, showing fast neurite outgrowth, axonal and dendritic speciation, strong synaptic connectivity in 3D networks, and rapidly-occurring and long-lasting coordinated electrical activity.

  2. DNA Interstrand Cross-Linking Activity of (1-Chloroethenyl)oxirane, a Metabolite of β-chloroprene

    PubMed Central

    Wadugu, Brian A.; Ng, Christopher; Bartley, Bethany L.; Rowe, Rebecca J.; Millard, Julie T.

    2010-01-01

    With the goal of elucidating the molecular and cellular mechanisms of chloroprene toxicity, we examined the potential DNA cross-linking of the bifunctional chloroprene metabolite, (1-chloroethenyl)oxirane (CEO). We used denaturing polyacrylamide gel electrophoresis to monitor possible formation of interstrand cross-links by CEO within synthetic DNA duplexes. Our data suggest interstrand cross-linking at deoxyguanosine residues within 5′-GC and 5′-GGC sites, with the rate of cross-linking depending on pH (pH 5.0 > pH 6.0 > pH 7.0). A comparison of the cross-linking efficiencies of CEO and the structurally similar cross-linkers diepoxybutane (DEB) and epichlorohydrin (ECH) revealed that DEB > CEO ≥ ECH. Furthermore, we found that cytotoxicity correlates with cross-linking efficiency, supporting a role for interstrand cross-links in the genotoxicology of chloroprene. PMID:20030381

  3. cAMP-induced actin cytoskeleton remodelling inhibits MKL1-dependent expression of the chemotactic and pro-proliferative factor, CCN1

    PubMed Central

    Duggirala, Aparna; Kimura, Tomomi E.; Sala-Newby, Graciela B.; Johnson, Jason L.; Wu, Yih-Jer; Newby, Andrew C.; Bond, Mark

    2015-01-01

    Elevation of intracellular cAMP concentration has numerous vascular protective effects that are in part mediated via actin cytoskeleton-remodelling and subsequent regulation of gene expression. However, the mechanisms are incompletely understood. Here we investigated whether cAMP-induced actin-cytoskeleton remodelling modulates VSMC behaviour by inhibiting expression of CCN1. In cultured rat VSMC, CCN1-silencing significantly inhibited BrdU incorporation and migration in a wound healing assay. Recombinant CCN1 enhanced chemotaxis in a Boyden chamber. Adding db-cAMP, or elevating cAMP using forskolin, significantly inhibited CCN1 mRNA and protein expression in vitro; transcriptional regulation was demonstrated by measuring pre-spliced CCN1 mRNA and CCN1-promoter activity. Forskolin also inhibited CCN1 expression in balloon injured rat carotid arteries in vivo. Inhibiting RhoA activity, which regulates actin-polymerisation, by cAMP-elevation or pharmacologically with C3-transferase, or inhibiting its downstream kinase, ROCK, with Y27632, significantly inhibited CCN1 expression. Conversely, expression of constitutively active RhoA reversed the inhibitory effects of forskolin on CCN1 mRNA. Furthermore, CCN1 mRNA levels were significantly decreased by inhibiting actin-polymerisation with latrunculin B or increased by stimulating actin-polymerisation with Jasplakinolide. We next tested the role of the actin-dependent SRF co-factor, MKL1, in CCN1 expression. Forskolin inhibited nuclear translocation of MKL1 and binding of MKL1 to the CCN1 promoter. Constitutively-active MKL1 enhanced basal promoter activity of wild-type but not SRE-mutated CCN1; and prevented forskolin inhibition. Furthermore, pharmacological MKL-inhibition with CCG-1423 significantly inhibited CCN1 promoter activity as well as mRNA and protein expression. Our data demonstrates that cAMP-induced actin-cytoskeleton remodelling regulates expression of CCN1 through MKL1: it highlights a novel c

  4. cAMP-induced actin cytoskeleton remodelling inhibits MKL1-dependent expression of the chemotactic and pro-proliferative factor, CCN1.

    PubMed

    Duggirala, Aparna; Kimura, Tomomi E; Sala-Newby, Graciela B; Johnson, Jason L; Wu, Yih-Jer; Newby, Andrew C; Bond, Mark

    2015-02-01

    Elevation of intracellular cAMP concentration has numerous vascular protective effects that are in part mediated via actin cytoskeleton-remodelling and subsequent regulation of gene expression. However, the mechanisms are incompletely understood. Here we investigated whether cAMP-induced actin-cytoskeleton remodelling modulates VSMC behaviour by inhibiting expression of CCN1. In cultured rat VSMC, CCN1-silencing significantly inhibited BrdU incorporation and migration in a wound healing assay. Recombinant CCN1 enhanced chemotaxis in a Boyden chamber. Adding db-cAMP, or elevating cAMP using forskolin, significantly inhibited CCN1 mRNA and protein expression in vitro; transcriptional regulation was demonstrated by measuring pre-spliced CCN1 mRNA and CCN1-promoter activity. Forskolin also inhibited CCN1 expression in balloon injured rat carotid arteries in vivo. Inhibiting RhoA activity, which regulates actin-polymerisation, by cAMP-elevation or pharmacologically with C3-transferase, or inhibiting its downstream kinase, ROCK, with Y27632, significantly inhibited CCN1 expression. Conversely, expression of constitutively active RhoA reversed the inhibitory effects of forskolin on CCN1 mRNA. Furthermore, CCN1 mRNA levels were significantly decreased by inhibiting actin-polymerisation with latrunculin B or increased by stimulating actin-polymerisation with Jasplakinolide. We next tested the role of the actin-dependent SRF co-factor, MKL1, in CCN1 expression. Forskolin inhibited nuclear translocation of MKL1 and binding of MKL1 to the CCN1 promoter. Constitutively-active MKL1 enhanced basal promoter activity of wild-type but not SRE-mutated CCN1; and prevented forskolin inhibition. Furthermore, pharmacological MKL-inhibition with CCG-1423 significantly inhibited CCN1 promoter activity as well as mRNA and protein expression. Our data demonstrates that cAMP-induced actin-cytoskeleton remodelling regulates expression of CCN1 through MKL1: it highlights a novel c

  5. Vitamin E-diffused highly cross-linked UHMWPE particles induce less osteolysis compared to highly cross-linked virgin UHMWPE particles in vivo.

    PubMed

    Bichara, David A; Malchau, Erik; Sillesen, Nanna H; Cakmak, Selami; Nielsen, G Petur; Muratoglu, Orhun K

    2014-09-01

    Recent in vitro findings suggest that UHMWPE wear particles containing vitamin E (VE) may have reduced biologic activity and decreased osteolytic potential. We hypothesized that particles from VE-stabilized, radiation cross-linked UHMWPE would cause less osteolysis in a murine calvarial bone model when compared to virgin gamma irradiated cross-linked UHMWPE. Groups received equal amount of particulate debris overlaying the calvarium for 10 days. Calvarial bone was examined using high resolution micro-CT and histomorphometric analyses. There was a statistically significant difference between virgin (12.2%±8%) and VE-UHMWPE (3%±1.4%) groups in regards to bone resorption (P=0.005) and inflammatory fibrous tissue overlaying the calvaria (0.48 vs. 0.20, P<0.0001). These results suggest that VE-UHMWPE particles have reduced osteolytic potential in vivo when compared to virgin UHMWPE.

  6. Investigation of the role of aromatic carboxylic acids in cross-linking processes in low-rank coals

    SciTech Connect

    Eskay, T.P.; Britt, P.F.; Buchanan, A.C. III

    1997-03-01

    In the pyrolysis and liquefaction of low-rank coals, low-temperature cross-linking reactions have been correlated with the loss of carboxyl groups and the evolution of CO{sub 2} and H{sub 2}O. It is not clearly understood how decarboxylation leads to cross-linking beyond the suggestion that decarboxylation could be a radical process that involves radical recombination or radical addition reactions. We have recently conducted a study of the pyrolysis of 1,2-(3,3{prime}-dicarboxyphenyl)ethane (1) and 1,2-(4,4{prime}-dicarboxyphenyl)ethane (2) and found that decarboxylation occurs readily between 350-425 {degrees}C with no evidence of coupling products or products representative of cross-links. We proposed that decarboxylation occurred primarily by an acid-promoted cationic pathway, and the source of acid was a second carboxylic acid. The decarboxylation of 1 and 2 was investigated in diphenyl ether and naphthalene as inert diluents. In each solvent, the rate of decarboxylation dropped by roughly a factor of 2 upon dilution from the neat liquid to ca. 0.4 mole fraction of acid, but further dilution had no effect on the rate. This could be a consequence of hydrogen bonding or an intramolecular protonation. Molecular mechanics calculations indicated that 1 and 2 can adopt an appropriate conformation for internal proton transfer from a carboxy group on one ring to the second aryl ring without a significant energy penalty. In addition, the dicarboxylic acid could internally hydrogen bond, which may further complicate the reaction mechanism. Therefore, we have conducted a study of the pyrolysis of a monocarboxybibenzyl, 1-(3-carboxyphenyl)-2-(4-biphenyl)ethane (3), to determine if decarboxylation occurs by an ionic pathway in the absence of intramolecular pathways.

  7. Fabrication of homogeneously cross-linked, functional alginate microcapsules validated by NMR-, CLSM- and AFM-imaging.

    PubMed

    Zimmermann, H; Hillgärtner, M; Manz, B; Feilen, P; Brunnenmeier, F; Leinfelder, U; Weber, M; Cramer, H; Schneider, S; Hendrich, C; Volke, F; Zimmermann, U

    2003-05-01

    Cross-linked alginate microcapsules of sufficient mechanical strength can immunoisolate cells for the long-term treatment of hormone and other deficiency diseases in human beings. However, gelation of alginate by external Ba(2+) (or other divalent cations) produces non-homogeneous cross-linking of the polymeric mannuronic (M) and guluronic (G) acid chains. The stability of such microcapsules is rather limited. Here, we show that homogeneous cross-linking can be achieved by injecting BaCl(2) crystals into alginate droplets before they come into contact with external BaCl(2). The high effectiveness of this crystal gun method is demonstrated by confocal laser scanning microscopy and by advanced nuclear magnetic resonance imaging. Both techniques gave clear-cut evidence that homogeneous cross-linkage throughout the microcapsule is only obtained with simultaneous internal and external gelation. Atomic force microscopy showed a very smooth surface topography for microcapsules made by the crystal gun method, provided that excess Ba(2+) ions were removed immediately after gelation. In vitro experiments showed greatly suppressed swelling for crystal gun microcapsules. Even alginate extracted from Lessonia nigrescens (highly biocompatible) yielded microcapsules with long-term mechanical stability not hitherto possible. Encapsulation of rat islets, human monoclonal antibodies secreting hybridoma cells and murine mesenchymal stem cells transfected with cDNA encoding for bone morphogenetic protein (BMP-4) revealed that injection of BaCl(2) crystals has no adverse side effects on cell viability and function. However, the release of low-molecular weight factors (such as insulin) may be delayed when using alginate concentrations in the usual range.

  8. Dissecting the Mechanisms of Tissue Transglutaminase-induced Cross-linking of α-Synuclein

    PubMed Central

    Schmid, Adrien W.; Chiappe, Diego; Pignat, Vérène; Grimminger, Valerie; Hang, Ivan; Moniatte, Marc; Lashuel, Hilal A.

    2009-01-01

    Tissue transglutaminase (tTG) has been implicated in the pathogenesis of Parkinson disease (PD). However, exactly how tTG modulates the structural and functional properties of α-synuclein (α-syn) and contributes to the pathogenesis of PD remains unknown. Using site-directed mutagenesis combined with detailed biophysical and mass spectrometry analyses, we sought to identify the exact residues involved in tTG-catalyzed cross-linking of wild-type α-syn and α-syn mutants associated with PD. To better understand the structural consequences of each cross-linking reaction, we determined the effect of tTG-catalyzed cross-linking on the oligomerization, fibrillization, and membrane binding of α-syn in vitro. Our findings show that tTG-catalyzed cross-linking of monomeric α-syn involves multiple cross-links (specifically 2-3). We subjected tTG-catalyzed cross-linked monomeric α-syn composed of either wild-type or Gln → Asn mutants to sequential proteolysis by multiple enzymes and peptide mapping by mass spectrometry. Using this approach, we identified the glutamine and lysine residues involved in tTG-catalyzed intramolecular cross-linking of α-syn. These studies demonstrate for the first time that Gln79 and Gln109 serve as the primary tTG reactive sites. Mutating both residues to asparagine abolishes tTG-catalyzed cross-linking of α-syn and tTG-induced inhibition of α-syn fibrillization in vitro. To further elucidate the sequence and structural basis underlying these effects, we identified the lysine residues that form isopeptide bonds with Gln79 and Gln109. This study provides mechanistic insight into the sequence and structural basis of the inhibitory effects of tTG on α-syn fibrillogenesis in vivo, and it sheds light on the potential role of tTG cross-linking on modulating the physiological and pathogenic properties of α-syn. PMID:19164286

  9. Improving the mechanical and thermal properties of gelatin hydrogels cross-linked by cellulose nanowhiskers.

    PubMed

    Dash, Rajalaxmi; Foston, Marcus; Ragauskas, Arthur J

    2013-01-16

    This study demonstrates the preparation of a renewable and biocompatible hydrogel with superior mechanical properties consisting of a gelatin matrix cross-linked with oxidized cellulose nanowhiskers. We found an increased degree of chemical cross-linking (0.14-17%) between gelatin and nanowhiskers with the increased amount of aldehyde contents (0.062-0.230mmolg(-1)). (1)H nuclear magnetic resonance (NMR) T(2) relaxation experiments on D(2)O swollen hydrogels demonstrated systems consisting of both gelatin and cellulose nanowhiskers displayed a higher percentage of "ridge" protons, attributed in part to increasing chemical cross-linking junction points between gelatin and nanowhiskers. This increase in hydrogel rigidity not only modified local chain dynamics but also influenced gel swelling, showing relatively reduced water uptake ability than that of the neat gelatin. Rheological measurements confirmed a 150% improvement in storage modulus (G') of the cross-linked hydrogels compared to neat gelatin. Chemical cross-linking also increased the resistance of the gels towards thermal degradation above the melting temperature of gelatin as observed by thermal scanning experiments.

  10. Tyrosine-Selective Functionalization for Bio-Orthogonal Cross-Linking of Engineered Protein Hydrogels.

    PubMed

    Madl, Christopher M; Heilshorn, Sarah C

    2017-02-02

    Engineered protein hydrogels have shown promise as artificial extracellular matrix materials for the 3D culture of stem cells due to the ability to decouple hydrogel biochemistry and mechanics. The modular design of these proteins allows for incorporation of various bioactive sequences to regulate cellular behavior. However, the chemistry used to cross-link the proteins into hydrogels can limit what bioactive sequences can be incorporated, in order to prevent nonspecific cross-linking within the bioactive region. Bio-orthogonal cross-linking chemistries may allow for the incorporation of any arbitrary bioactive sequence, but site-selective and scalable incorporation of bio-orthogonal reactive groups such as azides that do not rely on commonly used amine-reactive chemistry is often challenging. In response, we have optimized the reaction of an azide-bearing 4-phenyl-1,2,4-triazoline-3,5-dione (PTAD) with engineered elastin-like proteins (ELPs) to selectively azide-functionalize tyrosine residues within the proteins. The PTAD-azide functionalized ELPs cross-link with bicyclononyne (BCN) functionalized ELPs via the strain-promoted azide-alkyne cycloaddition (SPAAC) reaction to form hydrogels. Human mesenchymal stem cells and murine neural progenitor cells encapsulated within these hydrogels remain highly viable and maintain their phenotypes in culture. Tyrosine-specific modification may expand the number of bioactive sequences that can be designed into protein-engineered materials by permitting incorporation of lysine-containing sequences without concern for nonspecific cross-linking.

  11. Ice templated and cross-linked xylan/nanocrystalline cellulose hydrogels.

    PubMed

    Köhnke, Tobias; Elder, Thomas; Theliander, Hans; Ragauskas, Arthur J

    2014-01-16

    Structured xylan-based hydrogels, reinforced with cellulose nanocrystals (CNCs), have successfully been prepared from water suspensions by cross-linking during freeze-casting. In order to induce cross-linking during the solidification/sublimation operation, xylan was first oxidized using sodium periodate to introduce dialdehydes. The oxidized xylan was then mixed with CNCs after which the suspension was frozen unidirectionally in order to control the ice crystal formation and by that the pore morphology of the material. Finally the ice crystal templates were removed by freeze-drying. During the freeze-casting process hemiacetal bonds are formed between the aldehyde groups and hydroxyl groups, either on other xylan molecules or on CNCs, which cross-links the system. The proposed cross-linking reaction was confirmed by using cross-polarization magic angle spinning (CP/MAS) nuclear magnetic resonance (NMR) spectroscopy. The pore morphology of the obtained materials was analyzed by scanning electron microscopy (SEM). The materials were also tested for compressive strength properties, both in dry and water swollen state. All together this study describes a novel combined freeze-casting/cross-linking process which enables fabrication of nanoreinforced biopolymer-based hydrogels with controlled porosity and 3-D architecture.

  12. Wear of moderately cross-linked polyethylene in fixed-bearing total knee replacements.

    PubMed

    Brockett, Claire L; Jennings, Louise M; Hardaker, Catherine; Fisher, John

    2012-07-01

    Cross-linked polyethylene has been introduced into total joint replacement to improve wear resistance. Although the performance of highly cross-linked polyethylene is well documented clinically and experimentally for total hip replacements, the reduction in mechanical properties with increasing irradiation is of concern for application to total knee replacement. The aim of this study was to investigate the wear performance of a moderately cross-linked polyethylene material in a fixed-bearing total knee replacement. The study was conducted using two femoral geometries, a conventional cruciate-retaining femoral and a high-flexion femoral geometry. The femoral geometry appeared to have no effect on the wear of the knee replacement under standard gait conditions. A significant reduction in wear volume was measured with the moderately cross-linked polyethylene compared with the conventional polyethylene over a six-million-cycle wear study. This study indicates the use of a moderately cross-linked polyethylene in a fixed-bearing total knee replacement may provide a low wearing option for total knee replacement.

  13. Orientation birefringence of cross-linked rubber containing low-mass compound

    NASA Astrophysics Data System (ADS)

    Kiyama, Ayumi; Nobukawa, Shogo; Yamauchi, Masayuki

    2015-05-01

    Molecular orientation of low-mass compounds (LMCs) in a cross-linked rubber is studied in order to obtain the basic information on the dynamics of LMC molecules in a polymer beyond the glass transition temperature. A small amount of LMCs such as 4-cyano-4'-pentylbiphenyl (5CB), tricresylphosphate (TCP), and styrene-based tackifier (TF) is added into polybutadiene rubber (BR). After cross-linking reaction, the sheet samples are used to evaluate the orientation birefringence during stretching and stress relaxation. The rectangular films, cut out from the cross-linked sheets, are set in a uniaxial stretching machine equipped with an optical system to measure both birefringence and tensile stress simultaneously. It is confirmed that orientation birefringence is proportional to the stress for not only pure cross-linked BR, but also cross-linked BR containing an LMC in a wide range of strain. Even after stretching, the birefringence does not change as far as the sample is kept at a constant strain. The results suggest that the LMC molecules are forced to orient with polymer chains by the strong intermolecular orientation correlation. Because of the LMC orientation, the stress-optical coefficient CR is enhanced by the addition of 5CB and TCP, but depressed by TF. Therefore, the LMC doping can be used to control the birefringence of a retardation film.

  14. Alkali reversal of psoralen cross-link for the targeted delivery of psoralen monoadduct lesion

    SciTech Connect

    Yeung, A.T.; Dinehart, W.J.; Jones, B.K.

    1988-08-23

    Psoralen intercalates into double-stranded DNA and photoreacts mainly with thymines to form monoadducts and interstrand cross-links. The authors used an oligonucleotide model to demonstrate a novel mechanism: the reversal of psoralen cross-links by base-catalyzed rearrangement at 90/sup 0/C (BCR). The BCR reaction is more efficient than the photoreversal reaction. They show that the BCR occurs predominantly on the furan side of a psoralen cross-link. The cleavage does not result in the breaking of the DNA backbone, and the thymine based freed from the cross-link by the cleavage reaction appears to be unmodified. Similarly, BCR of the furan-side monoadduct of psoralen removed the psoralen molecule and regenerated the unaltered native oligonucleotide. The pyrone-side psoralen monoadduct is relatively resistant to BCR. One can use BCR to perform efficient oligonucleotide-directed, site-specific delivery of a psoralen monoadduct. As a demonstration of this approach, they have hybridized a 19 base long oligonucleotide vehicle containing a furan-side psoralen monoadduct to a 56 base long complementary oligonucleotide target strand and formed a specific cross-link at the target site with 365-nm UV. Subsequent BCR released the oligonucleotide vehicle and deposited the psoralen at the target site.

  15. Site specificity of psoralen-DNA interstrand cross-linking determined by nuclease Bal31 digestion

    SciTech Connect

    Zhen, W.; Buchardt, O.; Nielsen, H.; Nielsen, P.E.

    1986-10-21

    A novel method for determination of psoralen photo-cross-linking sites in double-stranded DNA is described, which is based on a pronounced inhibition of Bal31 exonuclease activity by psoralen-DNA interstrand cross-links. The results using a 51 base pair fragment of plasmid pUC19 and a 346 base pair fragment of pBR322 show that 5'-TA sequences are preferred cross-linking sites compared to 3'-TA sequences. They also indicate that sequences flanking the 5'-TA site influence the cross-linking efficiency at the site. The DNA photo-cross-linking by 4,5',8-trimethylpsoralen and 8-methoxypsoralen was analyzed, and these two psoralens showed identical site specificity. The 5'-TA preference is rationalized on the basis of the local DNA structure in terms of ..pi..-..pi.. electronic interaction between the thymines and the intercalated psoralens, as well as on the base tilt angles of the DNA.

  16. Synthesis, Characterization, and Antibacterial Activity of Cross-Linked Chitosan-Glutaraldehyde

    PubMed Central

    Li, Bin; Shan, Chang-Lin; Zhou, Qing; Fang, Yuan; Wang, Yang-Li; Xu, Fei; Han, Li-Rong; Ibrahim, Muhammad; Guo, Long-Biao; Xie, Guan-Lin; Sun, Guo-Chang

    2013-01-01

    This present study deals with synthesis, characterization and antibacterial activity of cross-linked chitosan-glutaraldehyde. Results from this study indicated that cross-linked chitosan-glutaraldehyde markedly inhibited the growth of antibiotic-resistant Burkholderia cepacia complex regardless of bacterial species and incubation time while bacterial growth was unaffected by solid chitosan. Furthermore, high temperature treated cross-linked chitosan-glutaraldehyde showed strong antibacterial activity against the selected strain 0901 although the inhibitory effects varied with different temperatures. In addition, physical-chemical and structural characterization revealed that the cross-linking of chitosan with glutaraldehyde resulted in a rougher surface morphology, a characteristic Fourier transform infrared (FTIR) band at 1559 cm−1, a specific X-ray diffraction peak centered at 2θ = 15°, a lower contents of carbon, hydrogen and nitrogen, and a higher stability of glucose units compared to chitosan based on scanning electron microscopic observation, FTIR spectra, X-ray diffraction pattern, as well as elemental and thermo gravimetric analysis. Overall, this study indicated that cross-linked chitosan-glutaraldehyde is promising to be developed as a new antibacterial drug. PMID:23670533

  17. A General Method for Targeted Quantitative Cross-Linking Mass Spectrometry

    PubMed Central

    Chavez, Juan D.; Eng, Jimmy K.; Schweppe, Devin K.; Cilia, Michelle; Rivera, Keith; Zhong, Xuefei; Wu, Xia; Allen, Terrence; Khurgel, Moshe; Kumar, Akhilesh; Lampropoulos, Athanasios; Larsson, Mårten; Maity, Shuvadeep; Morozov, Yaroslav; Pathmasiri, Wimal; Perez-Neut, Mathew; Pineyro-Ruiz, Coriness; Polina, Elizabeth; Post, Stephanie; Rider, Mark; Tokmina-Roszyk, Dorota; Tyson, Katherine; Vieira Parrine Sant'Ana, Debora; Bruce, James E.

    2016-01-01

    Chemical cross-linking mass spectrometry (XL-MS) provides protein structural information by identifying covalently linked proximal amino acid residues on protein surfaces. The information gained by this technique is complementary to other structural biology methods such as x-ray crystallography, NMR and cryo-electron microscopy[1]. The extension of traditional quantitative proteomics methods with chemical cross-linking can provide information on the structural dynamics of protein structures and protein complexes. The identification and quantitation of cross-linked peptides remains challenging for the general community, requiring specialized expertise ultimately limiting more widespread adoption of the technique. We describe a general method for targeted quantitative mass spectrometric analysis of cross-linked peptide pairs. We report the adaptation of the widely used, open source software package Skyline, for the analysis of quantitative XL-MS data as a means for data analysis and sharing of methods. We demonstrate the utility and robustness of the method with a cross-laboratory study and present data that is supported by and validates previously published data on quantified cross-linked peptide pairs. This advance provides an easy to use resource so that any lab with access to a LC-MS system capable of performing targeted quantitative analysis can quickly and accurately measure dynamic changes in protein structure and protein interactions. PMID:27997545

  18. Preparation of cross-linked hen-egg white lysozyme crystals free of cracks

    PubMed Central

    Yan, Er-Kai; Lu, Qin-Qin; Zhang, Chen-Yan; Liu, Ya-Li; He, Jin; Chen, Da; Wang, Bo; Zhou, Ren-Bin; Wu, Ping; Yin, Da-Chuan

    2016-01-01

    Cross-linked protein crystals (CLPCs) are very useful materials in applications such as biosensors, catalysis, and X-ray crystallography. Hence, preparation of CLPCs is an important research direction. During the preparation of CLPCs, an often encountered problem is that cracks may appear in the crystals, which may finally lead to shattering of the crystals into small pieces and cause problem in practical applications. To avoid cross-link induced cracking, it is necessary to study the cracking phenomenon in the preparation process. In this paper, we present an investigation on how to avoid cracking during preparation of CLPCs. An orthogonal experiment was designed to study the phenomenon of cross-link induced cracking of hen-egg white lysozyme (HEWL) crystals against five parameters (temperature, solution pH, crystal growth time, glutaraldehyde concentration, and cross-linking time). The experimental results showed that, the solution pH and crystal growth time can significantly affect cross-link induced cracking. The possible mechanism was studied, and optimized conditions for obtaining crack-free CLPCs were obtained and experimentally verified. PMID:27703210

  19. Effects of partial hydrolysis and subsequent cross-linking on wheat gluten physicochemical properties and structure.

    PubMed

    Wang, Kaiqiang; Luo, Shuizhong; Cai, Jing; Sun, Qiaoqiao; Zhao, Yanyan; Zhong, Xiyang; Jiang, Shaotong; Zheng, Zhi

    2016-04-15

    The rheological behavior and thermal properties of wheat gluten following partial hydrolysis using Alcalase and subsequent microbial transglutaminase (MTGase) cross-linking were investigated. The wheat gluten storage modulus (G') and thermal denaturation temperature (Tg) were significantly increased from 2.26 kPa and 54.43°C to 7.76 kPa and 57.69°C, respectively, by the combined action of partial hydrolysis (DH 0.187%) and cross-linking. The free SH content, surface hydrophobicity, and secondary structure analysis suggested that an appropriate degree of Alcalase-based hydrolysis allowed the compact wheat gluten structure to unfold, increasing the β-sheet content and surface hydrophobicity. This improved its molecular flexibility and exposed additional glutamine sites for MTGase cross-linking. SEM images showed that a compact 3D network formed, while SDS-PAGE profiles revealed that excessive hydrolysis resulted in high-molecular-weight subunits degrading to smaller peptides, unsuitable for cross-linking. It was also demonstrated that the combination of Alcalase-based partial hydrolysis with MTGase cross-linking might be an effective method for modifying wheat gluten rheological behavior and thermal properties.

  20. Availability of fluorescence spectroscopic in the accompaniment of formation of corneal cross-linking

    NASA Astrophysics Data System (ADS)

    Costa, M. M.; Kurachi, C.; Bagnato, V. S.; Faria e Sousa, S. J.; Ventura, L.

    2010-02-01

    The corneal cross-linking is a method that associates riboflavin and ultraviolet light to induce a larger mechanical resistance at cornea. This method has been used for the treatment of Keratoconus. Since cross-linking is recent as treatment, there is a need to verify the effectiveness of the method. Therefore, the viability of the fluorescence spectroscopy technique to follow the cross-linking formation at cornea was studied. Corneas were divided in two measuring procedures: M1 (cornea + riboflavin), and M2 (cornea + riboflavina + light irradiation, 365nm). For fluorescence measurements, a spectrofluorimeter was used, where several wavelengths were selected (between 320nm and 370nm) for cornea excitation. Several fluorescence spectra were collected, at 10 min-interval, during 60 min. Spectra allowed one to observe two very well defined bands of fluorescence: the first one at 400nm (collagen), and the second one at 520nm (riboflavin). After spectra analyses, a decrease of collagen fluorescence was observed for both groups. For riboflavin, on the other hand, there was a fluorescence increase for M1, and a decrease for M2. Thus, it is possible to conclude that it this technique is sensitive for the detection of tissue structural changes during cross-linking treatment, encouraging subsequent studies on quantification of cross-linking promotion in tissue.

  1. Biodegradable Chitosan-Based Ambroxol Hydrochloride Microspheres: Effect of Cross-Linking Agents

    PubMed Central

    Gangurde, HH; Chavan, NV; Mundada, AS; Derle, DV; Tamizharasi, S

    2011-01-01

    The objective of this study was to investigate the influence of type of cross-linking method used on the properties of ambroxol hydrochloride microspheres such as encapsulation efficiency, particle size, and drug release. Microspheres were prepared by solvent evaporation technique using chitosan as a matrix-forming agent and cross-linked using formaldehyde and heat treatment. Morphological and physicochemical properties of microspheres were then investigated by scanning electron microscopy (SEM), X-ray diffractometry (XRD), differential scanning calorimetry (DSC), and Fourier-transform infrared spectroscopy (FTIR) spectroscopy. The cross-linking of chitosan takes place at the free amino group because of formation of imine bond as evidenced by FTIR. The DSC, XRD, and FTIR analysis showed that chitosan microspheres cross linked by heating were superior in properties and performance as compared to the microspheres cross-linked using formaldehyde. SEM results revealed that heat-treated microspheres were spherical, discrete having smooth, and porous structure. The particle size and encapsulation efficiencies of the prepared chitosan microspheres ranged between 10.83–24.11 μm and 39.73μ80.56%, respectively. The drug release was extended up to 12 h, and the kinetics of the drug release was obeying Higuchi kinetic proving diffusion-controlled drug release. PMID:21607049

  2. Cross-linking connectivity in bone collagen fibrils: the COOH-terminal locus of free aldehyde

    NASA Technical Reports Server (NTRS)

    Otsubo, K.; Katz, E. P.; Mechanic, G. L.; Yamauchi, M.

    1992-01-01

    Quantitative analyses of the chemical state of the 16c residue of the alpha 1 chain of bone collagen were performed on samples from fetal (4-6-month embryo) and mature (2-3 year old) bovine animals. All of this residue could be accounted for in terms of three chemical states, in relative amounts which depended upon the age of the animal. Most of the residue was incorporated into either bifunctional or trifunctional cross-links. Some of it, however, was present as free aldehyde, and the content increased with maturation. This was established by isolating and characterizing the aldehyde-containing peptides generated by tryptic digestion of NaB3H4-reduced mature bone collagen. We have concluded that the connectivity of COOH-terminal cross-linking in bone collagen fibrils changes with maturation in the following way: at first, each 16c residue in each of the two alpha 1 chains of the collagen molecule is incorporated into a sheet-like pattern of intermolecular iminium cross-links, which stabilizes the young, nonmineralized fibril as a whole. In time, some of these labile cross-links maturate into pyridinoline while others dissociate back to their precursor form. The latter is likely due to changes in the molecular packing brought about by the mineralization of the collagen fibrils. The resultant reduction in cross-linking connectivity may provide a mechanism for enhancing certain mechanical characteristics of the skeleton of a mature animal.

  3. Organization of photosystem I polypeptides examined by chemical cross-linking

    NASA Technical Reports Server (NTRS)

    Armbrust, T. S.; Chitnis, P. R.; Guikema, J. A.; Spooner, B. S. (Principal Investigator)

    1996-01-01

    Photosystem I from the cyanobacterium Synechocystis sp. PCC 6803 was examined using the chemical cross-linkers glutaraldehyde and N-ethyl-1-3-[3-(dimethylamino)propyl]carbodiimide to investigate the organization of the polypeptide subunits. Thylakoid membranes and photosystem I, which was isolated by Triton X-100 fractionation, were treated with cross-linking reagents and were resolved using a Tricine/urea low-molecular-weight resolution gel system. Subunit-specific antibodies and western blotting analysis were used to identify the components of cross-linked species. These analyses identified glutaraldehyde-dependent cross-linking products composed of small amounts of PsaD and PsaC, PsaC and PsaE, and PsaE and PsaF. The novel cross-link between PsaE and PsaF was also observed following treatment with N-ethyl-1-3-[3-(dimethylamino)propyl]carbodiimide. These cross-linking results suggest a structural interaction between PsaE and PsaF and predict a transmembrane topology for PsaF.

  4. Photoaffinity Labeling Reveals Nuclear Proteins that Uniquely Recognize Cisplatin-DNA Interstrand Cross-Links

    PubMed Central

    Zhu, Guangyu; Lippard, Stephen J.

    2009-01-01

    The DNA-binding inorganic compound cisplatin is one of the most successful anticancer drugs. The detailed mechanism by which cells recognize and process of cisplatin-DNA damage is of great interest. Although the family of proteins that bind cisplatin 1,2- and 1,3-intrastrand cross-links has been identified, much less is known about cellular protein interactions with cisplatin interstrand cross-links (ICLs). In order to address this question, a photoreactive analogue of cisplatin, PtBP6, was used to construct a DNA duplex containing a site-specific platinum ICL. This DNA probe was characterized and used in photo-cross-linking experiments to separate and identify nuclear proteins that bind to the ICL by peptide mass fingerprint analysis. Several such proteins were discovered, including PARP-1, hMutSβ, DNA ligase III, XRCC1, and PNK. The photo-cross-linking approach was independently validated by an electrophoretic mobility shift assay demonstrating hMutSβ binding to a cisplatin ICL. Proteins that recognize the platinum ICL were also identified in cisplatin resistant cells, cells halted at various phases of the cell cycle, and in different carcinoma cells. Nuclear proteins that bind to the platinum ICL differ from those binding to intrastrand cross-links, indicating different mechanisms for disruption of cellular functions. PMID:19364127

  5. Mechanical and biocompatible characterization of a cross-linked collagen-hyaluronic acid wound dressing.

    PubMed

    Kirk, James F; Ritter, Gregg; Finger, Isaac; Sankar, Dhyana; Reddy, Joseph D; Talton, James D; Nataraj, Chandra; Narisawa, Sonoko; Millán, José Luis; Cobb, Ronald R

    2013-01-01

    Collagen scaffolds have been widely employed as a dermal equivalent to induce fibroblast infiltrations and dermal regeneration in the treatment of chronic wounds and diabetic foot ulcers. Cross-linking methods have been developed to address the disadvantages of the rapid degradation associated with collagen-based scaffolds. To eliminate the potential drawbacks associated with glutaraldehyde cross-linking, methods using a water soluble carbodiimide have been developed. In the present study, the glycosaminoglycan (GAG) hyaluronic acid (HA), was covalently attached to an equine tendon derived collagen scaffold using 1-ethyl-3-(3-dimethyl aminopropyl) carbodiimide (EDC) to create ntSPONGE The HA was shown to be homogeneously distributed throughout the collagen matrix. In vitro analyses of the scaffold indicated that the cross-linking enhanced the biological stability by decreasing the enzymatic degradation and increasing the thermal denaturation temperature. The material was shown to support the attachment and proliferation of mouse L929 fibroblast cells. In addition, the cross-linking decreased the resorption rate of the collagen as measured in an intramuscular implant model in rabbits. The material was also shown to be biocompatible in a variety of in vitro and in vivo assays. These results indicate that this cross-linked collagen-HA scaffold, ntSPONGE has the potential for use in chronic wound healing.

  6. Genipin cross-linked decellularized tracheal tubular matrix for tracheal tissue engineering applications

    PubMed Central

    Sun, Fei; Jiang, Yuan; Xu, Yanfei; Shi, Hongcan; Zhang, Siquan; Liu, Xingchen; Pan, Shu; Ye, Gang; Zhang, Weidong; Zhang, Fangbiao; Zhong, Chonghao

    2016-01-01

    Decellularization techniques have been widely used as an alternative strategy for organ reconstruction. This study investigated the mechanical, pro-angiogenic and in vivo biocompatibility properties of decellularized airway matrices cross-linked with genipin. New Zealand rabbit tracheae were decellularized and cross-linked with genipin, a naturally derived agent. The results demonstrated that, a significant (p < 0.05) increase in the secant modulus was computed for the cross-linked tracheae, compared to the decellularized samples. Angiogenic assays demonstrated that decellularized tracheal scaffolds and cross-linked tracheae treated with 1% genipin induce strong in vivo angiogenic responses (CAM analysis). Seven, 15 and 30 days after implantation, decreased (p < 0.01) inflammatory reactions were observed in the xenograft models for the genipin cross-linked tracheae matrices compared with control tracheae, and no increase in the IgM or IgG content was observed in rats. In conclusion, treatment with genipin improves the mechanical properties of decellularized airway matrices without altering the pro-angiogenic properties or eliciting an in vivo inflammatory response. PMID:27080716

  7. Tunable porosity and polarity of polar post-cross-linked resins and selective adsorption.

    PubMed

    Wang, Xiaomei; Zhang, Ting; Huo, Jiaqi; Huang, Jianhan; Liu, You-Nian

    2017-02-01

    Herein we synthesized three polar post-cross-linked resins by adjusting the initial cross-linking degree of the precursor copolymers, and found that the porosity and polarity of these resins could be effectively tuned. The polar post-cross-linked resin with the initial cross-linking degree of 10% (abbreviated as PVE_10%_pc) possessed a much greater BET surface area and almost all micro/mesopores, but lower content of ester groups, while that with the initial cross-linking degree of 60% (named as PVE_60%_pc) had a much less BET surface area, less micro/mesopores and considerable macropores, but higher content of ester groups. The different porosity and polarity of these resins endowed them with different selectivity for the adsorption of aromatic compounds. PVE_10%_pc owned the largest equilibrium capacity to phenol whilst PVE_40%_pc was the most efficient for adsorption of benzoic acid, and hydrogen bonding, hydrophobic interaction and π-π stacking were the main driving forces for the adsorption.

  8. Novel Technique of Transepithelial Corneal Cross-Linking Using Iontophoresis in Progressive Keratoconus

    PubMed Central

    Raffa, Paolo; Rosati, Marianna

    2016-01-01

    In this work, the authors presented the techniques and the preliminary results at 6 months of a randomized controlled trial (NCT02117999) comparing a novel transepithelial corneal cross-linking protocol using iontophoresis with the Dresden protocol for the treatment of progressive keratoconus. At 6 months, there was a significant average improvement with an average flattening of the maximum simulated keratometry reading of 0.72 ± 1.20 D (P = 0.01); in addition, corrected distance visual acuity improved significantly (P = 0.08) and spherical equivalent refraction was significantly less myopic (P = 0.02) 6 months after transepithelial corneal cross-linking with iontophoresis. The novel protocol using iontophoresis showed comparable results with standard corneal cross-linking to halt progression of keratoconus during 6-month follow-up. Investigation of the long-term RCT outcomes are ongoing to verify the efficacy of this transepithelial corneal cross-linking protocol and to determine if it may be comparable with standard corneal cross-linking in the management of progressive keratoconus. PMID:27597895

  9. Cross-linked acrylic hydrogel for the controlled delivery of hydrophobic drugs in cancer therapy

    PubMed Central

    Deepa, G; Thulasidasan, Arun Kumar T; Anto, Ruby John; Pillai, J Jisha; Kumar, GS Vinod

    2012-01-01

    Objective: To investigate cross-linked hydrogels prepared via inverse emulsion polymerization to entrap poorly aqueous soluble drugs. Polyethylene glycol cross-linked acrylic polymers were synthesized and the loading and release of curcumin, a model hydrophobic drug, was investigated. Methods: Physicochemical characteristics of hydrogels were studied with 13C nuclear magnetic resonance, Fourier transform infrared spectroscopy, transmission electron microscopy, scanning electron microscopy, differential scanning calorimetry, and swelling. Polymerization of the acrylic acid with cross-linked polyethylene glycol diacrylate was characterized with 13C nuclear magnetic resonance imaging and Fourier transform infrared spectroscopy. Results: The in vitro release rate of curcumin showed that there was a sustained release from the hydrogel with increased cross-linking; the release rate depended on the pH of the releasing medium. Intracellular and cytotoxicity studies were carried out in human cervical cancer cell lines. Conclusion: The results suggest cross-linked acrylic polymers can be used as efficient vectors for pH-sensitive, controlled delivery of hydrophobic drugs. PMID:22888244

  10. Effect of plasma and carboxylesterase on the stability, mutagenicity, and DNA cross-linking activity of some direct-acting N-nitroso compounds.

    PubMed

    Aukerman, S L; Brundrett, R B; Hilton, J; Hartman, P E

    1983-01-01

    The effects of mouse plasma, human plasma, and purified porcine liver carboxylesterase on nitrosourea, nitrosamide, and nitrosocarbamate chemical stability, mutagenicity, and DNA cross-linking activity were compared. These three classes of N-nitroso compounds are chemically similar but displayed different biological activities and were affected differently by plasma and carboxylesterase. Nitrosourea stability as well as mutagenicity and DNA cross-linking activity were affected negligibly by esterase or plasma. In contrast, nitrosamide and nitrosocarbamate stability, mutagenicity, and DNA cross-linking activity were rapidly decreased in the presence of plasma or carboxylesterase. For example, chemical half-lives were from 10- to 20-fold shorter for the nitrosamides and nitrosocarbamates in the presence of 5% mouse plasma. Similar decreases were seen for mutagenicity and DNA cross-linking activity. Preliminary studies indicated one active plasma component to be an enzyme, possibly an esterase. Additional factors such as sulfhydryls may also participate. Whereas some nitrosoureas are active antitumor agents, the lack of antitumor activity for analogous nitrosamides and nitrosocarbamates may reside predominantly in their rapid in vivo inactivation. These results may help to account for the high in vitro mutagenicity as compared with the low in vivo activities of nitrosamides and nitrosocarbamates.

  11. Cryo-imaging and modeling of the super molecular structure of cross-linked gelatin and its applications

    NASA Astrophysics Data System (ADS)

    Marmorat, Clement; Arinstein, Arkadi; Koifman, Naama; Talmon, Yeshayahu; Zussman, Eyal; Rafailovich, Miriam

    The need for naturally derived materials to synthetize bio-compatible scaffolds is growing. In its natural state, gelatin derives its properties from a network of structured, intertwined, triple helical chains. The mechanical properties can be further controlled by additional enzymatic cross-linking. But, in contrast to simple polymer systems, the response to an imposed deformation is then determined by two competing factors, the establishment of the cross-linked mesh vs. the self-assembly of the fibrils into larger and therefore stronger hierarchical structures. Properties deduced from the response functions to measurements; such as rheology or swelling, are then a combination of these two very different factors, hence impossible to model unless more precise knowledge is available regarding the internal structure. We applied cryogenic-temperature scanning electron microscopy (cryo-SEM) to image the networks. Based on these images, a theoretical model was developed, for which we obtained excellent agreement for the mesh size of both networks, and their mechanical properties. We then used these controlled scaffolds, embedding them with fluorescent beads, to image live cells traction forces at stake during cell migration.

  12. A statistical model of protein binding in parallel actin bundles

    NASA Astrophysics Data System (ADS)

    Shin, Homin; Grason, Gregory; Purdy Drew, Kirstin; Wong, Gerard

    2010-03-01

    We propose a coarse-grained lattice model of cross-linking proteins in parallel actin bundles. Based on this model that captures the interplay between geometrical frustration of binding and the intrinsic flexibility of filaments and linkers, we predict a unique regular ground-state structure of fully cross-linked bundles. We also discuss the linker-dependent thermodynamic transition of actin filaments from their native state to the overtwisted state and map out the ``twist-state'' phase diagram in terms of linker flexibility as well as the chemical potential. A flexible linker regime exhibits a continuous spectrum of intermediate twist states, while a stiff linker regime only allows for untwisted actin filaments and fully overtwisted bundles. Our predictions compare well with small-angle scattering studies of bundles formed in the presence of two types of reconstituted cross-linking proteins, fascin and espin. Additionally, this study reveals how subtle differences in crosslinking agents themselves may be used by cells to achieve self-organized bundles with dramatically different properties.

  13. Microtubule actin crosslinking factor 1 regulates the Balbiani body and animal-vegetal polarity of the zebrafish oocyte.

    PubMed

    Gupta, Tripti; Marlow, Florence L; Ferriola, Deborah; Mackiewicz, Katarzyna; Dapprich, Johannes; Monos, Dimitri; Mullins, Mary C

    2010-08-19

    Although of fundamental importance in developmental biology, the genetic basis for the symmetry breaking events that polarize the vertebrate oocyte and egg are largely unknown. In vertebrates, the first morphological asymmetry in the oocyte is the Balbiani body, a highly conserved, transient structure found in vertebrates and invertebrates including Drosophila, Xenopus, human, and mouse. We report the identification of the zebrafish magellan (mgn) mutant, which exhibits a novel enlarged Balbiani body phenotype and a disruption of oocyte polarity. To determine the molecular identity of the mgn gene, we positionally cloned the gene, employing a novel DNA capture method to target region-specific genomic DNA of 600 kb for massively parallel sequencing. Using this technique, we were able to enrich for the genomic region linked to our mutation within one week and then identify the mutation in mgn using massively parallel sequencing. This is one of the first successful uses of genomic DNA enrichment combined with massively parallel sequencing to determine the molecular identity of a gene associated with a mutant phenotype. We anticipate that the combination of these technologies will have wide applicability for the efficient identification of mutant genes in all organisms. We identified the mutation in mgn as a deletion in the coding sequence of the zebrafish microtubule actin crosslinking factor 1 (macf1) gene. macf1 is a member of the highly conserved spectraplakin family of cytoskeletal linker proteins, which play diverse roles in polarized cells such as neurons, muscle cells, and epithelial cells. In mgn mutants, the oocyte nucleus is mislocalized; and the Balbiani body, localized mRNAs, and organelles are absent from the periphery of the oocyte, consistent with a function for macf1 in nuclear anchoring and cortical localization. These data provide the first evidence for a role for spectraplakins in polarization of the vertebrate oocyte and egg.

  14. Preparation of cross-linked maize (Zea mays L.) starch in different reaction media.

    PubMed

    Hong, Jung Sun; Gomand, Sara V; Delcour, Jan A

    2015-06-25

    Granular normal maize starch was reacted with sodium trimetaphosphate in deionized water ( [Formula: see text] ), aqueous sodium sulfate solution ( [Formula: see text] ), aqueous ethanol (MSethanol) or aqueous acetone (MSacetone) under otherwise identical reaction conditions. Analysis of the resultant starches by Rapid Visco Analysis (RVA) showed that the starch was cross-linked to a higher degree in aqueous ethanol or aqueous acetone than in water or sodium sulfate solution, and with minimal starch leaching. While MSacetone and MSethanol had incorporated similar levels of phosphorous, RVA analysis and microscopic analysis showed that MSacetone granules were more effectively stabilized by cross-linking than MSethanol granules. Cross-linking in aqueous acetone is believed to either contain the greater numbers of distarch monophosphate (versus monostarch monophosphate), or occur more intensively at the granule outer layers than that in aqueous ethanol and, at the same time, to account for the greater granular strength of MSethanol than that of MSacetone.

  15. Genipin Cross-Linked Glucose Oxidase and Catalase Multi-enzyme for Gluconic Acid Synthesis.

    PubMed

    Cui, Caixia; Chen, Haibin; Chen, Biqiang; Tan, Tianwei

    2017-02-01

    In this work, glucose oxidase (GOD) and catalase (CAT) were used simultaneously to produce gluconic acid from glucose. In order to reduce the distance between the two enzymes, and therefore improve efficiency, GOD and CAT were cross-linked together using genipin. Improvements in gluconic acid production were due to quick removal of harmful intermediate hydrogen peroxide by CAT. GOD activity was significantly affected by the proportion of CAT in the system, with GOD activity in the cross-linked multi-enzyme (CLME) being 10 times higher than that in an un-cross-linked GOD/CAT mixture. The glucose conversion rate after 15 h using 15 % glucose was also 10 % higher using the CLME than was measured using a GOD/CAT mixture.

  16. Production of Materials with Spatially-Controlled Cross-Link Density via Vat Photopolymerization.

    PubMed

    Peterson, Gregory I; Schwartz, Johanna J; Zhang, Di; Weiss, Benjamin M; Ganter, Mark A; Storti, Duane W; Boydston, Andrew J

    2016-10-11

    We describe an efficient method to produce objects comprising spatially controlled and graded cross-link densities using vat photopolymerization additive manufacturing (AM). Using a commercially available diacrylate-based photoresin, 3D printer, and digital light processing (DLP) projector, we projected grayscale images to print objects in which the varied light intensity was correlated to controlled cross-link densities and associated mechanical properties. Cylinder and bar test specimens were used to establish correlations between light intensities used for printing and cross-link density in the resulting specimens. Mechanical testing of octet truss unit cells in which the properties of the crossbars and vertices were independently modified revealed unique mechanical responses from the different compositions. From the various test geometries, we measured changes in mechanical properties such as increased strain-to-break in inhomogeneous structures in comparison with homogeneous variants.

  17. Tuning of cross-linking and mechanical properties of laser-deposited poly (methyl methacrylate) films

    SciTech Connect

    Sueske, Erik; Scharf, Thorsten; Krebs, Hans-Ulrich; Panchenko, Elena; Junkers, Thomas; Egorov, Mark; Buback, Michael; Kijewski, Harald

    2005-03-15

    The chemical composition, amount of cross-linking and its influence on the mechanical properties of poly(methyl methacrylate) (PMMA) thin films produced by pulsed laser deposition (PLD) at a wavelength of 248 nm under ultrahigh vacuum were investigated by infrared spectroscopy, scanning electron microscopy, size-exclusion chromatography, thermogravimetric analysis, and nanoindentation experiments. The films consist of two components, one fraction with a molecular weight well below that of the target material and a second fraction, which is cross-linked. Compared to bulk material, the Young's modulus of the film is increased. The amount of cross-linking in the film can be tuned by the applied laser fluence leading to changes of the mechanical properties.

  18. Synthesis of acrylic and allylic bifunctional cross-linking monomers derived from PET waste

    NASA Astrophysics Data System (ADS)

    Cruz-Aguilar, A.; Herrera-González, A. M.; Vázquez-García, R. A.; Navarro-Rodríguez, D.; Coreño, J.

    2013-06-01

    An acrylic and two novel allylic monomers synthesized from bis (hydroxyethyl) terephthalate, BHET, are reported. This was obtained by glycolysis of post-consumer PET with boiling ethylene glycol. The bifunctional monomer bis(2-(acryloyloxy)ethyl) terephthalate was obtained from acryloyl chloride, while the allylic monomers 2-(((allyloxi)carbonyl)oxy) ethyl (2-hydroxyethyl) terephthalate and bis(2-(((allyloxi)carbonyl)oxy)ethyl) terephthalate, from allyl chloroformate. Cross-linking was studied in bulk polymerization using two different thermal initiators. Monomers were analyzed by means of 1H NMR and the cross-linked polymers by infrared spectroscopy. Gel content higher than 90% was obtained for the acrylic monomer. In the case of the mixture of the allylic monomers, the cross-linked polymer was 80 % using BPO initiator, being this mixture 24 times less reactive than the acrylic monomer.

  19. Three-dimensional multimodal microscopy of rabbit cornea after cross-linking treatment

    NASA Astrophysics Data System (ADS)

    Krüger, A.; Hovakimyan, M.; Ramírez, D. F.; Lorbeer, R.-A.; Kröger, M.; Stachs, O.; Wree, A.; Guthoff, R. F.; Lubatschowski, H.; Heisterkamp, A.

    2010-02-01

    Cross-linking of stromal collagen with Riboflavin and UVA radiation is an alternative treatment of keratoconus. After the cross-linking a wound healing process starts with the regeneration of the abraded epithelial layer and the stromal keratocyte-network. To clarify possible side effects by visualization we established an imaging platform for the multimodal three-dimensional imaging of the cornea and looked for differences between normal and cross-linked rabbit corneae. The microscopy system utilizes femtosecond laser light for two photon excitation of autofluorescent metabolic compounds, second harmonic imaging in forward and backward direction for the study of stromal collagen-I structure and confocal detection of the backscattered femtosecond laser light for cell detection. Preliminary results show signatures of treatment 5 weeks after the intervention in all imaging modalities.

  20. Photochromic cross-link polymer for color changing and sensing surface

    NASA Astrophysics Data System (ADS)

    Fu, Richard; Shi, Jianmin; Forsythe, Eric; Srour, Merric

    2016-12-01

    Photochromic cross-link polymers were developed using patented ultraviolet (UV) photoinitiator and commercial photochromic dyes. The photochromic dyes have been characterized by measuring absorbance before and after UV activation using UV-visible (Vis) spectrometry with varying activation intensities and wavelengths. Photochromic cross-link polymers were characterized by a dynamic xenon and UV light activation and fading system. The curing processes on cloth were established and tested to obtain effective photochromic responses. Both PulseForge photonic curing and PulseForge plus heat surface curing processes had much better photochromic responses (18% to 19%, 16% to 25%, respectively) than the xenon lamp treatment (8%). The newly developed photochromic cross-link polymer showed remarkable coloration contrasts and fast and comparable coloration and fading rates. Those intelligent, controlled color changing and sensing capabilities will be used on flexible and "drapeable" surfaces, which will incorporate ultra-low power sensors, sensor indicators, and identifiers.

  1. Localization of the dominant non-enzymatic intermolecular cross-linking sites on fibrous collagen.

    PubMed

    Chiue, Hiroko; Yamazoye, Tsutako; Matsumura, Sueo

    2015-06-05

    Previous studies have shown that fibrous collagen undergoes intermolecular cross-linking at multiple sites of the elongated triple-helical regions among adjacent juxtaposed collagen molecules on incubation with a very high concentration of reducing sugar such as 200 mM ribose, and the similarity of the changes in its physicochemical properties to that of senescent collagen aged in vivo has been emphasized. In the present study, however, it was found that when incubated with less than 30 mM ribose, fibrous collagen underwent intermolecular cross-linking primarily between the telopeptide region of a collagen molecule and the triple-helical region of another adjacent collagen molecule, and intermolecular cross-linking between the triple-helical regions of adjacent collagen molecules was very small. Physiological significance of the previous studies thus needs to be reevaluated.

  2. Tuning of cross-linking and mechanical properties of laser-deposited poly (methyl methacrylate) films

    NASA Astrophysics Data System (ADS)

    Süske, Erik; Scharf, Thorsten; Krebs, Hans-Ulrich; Panchenko, Elena; Junkers, Thomas; Egorov, Mark; Buback, Michael; Kijewski, Harald

    2005-03-01

    The chemical composition, amount of cross-linking and its influence on the mechanical properties of poly(methyl methacrylate) (PMMA) thin films produced by pulsed laser deposition (PLD) at a wavelength of 248nm under ultrahigh vacuum were investigated by infrared spectroscopy, scanning electron microscopy, size-exclusion chromatography, thermogravimetric analysis, and nanoindentation experiments. The films consist of two components, one fraction with a molecular weight well below that of the target material and a second fraction, which is cross-linked. Compared to bulk material, the Young's modulus of the film is increased. The amount of cross-linking in the film can be tuned by the applied laser fluence leading to changes of the mechanical properties.

  3. Cheese whey protein recovery by ultrafiltration through transglutaminase (TG) catalysis whey protein cross-linking.

    PubMed

    Wen-Qiong, Wang; Lan-Wei, Zhang; Xue, Han; Yi, Lu

    2017-01-15

    In whey ultrafiltration (UF) production, two main problems are whey protein recovery and membrane fouling. In this study, membrane coupling protein transglutaminase (TG) catalysis protein cross-linking was investigated under different conditions to find out the best treatment. We found that the optimal conditions for protein recovery involved catalyzing whey protein cross-linking with TG (40U/g whey proteins) at 40°C for 60min at pH 5.0. Under these conditions, the recovery rate was increased 15-20%, lactose rejection rate was decreased by 10%, and relative permeate flux was increase 30-40% compared to the sample without enzyme treatment (control). It was noticeable that the total resistance and cake resistance were decreased after enzyme catalysis. This was mainly due to the increased particle size and decreased zeta potential. Therefore, membrane coupling enzyme catalysis protein cross-linking is a potential means for further use.

  4. xiNET: Cross-link Network Maps With Residue Resolution*

    PubMed Central

    Combe, Colin W.; Fischer, Lutz; Rappsilber, Juri

    2015-01-01

    xiNET is a visualization tool for exploring cross-linking/mass spectrometry results. The interactive maps of the cross-link network that it generates are a type of node-link diagram. In these maps xiNET displays: (1) residue resolution positional information including linkage sites and linked peptides; (2) all types of cross-linking reaction product; (3) ambiguous results; and, (4) additional sequence information such as domains. xiNET runs in a browser and exports vector graphics which can be edited in common drawing packages to create publication quality figures. Availability: xiNET is open source, released under the Apache version 2 license. Results can be viewed by uploading data to http://crosslinkviewer.org/ or by downloading the software from http://github.com/colin-combe/crosslink-viewer and running it locally. PMID:25648531

  5. In vitro progesterone release from γ-irradiated cross-linked polydimethylsiloxane

    NASA Astrophysics Data System (ADS)

    Mashak, Arezou; Taghizadeh, S. Mojtaba

    2006-02-01

    Instead of conventional method such as thermal cross-linking method, γ-irradiation is used to improve the properties of polydimethylsiloxane (PDMS) as a matrix containing progesterone. The thermal cross-linking of PDMS monolithic systems containing drug is deleterious to the drug. Usually, all drugs are unstable both at high vulcanizing temperature and in the presence of peroxide catalysts. This novel method is found to be effective for the stability of the controlled drug delivery systems. The PDMS (three medical grades) matrices were exposed to γ-irradiation in ambient conditions with total doses of 50, 75 and 100 kGy. The mechanical properties confirmed that the samples are cross-linked. It is found that the progesterone release rate is affected by irradiation treatment. It is deduced, however that there is no significant difference in the release profile of progesterone by increasing the irradiation dose from 50 to 100 kGy.

  6. UV cross-linked, lithium-conducting ternary polymer electrolytes containing ionic liquids

    NASA Astrophysics Data System (ADS)

    Kim, G. T.; Appetecchi, G. B.; Carewska, M.; Joost, M.; Balducci, A.; Winter, M.; Passerini, S.

    In this manuscript is reported an attempt to prepare high ionic conductivity lithium polymer electrolytes by UV cross-linking the poly(ethyleneoxide) (briefly called PEO) polymer matrix in presence of the plasticizing lithium salt, lithium bis(trifluoromethanesulfonyl)imide (LiTFSI) and an ionic liquid of the pyrrolidinium family (N-alkyl- N-methylpyrrolidinium TFSI) having a common anion with the lithium salt. It is demonstrated that polymer electrolytes with room temperature ionic conductivities of nearly 10 -3 S cm -1 could be obtained as a result of the reduced crystallinity of the ternary electrolytes. The results clearly indicate that the cross-linked ternary electrolyte shows superior mechanical properties with respect to the non-cross-linked electrolytes and higher conductivities with respect to polymer electrolytes containing none or less ionic liquid.

  7. Cross-linking cellulose nanofibrils for potential elastic cryo-structured gels

    NASA Astrophysics Data System (ADS)

    Syverud, Kristin; Kirsebom, Harald; Hajizadeh, Solmaz; Chinga-Carrasco, Gary

    2011-12-01

    Cellulose nanofibrils were produced from P. radiata kraft pulp fibers. The nanofibrillation was facilitated by applying 2,2,6,6-tetramethylpiperidinyl-1-oxyl-mediated oxidation as pretreatment. The oxidized nanofibrils were cross-linked with polyethyleneimine and poly N-isopropylacrylamide- co-allylamine- co-methylenebisacrylamide particles and were frozen to form cryo-structured gels. Samples of the gels were critical-point dried, and the corresponding structures were assessed with scanning electron microscopy. It appears that the aldehyde groups in the oxidized nanofibrils are suitable reaction sites for cross-linking. The cryo-structured materials were spongy, elastic, and thus capable of regaining their shape after a given pressure was released, indicating a successful cross-linking. These novel types of gels are considered potential candidates in biomedical and biotechnological applications.

  8. Carbon Nanofiber Incorporated Silica Based Aerogels with Di-Isocyanate Cross-Linking

    NASA Technical Reports Server (NTRS)

    Vivod, Stephanie L.; Meador, Mary Ann B.; Capadona, Lynn A.; Sullivan, Roy M.; Ghosn, Louis J.; Clark, Nicholas; McCorkle, Linda

    2008-01-01

    Lightweight materials with excellent thermal insulating properties are highly sought after for a variety of aerospace and aeronautic applications. (1) Silica based aerogels with their high surface area and low relative densities are ideal for applications in extreme environments such as insulators for the Mars Rover battery. (2) However, the fragile nature of aerogel monoliths prevents their widespread use in more down to earth applications. We have shown that the fragile aerogel network can be cross-linked with a di-isocyanate via amine decorated surfaces to form a conformal coating. (3) This coating reinforces the neck regions between secondary silica particles and significantly strengthens the aerogels with only a small effect on density or porosity. Scheme 1 depicts the cross-linking reaction with the di-isocyanate and exhibits the stages that result in polymer cross-linked aerogel monoliths.

  9. Cross-linked block copolymer templated assembly of nanoparticle arrays with high density and position selectivity

    NASA Astrophysics Data System (ADS)

    Liu, Zhicheng; Chang, Tongxin; Huang, Haiying; Bai, Lu

    2016-10-01

    Patterning ordered nanoparticle arrays is crucial for the fascinating collective properties of nanoparticles. Block copolymer template provides us a platform for the simple and efficient assembly of nanoparticle arrays. In this work, cylinder-forming poly(styrene-block-2-vinylpyridine) thin film was firstly plasma-etched to expose poly(2-vinylpyridine) cylinders. Then the templates were cross-linked by small molecules so as to access gold nanoparticle arrays with both high density and excellent position selectivity. The cross-linking process significantly restrains the unfavorable surface reconstruction of the thin film. It is demonstrated that the quality of the nanoparticle array was affected by the degree of the cross-linking and the immersion time in nanoparticle solution. The highly ordered gold nanoparticle arrays are promising in several fields such as optics and surface enhanced Raman scattering (SERS).

  10. In vitro calcification and in vivo biocompatibility of the cross-linked polypentapeptide of elastin

    SciTech Connect

    Wood, S.A.; Lemons, J.E.; Prasad, K.U.; Urry, D.W.

    1986-03-01

    The in vitro calcifiability and molecular weight dependence of calcification of the polypentapeptide, (L X Val1-L X Pro2-Gly3-L X Val4-Gly5)n, which had been gamma-irradiation cross-linked have been determined when exposed to dialyzates of normal, nonaugmented fetal bovine serum. The material was found to calcify: calcifiability was found to be highly molecular weight dependent and to be most favored when the highest molecular weight polymers (n approximately equal to 240) had been used for cross-linking. The in vivo biocompatibility, biodegradability, and calcifiability of the gamma-irradiation cross-linked polypentapeptide were examined in rabbits in both soft and hard tissue sites. The material was found to be biocompatible irrespective of its physical form and to be biodegradable but with n of 200 or less it was not shown to calcify or ossify in the rabbit tibial nonunion model.

  11. Cross-link analysis of the C-telopeptide domain from type III collagen.

    PubMed Central

    Henkel, W

    1996-01-01

    Several peptides were isolated from tryptic digests of insoluble calf aorta matrix by chromatography. Reductive pyridylethylation of a tryptic 15 kDa pool released fragments deriving from the C-terminus of type III collagen. A 50-residue peptide Tc(III) was shown by sequence analysis to be the C-terminal peptide from the alpha 1(III)-chain, containing a helical and non-helical region of equal sizes. The peptide was further digested with collagenase to give Colc(III), comprising the complete C-terminal non-helical region of alpha 1(III) including a hydroxylysine in position 16c. The peptide Tc(III) x TN(III) was isolated, demonstrating covalent cross-linking between the C-terminal non-helical region of one type III molecule and the N-terminal helical cross-linking region of another. Its digestion with cyanogen bromide yielded the small fragments alpha 1(III)CB3B* and alpha 1(III)CB3C, confirming TN(III) as an N-terminal helical crosslink site. Sequence analysis of both Tc(III) x TN(III) and its collagenase-derived cross-linked peptide Colc(III) x TN(III) established the 4D-staggered alignment of adjacent collagen III molecules. The cross-link structure of both peptides was mainly dihydroxylysinonorleucine with a small amount of hydroxylysinonorleucine, indicating that the lysine residues involved in formation of the cross-links are both hydroxylated. No pyridinoline or histidinohydroxylysinonorleucine cross-links were found within the non-reduced C-telopeptide region of type III collagen. PMID:8809038

  12. A novel 49-kilodalton protein from Artemia cross-links microtubules in vitro.

    PubMed

    Zhang, J; MacRae, T H

    1992-01-01

    A 49 kilodalton (kDa) protein, previously proposed to cross-link microtubules, was purified to apparent homogeneity from cell-free extracts of the brine shrimp Artemia. When incubated with tubulin under assembly conditions, the purified 49-kDa protein cross-linked the resulting microtubules. Preformed microtubules were also cross-linked when incubated with the 49-kDa protein. Upon centrifugation through sucrose cushions the 49-kDa protein cosedimented with microtubules, suggesting a stable association between the cross-linking protein and tubulin. Such microtubules were interconnected by particles which were circular, bilobed, or elongated in shape. Disruption of microtubule cross-linking and dissociation of the 49-kDa protein from microtubules occurred in the presence of ATP and 5'-adenylyl-imidodiphosphate (AMP-PNP), a nonhydrolyzable analogue of ATP. The 49-kDa protein was moderately resistant to heat, it did not stimulate tubulin assembly, and it did not react with antibodies to neural microtubule-associated proteins (MAPs) and kinesin. These observations indicate that the 49-kDa protein is different from many known MAPs, a conclusion strengthened by the inability of antibodies raised to the 49-kDa protein to recognize these proteins. The amino terminal 15 amino acid residues of the 49-kDa protein were determined by Edman digestion and an antibody raised to this peptide reacted with the 49-kDa protein on Western blots. Microtubule cross-linking was unaffected by the synthetic amino-terminal peptide, even when it was present at a fivefold molar excess over the 49-kDa protein. A search of three protein databanks revealed that the amino terminus of the 49-kDa protein is unique among published sequences.(ABSTRACT TRUNCATED AT 250 WORDS)

  13. Carbodiimide cross-linked amniotic membranes for cultivation of limbal epithelial cells.

    PubMed

    Ma, David Hui-Kang; Lai, Jui-Yang; Cheng, Hsiao-Yun; Tsai, Chen-Chi; Yeh, Lung-Kun

    2010-09-01

    In ophthalmic tissue engineering, amniotic membrane (AM) is one of the most prevalent natural matrices used for limbal epithelial cell (LEC) cultivation and transplantation. However, the application of AM as a scaffold is limited by its low biomechanical strength and rapid biodegradation. The present study reports the development of 1-ethyl-3-(3-dimethyl aminopropyl) carbodiimide hydrochloride (EDC)/N-hydroxysuccinimide (NHS) cross-linked AM as an LEC carrier. The collagenous tissue materials were modified with varying cross-linker concentrations (0-0.25 mmol EDC/mg AM) and were characterized by attenuated total reflection-Fourier transform infrared spectroscopy (ATR-FTIR), differential scanning calorimetry (DSC), ninhydrin assays, electron microscopy, light transmission measurements, mechanical and in vitro degradation tests, as well as diffusion permeability and cell culture studies. Our results showed that chemical cross-linking approaches saturation at concentrations of 0.05 mmol EDC/mg AM. The formation of cross-links (i.e., amide bonds) in the samples treated with 0.05 mmol EDC/mg AM may cause significant aggregation of tropocollagen molecules and collagen microfibrils without affecting cell morphology of biological tissues. With the optimum concentration of 0.05 mmol EDC/mg AM, chemical cross-linker could significantly enhance the mechanical and thermal stability, optical transparency, and resistance to collagenase digestion. Continuous permeation of albumin through the cross-linked AM would be helpful to cell growth over the matrix surface. In addition, the EDC cross-linked samples were able to support LEC proliferation and preserve epithelial progenitor cells in vitro and in vivo. It is concluded that the AM cross-linked with 0.05 mmol EDC/mg AM may be a potential biomaterial for regenerative medicine.

  14. Collagen telopeptides (cross-linking sites) play a role in collagen gel lattice contraction

    NASA Technical Reports Server (NTRS)

    Woodley, D. T.; Yamauchi, M.; Wynn, K. C.; Mechanic, G.; Briggaman, R. A.

    1991-01-01

    Solubilized interstitial collagens will form a fibrillar, gel-like lattice when brought to physiologic conditions. In the presence of human dermal fibroblasts the collagen lattice will contract. The rate of contraction can be determined by computer-assisted planemetry. The mechanisms involved in contraction are as yet unknown. Using this system it was found that the rate of contraction was markedly decreased when collagen lacking telopeptides was substituted for native collagen. Histidinohydroxylysinonorleucine (HHL) is a major stable trifunctional collagen cross-link in mature skin that involves a carboxyl terminal, telopeptide site 16c, the sixteenth amino acid residue from the carboxy terminal of the telopeptide region of alpha 1 (I) in type I collagen. Little, if any, HHL was present in native, purified, reconstituted, soluble collagen fibrils from 1% acetic acid-extracted 2-year-old bovine skin. In contrast, HHL cross-links were present (0.22 moles of cross-link per mole of collagen) in lattices of the same collagen contracted by fibroblasts. However, rat tail tendon does not contain HHL cross-links, and collagen lattices made of rat tail tendon collagen are capable of contraction. This suggests that telopeptide sites, and not mature HHL cross-links per se, are essential for fibroblasts to contract collagen lattices. Beta-aminopropionitrile fumarate (BAPN), a potent lathyrogen that perturbs collagen cross-linking by inhibition of lysyl oxidase, also inhibited the rate of lattice cell contraction in lattices composed of native collagen. However, the concentrations of BAPN that were necessary to inhibit the contraction of collagen lattices also inhibited fibroblast growth suggestive of cellular toxicity. In accordance with other studies, we found no inhibition of the rate of lattice contraction when fibronectin-depleted serum was used. Electron microscopy of contracted gels revealed typical collagen fibers with a characteristic axial periodicity. The data

  15. Actin and Septin Ultrastructures at the Budding Yeast Cell Cortex

    PubMed Central

    Rodal, Avital A.; Kozubowski, Lukasz; Goode, Bruce L.; Drubin, David G.; Hartwig, John H.

    2005-01-01

    Budding yeast has been a powerful model organism for studies of the roles of actin in endocytosis and septins in cell division and in signaling. However, the depth of mechanistic understanding that can be obtained from such studies has been severely hindered by a lack of ultrastructural information about how actin and septins are organized at the cell cortex. To address this problem, we developed rapid-freeze and deep-etch techniques to image the yeast cell cortex in spheroplasted cells at high resolution. The cortical actin cytoskeleton assembles into conical or mound-like structures composed of short, cross-linked filaments. The Arp2/3 complex localizes near the apex of these structures, suggesting that actin patch assembly may be initiated from the apex. Mutants in cortical actin patch components with defined defects in endocytosis disrupted different stages of cortical actin patch assembly. Based on these results, we propose a model for actin function during endocytosis. In addition to actin structures, we found that septin-containing filaments assemble into two kinds of higher order structures at the cell cortex: rings and ordered gauzes. These images provide the first high-resolution views of septin organization in cells. PMID:15525671

  16. Mutational Analysis of a Conserved Glutamic Acid Required for Self-Catalyzed Cross-Linking of Bacteriophage HK97 Capsids▿

    PubMed Central

    Dierkes, Lindsay E.; Peebles, Craig L.; Firek, Brian A.; Hendrix, Roger W.; Duda, Robert L.

    2009-01-01

    The capsid of bacteriophage HK97 is stabilized by ∼400 covalent cross-links between subunits which form without any action by external enzymes or cofactors. Cross-linking only occurs in fully assembled particles after large-scale structural changes bring together side chains from three subunits at each cross-linking site. Isopeptide cross-links form between asparagine and lysine side chains on two subunits. The carboxylate of glutamic acid 363 (E363) from a third subunit is found ∼2.4 Å from the isopeptide bond in the partly hydrophobic pocket that contains the cross-link. It was previously reported without supporting data that changing E363 to alanine abolishes cross-linking, suggesting that E363 plays a role in cross-linking. This alanine mutant and six additional substitutions for E363 were fully characterized and the proheads produced by the mutants were tested for their ability to cross-link under a variety of conditions. Aspartic acid and histidine substitutions supported cross-linking to a significant extent, while alanine, asparagine, glutamine, and tyrosine did not, suggesting that residue 363 acts as a proton acceptor during cross-linking. These results support a chemical mechanism, not yet fully tested, that incorporates this suggestion, as well as features of the structure at the cross-link site. The chemically identical isopeptide bonds recently documented in bacterial pili have a strikingly similar chemical geometry at their cross-linking sites, suggesting a common chemical mechanism with the phage protein, but the completely different structures and folds of the two proteins argues that the phage capsid and bacterial pilus proteins have achieved shared cross-linking chemistry by convergent evolution. PMID:19091865

  17. A novel DTPA cross-linking of hyaluronic acid and metal complexation thereof.

    PubMed

    Buffa, Radovan; Běťák, Jiří; Kettou, Sofiane; Hermannová, Martina; Pospíšilová, Lucie; Velebný, Vladimír

    2011-09-27

    Macromolecular conjugates of a natural polysaccharide, hyaluronic acid, with diethylenetriaminepentaacetic acid (DTPA)-metal complexes were synthesized and characterized by FTIR, NMR, SEC-MALLS and ICP analysis. Several parameters of the cross-linking reaction as molecular weight of starting HA, temperature, equivalent of DTPA bis-anhydride, concentration of HA, presence of transacylation catalyst DMAP and reaction time were studied. The mechanism for the reaction was suggested and relationship between the molecular weight assigned by SEC-MALLS, reaction parameters and rheological properties of the final cross-linked products were investigated.

  18. Durability of highly cross-linked polyethylene in total hip and total knee arthroplasty.

    PubMed

    Dion, Neil T; Bragdon, Charles; Muratoglu, Orhun; Freiberg, Andrew A

    2015-07-01

    This article reviews the history of the development of highly cross-linked polyethylene and provides an in-depth review of the clinical results regarding the durability of highly cross-linked polyethylene (HXLPE) used in total hip arthroplasty (THA) and total knee arthroplasty (TKA). The use of polyethylene as a bearing surface has contributed to the success of THA and TKA; however, polyethylene wear and osteolysis can lead to failure. Ongoing clinical and retrieval studies are required to analyze outcomes at longer-term follow-up.

  19. Cellular Uptake of Gold Nanoparticles Directly Cross-linked with Carrier Peptides by Osteosarcoma Cells

    PubMed Central

    Mandal, Deendayal; Maran, Avudaippan; Yaszemski, Michael J.; Bolander, Mark E; Sarkar, Gobinda

    2010-01-01

    Nanoparticles have been extensively used for a variety of biomedical applications and there is a growing need for highly specific and efficient delivery of the nanoparticles into target cells and subcellular location. We attempted to accomplish this goal by modifying gold particles with peptide motif’s that are known to deliver a ‘cargo’ into chosen cellular location specifically, we intended to deliver nanogold particles into cells through chemical cross-linking with different peptides known to carry protein into cells. Our results suggest that specific sequence of such ‘carrier peptides’ can efficiently deliver gold nanoparticles into cells when chemically cross-linked with the metal particles. PMID:18807262

  20. Method of Cross-Linking Aerogels Using a One-Pot Reaction Scheme

    NASA Technical Reports Server (NTRS)

    Meador, Ann B.; Capadona, Lynn A.

    2008-01-01

    A document discusses a new, simplified method for cross-linking silica and other oxide aerogels, with a polymeric material to increase strength of such materials without adversely affecting porosity or low density. This innovation introduces the polymer precursor into the sol before gelation either as an agent, which co-reacts with the oxide gel, or as soluble polymer precursors, which do not interact with the oxide gel in any way. Subsequent exposure to heat, light, catalyst or other method of promoting polymerization causes cross-linking without any additional infiltration steps.

  1. Immunogenic Display of Purified Chemically Cross-Linked HIV-1 Spikes

    PubMed Central

    Leaman, Daniel P.; Lee, Jeong Hyun; Ward, Andrew B.

    2015-01-01

    ABSTRACT HIV-1 envelope glycoprotein (Env) spikes are prime vaccine candidates, at least in principle, but suffer from instability, molecular heterogeneity and a low copy number on virions. We anticipated that chemical cross-linking of HIV-1 would allow purification and molecular characterization of trimeric Env spikes, as well as high copy number immunization. Broadly neutralizing antibodies bound tightly to all major quaternary epitopes on cross-linked spikes. Covalent cross-linking of the trimer also stabilized broadly neutralizing epitopes, although surprisingly some individual epitopes were still somewhat sensitive to heat or reducing agent. Immunodepletion using non-neutralizing antibodies to gp120 and gp41 was an effective method for removing non-native-like Env. Cross-linked spikes, purified via an engineered C-terminal tag, were shown by negative stain EM to have well-ordered, trilobed structure. An immunization was performed comparing a boost with Env spikes on virions to spikes cross-linked and captured onto nanoparticles, each following a gp160 DNA prime. Although differences in neutralization did not reach statistical significance, cross-linked Env spikes elicited a more diverse and sporadically neutralizing antibody response against Tier 1b and 2 isolates when displayed on nanoparticles, despite attenuated binding titers to gp120 and V3 crown peptides. Our study demonstrates display of cross-linked trimeric Env spikes on nanoparticles, while showing a level of control over antigenicity, purity and density of virion-associated Env, which may have relevance for Env based vaccine strategies for HIV-1. IMPORTANCE The envelope spike (Env) is the target of HIV-1 neutralizing antibodies, which a successful vaccine will need to elicit. However, native Env on virions is innately labile, as well as heterogeneously and sparsely displayed. We therefore stabilized Env spikes using a chemical cross-linker and removed non-native Env by immunodepletion with non

  2. DNA oligonucleotide duplexes containing intramolecular platinated cross-links: energetics, hydration, sequence, and ionic effects.

    PubMed

    Kankia, Besik I; Soto, Ana Maria; Burns, Nicole; Shikiya, Ronald; Tung, Chang-Shung; Marky, Luis A

    2002-11-05

    The anticancer activity of cisplatin arises from its ability to bind covalently to DNA, forming primarily intrastrand cross-links to adjacent purine residues; the most common adducts involve d(GpG) (65%) and d(ApG) (25%) intrastrand cross-links. The incorporation of these platinum adducts in a B-DNA helix induces local distortions, causing bending and unwinding of the DNA. In this work, we used temperature-dependent UV spectroscopy to investigate the unfolding thermodynamics, and associated ionic effects, of two sets of DNA decamer duplexes containing either cis-[Pt(NH(3))(2)[d(GpG

  3. Dynamically vulcanized biobased polylactide/natural rubber blend material with continuous cross-linked rubber phase.

    PubMed

    Chen, Yukun; Yuan, Daosheng; Xu, Chuanhui

    2014-03-26

    We prepared a biobased material, dynamically vulcanized polylactide (PLA)/natural rubber (NR) blend in which the cross-linked NR phase owned a continuous network-like dispersion. This finding breaks the traditional concept of a sea-island morphology formed after dynamic vulcanization of the blends. The scan electron microscopy and dissolution/swell experiments provided the direct proof of the continuous cross-linked NR phase. This new biobased PLA/NR blend material with the novel structure is reported for the first time in the field of dynamic vulcanization and shows promise for development for various functional applications.

  4. Bioreducible cross-linked nanoshell enhances gene transfection of polycation/DNA polyplex in vivo.

    PubMed

    Piao, Ji-Gang; Ding, Sheng-Gang; Yang, Lu; Hong, Chun-Yan; You, Ye-Zi

    2014-08-11

    In this study, we have prepared a self-cross-linking PEG-based branched polymer, which easily forms a bioreducible nanoshell around polyplexes of cationic polymer and DNA, simply via heating the polyplex dispersions in the presence of this self-cross-linking branched polymer. This nanoshell can prevent the polyplex from dissociation and aggregation in physiological fluids without inhibiting the electrostatic interactions between the polymer and DNA. Furthermore, glutathione (GSH) can act as a stimulus to open the nanoshell after it has entered the cell. The polyplexes coated with the bioreducible nanoshell show an obvious enhancement in gene transfection in vivo compared with bare polyplexes.

  5. Probing the active site of a diels-alderase ribozyme by photoaffinity cross-linking.

    PubMed

    Wombacher, Richard; Jäschke, Andres

    2008-07-09

    The active site of a Diels-Alderase ribozyme is located in solution by photoaffinity cross-linking using a productlike azidobenzyl probe. Two key nucleotides are identified that contact the Diels-Alder product in a conformation-dependent fashion. The design of such probes does not require knowledge of the three-dimensional structure of the ribozyme, and the technique yields both static and dynamic structural information. This work establishes photoaffinity cross-linking as an empirical approach that is applied here for the first time to an artificial ribozyme.

  6. Nuclear Actin in Development and Transcriptional Reprogramming.

    PubMed

    Misu, Shinji; Takebayashi, Marina; Miyamoto, Kei

    2017-01-01

    Actin is a highly abundant protein in eukaryotic cells and dynamically changes its polymerized states with the help of actin-binding proteins. Its critical function as a constituent of cytoskeleton has been well-documented. Growing evidence demonstrates that actin is also present in nuclei, referred to as nuclear actin, and is involved in a number of nuclear processes, including transcriptional regulation and chromatin remodeling. The contribution of nuclear actin to transcriptional regulation can be explained by its direct interaction with transcription machineries and chromatin remodeling factors and by controlling the activities of transcription factors. In both cases, polymerized states of nuclear actin affect the transcriptional outcome. Nuclear actin also plays an important role in activating strongly silenced genes in somatic cells for transcriptional reprogramming. When these nuclear functions of actin are considered, it is plausible to speculate that nuclear actin is also implicated in embryonic development, in which numerous genes need to be activated in a well-coordinated manner. In this review, we especially focus on nuclear actin's roles in transcriptional activation, reprogramming and development, including stem cell differentiation and we discuss how nuclear actin can be an important player in development and cell differentiation.

  7. Reduction of total knee replacement wear with vitamin E blended highly cross-linked ultra-high molecular weight polyethylene.

    PubMed

    Vaidya, C; Alvarez, E; Vinciguerra, J; Bruce, D A; DesJardins, J D

    2011-01-01

    Ultra-high molecular weight polyethylene (UHMWPE) is a common bearing component in total knee replacement (TKR) implants, and its susceptibility to wear continues to be the long-term limiting factor in the life of these implants. This study hypothesized that in TKR systems, a highly cross-linked (HXL) UHMWPE blended with vitamin E will result in reduced wear as compared to a direct compression-moulded (DCM) UHMWPE. A wear simulation study was conducted using an asymmetric lateral pivoting '3D Knee' design to compare the two inserts. The highly cross-linked UHMWPE was aged prior to the testing and force-controlled wear testing was carried out for 5 million cycles using a load-controlled ISO-14243 standard at a frequency of 1 Hz on both groups. Gravimetric measurements of DCM UHMWPE (4.4 +/- 3.0 mg/million cycles) and HXL UHMWPE with vitamin E (1.9 +/- 1.9 mg/million cycles) showed significant statistical differences (p < 0.01) between the wear rates. Wear modes and surface roughness for both groups revealed no significant dissimilarities.

  8. Actinic Prurigo.

    PubMed

    Rodríguez-Carreón, Alma Angélica; Rodríguez-Lobato, Erika; Rodríguez-Gutiérrez, Georgina; Cuevas-González, Juan Carlos; Mancheno-Valencia, Alexandra; Solís-Arias, Martha Patricia; Vega-Memije, María Elisa; Hojyo-Tomoka, María Teresa; Domínguez-Soto, Luciano

    2015-01-01

    Actinic prurigo is an idiopathic photodermatosis that affects the skin, as well as the labial and conjunctival mucosa in indigenous and mestizo populations of Latin America. It starts predominantly in childhood, has a chronic course, and is exacerbated with solar exposure. Little is known of its pathophysiology, including the known mechanisms of the participation of HLA-DR4 and an abnormal immunologic response with increase of T CD4+ lymphocytes. The presence of IgE, eosinophils, and mast cells suggests that it is a hypersensitivity reaction (likely type IVa or b). The diagnosis is clinical, and the presence of lymphoid follicles in the mucosal histopathologic study of mucosa is pathognomonic. The best available treatment to date is thalidomide, despite its secondary effects.

  9. Vault-poly-ADP-ribose polymerase in the Octopus vulgaris brain: a regulatory factor of actin polymerization dynamic.

    PubMed

    De Maio, Anna; Natale, Emiliana; Rotondo, Sergio; Di Cosmo, Anna; Faraone-Mennella, Maria Rosaria

    2013-09-01

    Our previous behavioural, biochemical and immunohistochemical analyses conducted in selected regions (supra/sub oesophageal masses) of the Octopus vulgaris brain detected a cytoplasmic poly-ADP-ribose polymerase (more than 90% of total enzyme activity). The protein was identified as the vault-free form of vault-poly-ADP-ribose polymerase. The present research extends and integrates the biochemical characterization of poly-ADP-ribosylation system, namely, reaction product, i.e., poly-ADP-ribose, and acceptor proteins, in the O. vulgaris brain. Immunochemical analyses evidenced that the sole poly-ADP-ribose acceptor was the octopus cytoskeleton 50-kDa actin. It was present in both free, endogenously poly-ADP-ribosylated form (70kDa) and in complex with V-poly-ADP-ribose polymerase and poly-ADP-ribose (260kDa). The components of this complex, alkali and high salt sensitive, were purified and characterized. The kind and the length of poly-ADP-ribose corresponded to linear chains of 30-35 ADP-ribose units, in accordance with the features of the polymer synthesized by the known vault-poly-ADP-ribose polymerase. In vitro experiments showed that V-poly-ADP-ribose polymerase activity of brain cytoplasmic fraction containing endogenous actin increased upon the addition of commercial actin and was highly reduced by ATP. Anti-actin immunoblot of the mixture in the presence and absence of ATP showed that the poly-ADP-ribosylation of octopus actin is a dynamic process balanced by the ATP-dependent polymerization of the cytoskeleton protein, a fundamental mechanism for synaptic plasticity.

  10. IDENTIFYING GENES CONTROLLING FERULATE CROSS-LINKING FORMATION IN GRASS CELL WALLS

    SciTech Connect

    de O Buanafina, Marcia Maria

    2013-10-16

    DESCRIPTION/ABSTRACT This proposal focuses on cell wall feruloylation and our long term goal is to identify and isolate novel genes controlling feruloylation and to characterize the phenotype of mutants in this pathway, with a spotlight on cell wall properties. Currently, the genes underlying AX feruloylation have not been identified and the isolation of such genes could be of great importance in manipulating ferulates accretion to the wall. Mutation of the feruloyl transferase gene(s) should lead to less ferulates secreted to the cell wall and reduced ferulate cross-linking. Our current research is based on the hypothesis that controlling the level of total feruloylation will have a direct impact on the level of cross-linking and in turn impact biomass utility for forage and biofuel production. Our results/accomplishments for this project so far include: 1. Mutagenised Brachypodium population. We have developed EMS mutagenized populations of model grass species Brachypodium distachyon. EMS populations have been developed from over 28,000 mutagenized seeds generating 5,184 M2 families. A total of 20,793 plants have been screened and 1,233 were originally selected. 2. Selected Brachypodium mutants: Potential mutants on their levels of cell wall ferulates and cell wall AX ? have been selected from 708 M2 families. A total of 303 back-crosses to no-mutagenized parental stock have been done, followed by selfing selected genotypes in order to confirm heritability of traits and to remove extraneous mutations generated by EMS mutagenesis. We are currently growing 12 F5 and F6 populations in order to assess CW composition. If low level of ferulates are confirmed in the candidate lines selected the mutation could be altered in different in one or several kinds of genes such as genes encoding an AX feruloyl transferase; genes encoding the arabinosyl transferase; genes encoding the synthesis of the xylan backbone; genes encoding enzymes of the monolignol pathway affecting FA

  11. Actin Dynamics: From Nanoscale to Microscale

    PubMed Central

    Carlsson, Anders E.

    2010-01-01

    The dynamic nature of actin in cells manifests itself in many ways: Polymerization near the cell edge is balanced by depolymerization in the interior, externally induced actin polymerization is followed by depolymerization, and spontaneous oscillations of the cell periphery are frequently seen. I discuss how mathematical modeling relates quantitative measures of actin dynamics to the rates of underlying molecular level processes. The rate of actin incorporation at the leading edge of a moving cell is roughly consistent with existing theories, and the factors determining the characteristic time of actin polymerization are fairly well understood. However, our understanding of actin disassembly is limited, in particular the interplay between severing and depolymerization and the role of specific combinations of proteins in implementing disassembly events. The origins of cell-edge oscillations, and their possible relation to actin waves, are a fruitful area of future research. PMID:20462375

  12. Labeling F-actin barbed ends with rhodamine-actin in permeabilized neuronal growth cones.

    PubMed

    Marsick, Bonnie M; Letourneau, Paul C

    2011-03-17

    The motile tips of growing axons are called growth cones. Growth cones lead navigating axons through developing tissues by interacting with locally expressed molecular guidance cues that bind growth cone receptors and regulate the dynamics and organization of the growth cone cytoskeleton. The main target of these navigational signals is the actin filament meshwork that fills the growth cone periphery and that drives growth cone motility through continual actin polymerization and dynamic remodeling. Positive or attractive guidance cues induce growth cone turning by stimulating actin filament (F-actin) polymerization in the region of the growth cone periphery that is nearer the source of the attractant cue. This actin polymerization drives local growth cone protrusion, adhesion of the leading margin and axonal elongation toward the attractant. Actin filament polymerization depends on the availability of sufficient actin monomer and on polymerization nuclei or actin filament barbed ends for the addition of monomer. Actin monomer is abundantly available in chick retinal and dorsal root ganglion (DRG) growth cones. Consequently, polymerization increases rapidly when free F-actin barbed ends become available for monomer addition. This occurs in chick DRG and retinal growth cones via the local activation of the F-actin severing protein actin depolymerizing factor (ADF/cofilin) in the growth cone region closer to an attractant. This heightened ADF/cofilin activity severs actin filaments to create new F-actin barbed ends for polymerization. The following method demonstrates this mechanism. Total content of F-actin is visualized by staining with fluorescent phalloidin. F-actin barbed ends are visualized by the incorporation of rhodamine-actin within growth cones that are permeabilized with the procedure described in the following, which is adapted from previous studies of other motile cells. When rhodamine-actin is added at a concentration above the critical concentration

  13. Structure elucidation of DNA interstrand cross-link by a combination of nuclease P1 digestion with mass spectrometry.

    PubMed

    Wang, Yuesong; Wang, Yinsheng

    2003-11-15

    DNA interstrand cross-link reagents are among the most powerful agents for cancer treatment. Here we report a combined nuclease P1 digestion/mass spectrometry method for the structure elucidation of duplex oligodeoxynucleotides (ODNs) containing an interstrand cross-link. Our results demonstrate that nuclease P1 digestion of a double-stranded ODN containing an interstrand cross-link (ICL) of 4,5',8-trimethylpsoralen or mitomycin C gives a tetranucleotide bearing the cross-linked nucleobase moiety. Product ion spectra of the deprotonated ions of the tetranucleotides provide information about the structure of the cross-link. Furthermore, product-ion spectra of tetranucleotides containing two orientation isomers of mitomycin C interstrand cross-link are distinctive. We believe that the method described in this paper can be generally applicable for investigating the structures of other DNA ICLs.

  14. Physicochemical properties and micro-structural characteristics in starch from kudzu root as affected by cross-linking.

    PubMed

    Chen, Boru; Dang, Leping; Zhang, Xiao; Fang, Wenzhi; Hou, Mengna; Liu, Tiankuo; Wang, Zhanzhong

    2017-03-15

    Kudzu starch was cross-linked with sodium trimetaphosphate (STMP) at different temperatures, time and of STMP concentrations in this work. The cross-linked starches (CLSs) were fractionated further into cross-linked amylose and amylopectin in order to compare the effect of cross-linking on the microstructure. According to scanning electron microscope (SEM), CLSs displayed the resemble appearance of spherical and polygonal shapes like NS. X-ray diffraction (XRD) revealed that amylose of native starch (A), NS and CLS displayed a combination of A-type and B-type structure, while that was not found in amylose of cross-linked starch (CLA). The deconvoluted fourier transform infrared (FT-IR) indicated that crystal structure of kudzu starch was losing with the proceeding of cross-linking reaction. The CLSs exhibited a higher retrogradation and freeze-thaw stability than NS. This was accompanied by a significant decrease in sedimentation, transparency, swelling power and solubility.

  15. Curvature and torsion in growing actin networks

    PubMed Central

    Shaevitz, Joshua W; Fletcher, Daniel A

    2011-01-01

    Intracellular pathogens such as Listeria monocytogenes and Rickettsia rickettsii move within a host cell by polymerizing a comet-tail of actin fibers that ultimately pushes the cell forward. This dense network of cross-linked actin polymers typically exhibits a striking curvature that causes bacteria to move in gently looping paths. Theoretically, tail curvature has been linked to details of motility by considering force and torque balances from a finite number of polymerizing filaments. Here we track beads coated with a prokaryotic activator of actin polymerization in three dimensions to directly quantify the curvature and torsion of bead motility paths. We find that bead paths are more likely to have low rather than high curvature at any given time. Furthermore, path curvature changes very slowly in time, with an autocorrelation decay time of 200 s. Paths with a small radius of curvature, therefore, remain so for an extended period resulting in loops when confined to two dimensions. When allowed to explore a three-dimensional (3D) space, path loops are less evident. Finally, we quantify the torsion in the bead paths and show that beads do not exhibit a significant left- or right-handed bias to their motion in 3D. These results suggest that paths of actin-propelled objects may be attributed to slow changes in curvature, possibly associated with filament debranching, rather than a fixed torque. PMID:18560043

  16. Curvature and torsion in growing actin networks

    NASA Astrophysics Data System (ADS)

    Shaevitz, Joshua W.; Fletcher, Daniel A.

    2008-06-01

    Intracellular pathogens such as Listeria monocytogenes and Rickettsia rickettsii move within a host cell by polymerizing a comet-tail of actin fibers that ultimately pushes the cell forward. This dense network of cross-linked actin polymers typically exhibits a striking curvature that causes bacteria to move in gently looping paths. Theoretically, tail curvature has been linked to details of motility by considering force and torque balances from a finite number of polymerizing filaments. Here we track beads coated with a prokaryotic activator of actin polymerization in three dimensions to directly quantify the curvature and torsion of bead motility paths. We find that bead paths are more likely to have low rather than high curvature at any given time. Furthermore, path curvature changes very slowly in time, with an autocorrelation decay time of 200 s. Paths with a small radius of curvature, therefore, remain so for an extended period resulting in loops when confined to two dimensions. When allowed to explore a three-dimensional (3D) space, path loops are less evident. Finally, we quantify the torsion in the bead paths and show that beads do not exhibit a significant left- or right-handed bias to their motion in 3D. These results suggest that paths of actin-propelled objects may be attributed to slow changes in curvature, possibly associated with filament debranching, rather than a fixed torque.

  17. Preparation and characterization of cross-linked carboxymethyl chitin porous membrane scaffold for biomedical applications.

    PubMed

    Zhao, Liqing; Wu, Yiguang; Chen, Shu; Xing, Tao

    2015-08-01

    Porous dermal scaffold membrane (PDSM) was successfully prepared by using a so-called sol-gel freeze-drying method. In this method, the carboxymethyl chitin (CMC) hydrosol was first cross-linked by 1-ethyl-3-[3-dimethylaminopropyl] carbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS), and then lyophilized to form the PDSM. For the first time, this research focused on the cross-linked CMC as the only component for three-dimensional PDSM. The effects of cross-linking conditions on the performance of the PDSM were investigated. And PDSM with optimal performance was obtained through 4-h cross-linking at 4 wt% of CMC concentration in the hydrosol, where the mass ratio of EDC to NHS to CMC was 5:3:10. The porosity of the optimized PDSM was more than 90% and the water swelling rate was above 4000%. The pore size was well distributed and was between 100 μm and 200 μm. And the tensile strength was above 0.09 MPa. The as-made PDSM could be degraded above 80% in 12 days in the presence of a 0.2mg/mL lysozyme solution. Very importantly, the PDSM had no cytotoxicity and good biocompatibility from MTT assays. Our results showed the application possibility of the as-prepared PDSM as dermal scaffold for skin tissue engineering.

  18. Highly conductive carbon nanotube buckypapers with improved doping stability via conjugational cross-linking.

    PubMed

    Chen, I-Wen Peter; Liang, Richard; Zhao, Haibo; Wang, Ben; Zhang, Chuck

    2011-12-02

    Carbon nanotube (CNT) sheets or buckypapers have demonstrated promising electrical conductivity and mechanical performance. However, their electrical conductivity is still far below the requirements for engineering applications, such as using as a substitute for copper mesh, which is currently used in composite aircraft structures for lightning strike protection. In this study, different CNT buckypapers were stretched to increase their alignment, and then subjected to conjugational cross-linking via chemical functionalization. The conjugationally cross-linked buckypapers (CCL-BPs) demonstrated higher electrical conductivity of up to 6200 S cm( - 1), which is more than one order increase compared to the pristine buckypapers. The CCL-BPs also showed excellent doping stability in over 300 h in atmosphere and were resistant to degradation at elevated temperatures. The tensile strength of the stretched CCL-BPs reached 220 MPa, which is about three times that of pristine buckypapers. We attribute these property improvements to the effective and stable conjugational cross-links of CNTs, which can simultaneously improve the electrical conductivity, doping stability and mechanical properties. Specifically, the electrical conductivity increase resulted from improving the CNT alignment and inter-tube electron transport capability. The conjugational cross-links provide effective 3D conductive paths to increase the mobility of electrons among individual nanotubes. The stable covalent bonding also enhances the thermal stability and load transfer. The significant electrical and mechanical property improvement renders buckypaper a multifunctional material for various applications, such as conducting composites, battery electrodes, capacitors, etc.

  19. Protective effect of Withania somnifera (Solanaceae) on collagen glycation and cross-linking.

    PubMed

    Babu, Pon Velayutham Anandh; Gokulakrishnan, Adikesavan; Dhandayuthabani, Rajendra; Ameethkhan, Dowlath; Kumar, Chandrasekara Vimal Pradeep; Ahamed, Md Iqbal Niyas

    2007-06-01

    Modification of collagen such as non-enzymatic glycation and cross-linking plays an important role in diabetic complications and age-related diseases. We evaluate the effect of Withania somnifera on glucose-mediated collagen glycation and cross-linking in vitro. Extent of glycation, viscosity, collagen-linked fluorescence and pepsin solubility were assessed in different experimental procedures to investigate the effect of W. somnifera. Tail tendons obtained from rats (Rattus norvegicus) weighing 250-275 g were incubated with 50 mM glucose and 100 mg of metformin or Withania root powder or ethanolic extract of Withania under physiological conditions of temperature and pH for 30 days. Formation of advanced glycation end products (AGE) was measured by fluorescent method whereas the cross-linking of collagen was assessed by pepsin digestion and viscosity measurements. Tendon collagen incubated with glucose showed an increase in glycation, AGE and cross-linking of collagen. The collagen incubated with W. somnifera and metformin ameliorates these modifications. The ethanolic extract of Withania showed more prominent effect than Withania root powder. The activity of ethanolic extract of Withania is comparable to metformin, a known antiglycating agent. In conclusion, Withania could have therapeutic role in the prevention of glycation induced pathogenesis in diabetes mellitus and aging.

  20. Cross-linked gelatin microspheres with continuously tunable degradation profiles for renal tissue regeneration.

    PubMed

    Serban, Monica A; Knight, Toyin; Payne, Richard G; Basu, Joydeep; Rivera, Elias A; Robbins, Neil; McCoy, Darell; Halberstadt, Craig; Jain, Deepak; Bertram, Timothy A

    2014-01-01

    Collagen and gelatin-based biomaterials are widely used in tissue engineering applications. Various methods have been reported for the cross-linking of these macromolecules for the purpose of delaying their biodegradation to prolong their in vivo residence (in tissue engineering applications) or tailoring their drug releasing capacity (when used as drug carriers). In this study, a carbodiimide-based cross-linking method, also used in the production of United States Food and Drug Administration-approved products, was employed to obtain differentially cross-linked gelatin beads. The colorimetric determination of the in vitro enzymatic susceptibility of the beads indicated that the resistance to degradation linearly correlated with the concentration of carbodiimide used for the cross-linking reaction. This result was also confirmed in vivo by the histological evaluation of the residence time of orthotopically injected cell-seeded beads. These data would indicate that the production of gelatin-based microbeads with tunable degradation profiles might be applicable toward the development of products that catalyze regeneration of kidney and other solid organs.

  1. Spirochete flagella hook protein self-catalyze a lysinoalanine covalent cross-link for motility

    PubMed Central

    Miller, Michael R.; Miller, Kelly A.; Bian, Jiang; James, Milinda E.; Zhang, Sheng; Lynch, Michael; Callery, Patrick S.; Hettick, Justin M.; Cockburn, Andrew; Liu, Jun; Li, Chunhao; Crane, Brian R.; Charon, Nyles W.

    2016-01-01

    Spirochetes are bacteria responsible for several serious diseases that include Lyme disease (Borrelia burgdorferi), syphilis (Treponema pallidum), leptospirosis (Leptospira interrogans), and contribute to periodontal diseases (Treponema denticola)1. These spirochetes employ an unusual form of flagella-based motility necessary for pathogenicity; indeed, spirochete flagella (periplasmic flagella, PFs) reside and rotate within the periplasmic space2–11. The universal joint or hook that links the rotary motor to the filament is composed of approximately 120–130 FlgE proteins, which in spirochetes form an unusually stable, high-molecular weight complex (HMWC)9,12–17. In other bacteria, the hook can be readily dissociated by treatments such as heat18. In contrast, spirochete hooks are resistant to these treatments, and several lines of evidence indicate that the HMWC is the consequence of covalent cross-linking12,13,17. Here we show that T. denticola FlgE self-catalyzes an interpeptide cross-linking reaction between conserved lysine and cysteine resulting in the formation of an unusual lysinoalanine adduct that polymerizes the hook subunits. Lysinoalanine cross-links are not needed for flagellar assembly, but they are required for cell motility, and hence infection. The self-catalytic nature of FlgE cross-linking has important implications for protein engineering, and its sensitivity to chemical inhibitors provides a new avenue for the development of antimicrobials targeting spirochetes. PMID:27670115

  2. Preparation of Nanocellulose Reinforced Chitosan Films, Cross-Linked by Adipic Acid

    PubMed Central

    Falamarzpour, Pouria; Behzad, Tayebeh; Zamani, Akram

    2017-01-01

    Adipic acid, an abundant and nontoxic compound, was used to dissolve and cross-link chitosan. After the preparation of chitosan films through casting technique, the in situ amidation reaction was performed at 80–100 °C as verified by Fourier transform infrared (FT-IR). The reaction was accompanied by the release of water which was employed to investigate the reaction kinetics. Accordingly, the reaction rate followed the first-order model and Arrhenius equation, and the activation energy was calculated to be 18 kJ/mol. Furthermore, the mechanical properties of the chitosan films were comprehensively studied. First, optimal curing conditions (84 °C, 93 min) were introduced through a central composite design. In order to evaluate the effects of adipic acid, the mechanical properties of physically cross-linked (uncured), chemically cross-linked (cured), and uncross-linked (prepared by acetic acid) films were compared. The use of adipic acid improved the tensile strength of uncured and chemically cross-linked films more than 60% and 113%, respectively. Finally, the effect of cellulose nanofibrils (CNFs) on the mechanical performance of cured films, in the presence of glycerol as a plasticizer, was investigated. The plasticized chitosan films reinforced by 5 wt % CNFs showed superior properties as a promising material for the development of chitosan-based biomaterials. PMID:28208822

  3. Modification and cross-linking parameters in hyaluronic acid hydrogels--definitions and analytical methods.

    PubMed

    Kenne, Lennart; Gohil, Suresh; Nilsson, Eva M; Karlsson, Anders; Ericsson, David; Helander Kenne, Anne; Nord, Lars I

    2013-01-02

    Definitions and methods for the quantification of degree of modification and cross-linking in cross-linked hyaluronic acid (HA) hydrogels are outlined. A novel method is presented in which the HA hydrogel is degraded by the enzyme chondroitinase AC and the digest product analyzed by size exclusion chromatography combined with electrospray ionization mass spectrometry (SEC-ESI-MS). This method allows for the determination of effective cross-linker ratio (CrR) which together with the degree of modification (MoD), determined by, e.g. (1)H NMR spectroscopy, enables the calculation of the degree of substitution (DS) and degree of cross-linking (CrD). The method, could be applicable to the major cross-linked HA hydrogels currently on the market, and is exemplified here by application to two HA hydrogels. The definitions and methods presented are important contributions in attempts to find relationships between MoD, DS and CrD to mechanical properties as well as to biocompatibility of HA hydrogels.

  4. Mitochondrial protein interactome elucidated by chemical cross-linking mass spectrometry.

    PubMed

    Schweppe, Devin K; Chavez, Juan D; Lee, Chi Fung; Caudal, Arianne; Kruse, Shane E; Stuppard, Rudy; Marcinek, David J; Shadel, Gerald S; Tian, Rong; Bruce, James E

    2017-02-14

    Mitochondrial protein interactions and complexes facilitate mitochondrial function. These complexes range from simple dimers to the respirasome supercomplex consisting of oxidative phosphorylation complexes I, III, and IV. To improve understanding of mitochondrial function, we used chemical cross-linking mass spectrometry to identify 2,427 cross-linked peptide pairs from 327 mitochondrial proteins in whole, respiring murine mitochondria. In situ interactions were observed in proteins throughout the electron transport chain membrane complexes, ATP synthase, and the mitochondrial contact site and cristae organizing system (MICOS) complex. Cross-linked sites showed excellent agreement with empirical protein structures and delivered complementary constraints for in silico protein docking. These data established direct physical evidence of the assembly of the complex I-III respirasome and enabled prediction of in situ interfacial regions of the complexes. Finally, we established a database and tools to harness the cross-linked interactions we observed as molecular probes, allowing quantification of conformation-dependent protein interfaces and dynamic protein complex assembly.

  5. Mechlorethamine-Induced DNA-Protein Cross-Linking in Human Fibrosarcoma (HT1080) Cells

    PubMed Central

    Michaelson-Richie, Erin D.; Ming, Xun; Codreanu, Simona G.; Loeber, Rachel L.; Liebler, Daniel C.; Campbell, Colin; Tretyakova, Natalia Y.

    2011-01-01

    Antitumor nitrogen mustards, such as bis(2-chloroethyl)methylamine (mechlorethamine), are useful chemotherapeutic agents with a long history of clinical application. The antitumor effects of nitrogen mustards are attributed to their ability to induce DNA-DNA and DNA-protein cross-links (DPCs) that block DNA replication. In the present work, a mass spectrometry based methodology was employed to characterize in vivo DNA-protein cross-linking following treatment of human fibrosarcoma (HT1080) cells with cytotoxic concentrations of mechlorethamine. A combination of mass spectrometry-based proteomics and immunological detection was used to identify 38 nuclear proteins which were covalently cross-linked to chromosomal DNA following treatment with mechlorethamine. Isotope dilution HPLC-ESI+-MS/MS analysis of total proteolytic digests revealed a concentration-dependent formation of N-[2-(S-cysteinyl)ethyl]-N-[2-(guan-7-yl)ethyl]methylamine (Cys-N7G-EMA) conjugates, indicating that mechlorethamine cross-links cysteine thiols within proteins to N-7 positions of guanine in DNA. PMID:21486066

  6. Encapsulation and controlled release of hydrophilic pesticide in shell cross-linked nanocapsules containing aqueous core.

    PubMed

    Sun, Chuxiang; Shu, Ke; Wang, Wei; Ye, Zhao; Liu, Ting; Gao, Yuxiang; Zheng, Hua; He, Guanghua; Yin, Yihua

    2014-03-10

    In this study, amphiphilic biocopolymers, synthesized by mixing azidobenzaldehyde (Az) and an aqueous solution of carboxymethyl chitosan (CMCS), which self-assemble into nanocapsules with a aqueous core (ACN) in aqueous media followed by photo-cross-linking to obtain shell cross-linked nanocapsules, were used to develop a controlled release pesticide system. The system was characterized by TEM and DLS. Its encapsulation efficiency was determined. The obtained result showed that it is efficient to encapsulate methomyl reaching encapsulation efficiency as high as 90% in an aqueous medium at pH 4.0, which is mainly attributed to the hydrogen bonding adsorption between methomyl molecules and the inner surface of nanocapsules. Release profiles of methomyl from methomyl-loaded nanocapsules in an aqueous solution at pH 6.0 were shown to be diffusion controlled with a half-release time (t(½)) of 36.3-69.5h from different samples. The shell cross-linking and its degree of cross-linking are assumed to be responsible for this diffusion behavior. The insecticidal activity test in laboratory showed that the control efficacy of methomyl-loaded nanocapsules against the armyworm larvae was significantly superior to the original. The relative control efficacy still maintained 100% over 7 days.

  7. Temperature dependence of creep compliance of highly cross-linked epoxy: A molecular simulation study

    SciTech Connect

    Khabaz, Fardin Khare, Ketan S. Khare, Rajesh

    2014-05-15

    We have used molecular dynamics (MD) simulations to study the effect of temperature on the creep compliance of neat cross-linked epoxy. Experimental studies of mechanical behavior of cross-linked epoxy in literature commonly report creep compliance values, whereas molecular simulations of these systems have primarily focused on the Young’s modulus. In this work, in order to obtain a more direct comparison between experiments and simulations, atomistically detailed models of the cross-linked epoxy are used to study their creep compliance as a function of temperature using MD simulations. The creep tests are performed by applying a constant tensile stress and monitoring the resulting strain in the system. Our results show that simulated values of creep compliance increase with an increase in both time and temperature. We believe that such calculations of the creep compliance, along with the use of time temperature superposition, hold great promise in connecting the molecular insight obtained from molecular simulation at small length- and time-scales with the experimental behavior of such materials. To the best of our knowledge, this work is the first reported effort that investigates the creep compliance behavior of cross-linked epoxy using MD simulations.

  8. Effect of dual modification with hydroxypropylation and cross-linking on physicochemical properties of taro starch.

    PubMed

    Hazarika, Bidyut Jyoti; Sit, Nandan

    2016-04-20

    Dual modification of taro starch by hydroxypropylation and cross-linking was carried out and the properties of the modified starches were investigated. Two different levels of hydroxypropylation (5 and 10%) and cross-linking (0.05 and 0.10%) were used in different sequences. The amylose contents of the starch decreased due to single and dual modification. For the dual-modified starches, the swelling, solubility and clarity was found to increase with level of hydroxypropylation and decrease with level of cross-linking. The freeze-thaw stability of the dual-modified starches was also affected by the sequence of modification. The viscosities of the cross-linked and dual-modified starches were more than native and hydroxypropylated starches. The firmness of the dual-modified starches was also higher than native and single modified starches. The dual-modified starches have benefits of both type of modifications and could be used for specific purposes e.g. food products requiring high viscosity as well as freeze-thaw stability.

  9. Kinetics of enzyme-catalyzed cross-linking of feruloylated arabinan from sugar beet.

    PubMed

    Zaidel, Dayang Norulfairuz Abang; Arnous, Anis; Holck, Jesper; Meyer, Anne S

    2011-11-09

    Ferulic acid (FA) groups esterified to the arabinan side chains of pectic polysaccharides can be oxidatively cross-linked in vitro by horseradish peroxidase (HRP) catalysis in the presence of hydrogen peroxide (H(2)O(2)) to form ferulic acid dehydrodimers (diFAs). The present work investigated whether the kinetics of HRP catalyzed cross-linking of FA esterified to α-(1,5)-linked arabinans are affected by the length of the arabinan chains carrying the feruloyl substitutions. The kinetics of the HRP-catalyzed cross-linking of four sets of arabinan samples from sugar beet pulp, having different molecular weights and hence different degrees of polymerization, were monitored by the disappearance of FA absorbance at 316 nm. MALDI-TOF/TOF-MS analysis confirmed that the sugar beet arabinans were feruloyl-substituted, and HPLC analysis verified that the amounts of diFAs increased when FA levels decreased as a result of the enzymatic oxidation treatment with HRP and H(2)O(2). At equimolar levels of FA (0.0025-0.05 mM) in the arabinan samples, the initial rates of the HRP-catalyzed cross-linking of the longer chain arabinans were slower than those of the shorter chain arabinans. The lower initial rates may be the result of the slower movement of larger molecules coupled with steric phenomena, making the required initial reaction of two FAs on longer chain arabinans slower than on shorter arabinans.

  10. Baking Performance of Phosphorylated Cross-Linked Resistant Starch in Low-Moisture Bakery Goods

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Phosphorylated cross-linked resistant starch (RS) is a type 4 RS, which can be used for enhancing the benefits of dietary fiber. The baking performance of the RS was explored using wire-cut cookie baking and benchtop chemically-leavened cracker baking methods to produce low-moisture baked goods (coo...

  11. Cross-Linked Nanoporous Materials from Reactive and Multifunctional Block Polymers

    SciTech Connect

    Seo, Myungeun; Amendt, Mark A.; Hillmyer, Marc A.

    2012-10-10

    Polylactide-b-poly(styrene-co-2-hydroxyethylmethacrylate) (PLA-b-P(S-co-HEMA)) and polylactide-b-poly(styrene-co-2-hydroxyethylacrylate) (PLA-b-P(S-co-HEA)) were synthesized by combination of ring-opening polymerization and reversible addition-fragmentation chain transfer polymerization. {sup 1}H nuclear magnetic resonance spectroscopy and size exclusion chromatography data indicated that the polymerizations were controlled and that hydroxyl groups were successfully incorporated into the block polymers. The polymers were reacted with 4,4{prime}-methylenebis(phenyl isocyanate) (MDI) to form the corresponding cross-linked materials. The materials were annealed at 150 C to complete the coupling reaction. Robust nanoporous materials were obtained from the cross-linked polymers by treatment with aqueous base to hydrolyze the PLA phase. Small-angle X-ray scattering study combined with scanning electron microscopy showed that MDI-cross-linked PLA-b-P(S-co-HEMA)/PLA-b-P(S-co-HEA) can adopt lamellar, hexagonally perforated lamellar, and hexagonally packed cylindrical morphologies after annealing. In particular, the HPL morphology was found to evolve from lamellae due to increase in volume fraction of PS phase as MDI reacted with hydroxyl groups. The reaction also kinetically trapped the morphology by cross-linking. Bicontinuous morphologies were also observed when dibutyltin dilaurate was added to accelerate reaction between the polymer and MDI.

  12. 21 CFR 177.2710 - Styrene-divinylbenzene resins, cross-linked.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... SERVICES (CONTINUED) FOOD FOR HUMAN CONSUMPTION (CONTINUED) INDIRECT FOOD ADDITIVES: POLYMERS Substances..., preparing, treating, packaging, transporting, or holding food, in accordance with the following prescribed... 21 Food and Drugs 3 2010-04-01 2009-04-01 true Styrene-divinylbenzene resins, cross-linked....

  13. Quantitative cross-linking/mass spectrometry reveals subtle protein conformational changes

    PubMed Central

    2016-01-01

    Quantitative cross-linking/mass spectrometry (QCLMS) probes protein structural dynamics in solution by quantitatively comparing the yields of cross-links between different conformational statuses. We have used QCLMS to understand the final maturation step of the proteasome lid and also to elucidate the structure of complement C3(H2O). Here we benchmark our workflow using a structurally well-described reference system, the human complement protein C3 and its activated cleavage product C3b. We found that small local conformational changes affect the yields of cross-linking residues that are near in space while larger conformational changes affect the detectability of cross-links. Distinguishing between minor and major changes required robust analysis based on replica analysis and a label-swapping procedure. By providing workflow, code of practice and a framework for semi-automated data processing, we lay the foundation for QCLMS as a tool to monitor the domain choreography that drives binary switching in many protein-protein interaction networks. PMID:27976756

  14. Cross-Linking Reactions for the Conversion of Polyphosphazenes into Useful Materials

    DTIC Science & Technology

    1994-05-18

    dissolve. Such solvent-swelled "gels" are known as hydrogels if the solvent is water, or organogels if an organic olvent is employed. Materials of this...An IPN consists of an organogel in which the monomer of one polymer is dissolved in the cross-linked matrix of the other. Polymerization of the

  15. Preparation of Nanocellulose Reinforced Chitosan Films, Cross-Linked by Adipic Acid.

    PubMed

    Falamarzpour, Pouria; Behzad, Tayebeh; Zamani, Akram

    2017-02-13

    Adipic acid, an abundant and nontoxic compound, was used to dissolve and cross-link chitosan. After the preparation of chitosan films through casting technique, the in situ amidation reaction was performed at 80-100 °C as verified by Fourier transform infrared (FT-IR). The reaction was accompanied by the release of water which was employed to investigate the reaction kinetics. Accordingly, the reaction rate followed the first-order model and Arrhenius equation, and the activation energy was calculated to be 18 kJ/mol. Furthermore, the mechanical properties of the chitosan films were comprehensively studied. First, optimal curing conditions (84 °C, 93 min) were introduced through a central composite design. In order to evaluate the effects of adipic acid, the mechanical properties of physically cross-linked (uncured), chemically cross-linked (cured), and uncross-linked (prepared by acetic acid) films were compared. The use of adipic acid improved the tensile strength of uncured and chemically cross-linked films more than 60% and 113%, respectively. Finally, the effect of cellulose nanofibrils (CNFs) on the mechanical performance of cured films, in the presence of glycerol as a plasticizer, was investigated. The plasticized chitosan films rein