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Sample records for actin cross-linking activity

  1. 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.

  2. 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

  3. 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.

  4. 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

  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. 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.

  7. 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.

  8. 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.

  9. 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.

  10. 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

  11. 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.

  12. 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.

  13. 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

  14. 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

  15. 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.

  16. 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.

  17. 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

  18. 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.

  19. 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

  20. 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.

  1. 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.

  2. 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.

  3. 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

  4. 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.

  5. 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

  6. 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.

  7. 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

  8. 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

  9. 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.

  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. 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

  13. 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.

  14. 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

  15. 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.

  16. 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.

  17. 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.

  18. 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

  19. 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

  20. Active force generation in cross-linked filament bundles without motor proteins.

    PubMed

    Walcott, Sam; Sun, Sean X

    2010-11-01

    Cytoskeletal filaments often interact laterally through cross-linking proteins, contributing to passive cellular viscoelasticity and, perhaps surprisingly, active force generation. We present a theory, based on the formation and rupture of cross-linker bonds, that relates molecular properties of those interactions to the macroscale mechanics of filament bundles. Computing the force-velocity relation for such a bundle, we demonstrate significant contractile forces in the absence of molecular motors. This theory provides insight into cytokinesis, cytoskeletal mechanics, and stress-fiber contraction.

  1. Synthesis and Antimicrobial Activity of Some Novel Cross-Linked Chitosan Hydrogels

    PubMed Central

    Mohamed, Nadia Ahmed; Fahmy, Mona Mohamed

    2012-01-01

    Four novel hydrogels based on chitosan were synthesized via a cross-linking reaction of chitosan with different concentrations of oxalyl bis 4-(2,5-dioxo-2H-pyrrol- 1(5H)-yl)benzamide. Their structures were confirmed by fourier transform infrared X-ray (FTIR), scanning electron microscopy (SEM) and X-ray diffraction. The antimicrobial activities of the hydrogels against two crop-threatening pathogenic fungi namely: Aspergillus fumigatus (A. fumigatus, RCMBA 06002), and Aspergillus niger (A. niger, RCMBA 06106), and five bacterial species namely: Bacillis subtilis (B. subtilis, RCMBA 6005), Staphylococcus aureus (S. aureus, RCMBA 2004), Streptococcus pneumoniae (S. pneumonia, RCMB 000101) as Gram positive bacteria, and Salmonella typhimurium (S. typhimurium, RCMB 000104), and Escherichia coli (E. coli, RCMBA 5003) as Gram negative bacteria have been investigated. The prepared hydrogels showed much higher antimicrobial activities than that of the parent chitosan. The hydrogels were more potent in case of Gram-positive bacteria than Gram-negative bacteria. Increasing the degree of cross-linking in the hydrogels resulted in a weaker antimicrobial activity. PMID:23109847

  2. 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

  3. Aromatic nitrogen mustard-based prodrugs: activity, selectivity, and the mechanism of DNA cross-linking.

    PubMed

    Chen, Wenbing; Han, Yanyan; Peng, Xiaohua

    2014-06-10

    Three novel H2O2-activated aromatic nitrogen mustard prodrugs (6-8) are reported. These compounds contain a DNA alkylating agent connected to a H2O2-responsive trigger by different electron-withdrawing linkers so that they are inactive towards DNA but can be triggered by H2O2 to release active species. The activity and selectivity of these compounds towards DNA were investigated by measuring DNA interstrand cross-link (ICL) formation in the presence or absence of H2O2. An electron-withdrawing linker unit, such as a quaternary ammonia salt (6), a carboxyamide (7), and a carbonate group (8), is sufficient to deactivate the aromatic nitrogen mustard resulting in less than 1.5 % cross-linking formation. However, H2O2 can restore the activity of the effectors by converting a withdrawing group to a donating group, therefore increasing the cross-linking efficiency (>20 %). The stability and reaction sites of the ICL products were determined, which revealed that alkylation induced by 7 and 8 not only occurred at the purine sites but also at the pyrimidine site. For the first time, we isolated and characterized the monomer adducts formed between the canonical nucleosides and the aromatic nitrogen mustard (15) which supported that nitrogen mustards reacted with dG, dA, and dC. The activation mechanism was studied by NMR spectroscopic analysis. An in vitro cytotoxicity assay demonstrated that compound 7 with a carboxyamide linker dramatically inhibited the growth of various cancer cells with a GI50 of less than 1 μM, whereas compound 6 with a charged linker did not show any obvious toxicity in all cell lines tested. These data indicated that a neutral carboxyamide linker is preferable for developing nitrogen mustard prodrugs. Our results showed that 7 is a potent anticancer prodrug that can serve as a model compound for further development. We believe these novel aromatic nitrogen mustards will inspire further and effective applications.

  4. Disulfide cross-linking influences symbiotic activities of nodule peptide NCR247.

    PubMed

    Shabab, Mohammed; Arnold, Markus F F; Penterman, Jon; Wommack, Andrew J; Bocker, Hartmut T; Price, Paul A; Griffitts, Joel S; Nolan, Elizabeth M; Walker, Graham C

    2016-09-06

    Interactions of rhizobia with legumes establish the chronic intracellular infection that underlies symbiosis. Within nodules of inverted repeat-lacking clade (IRLC) legumes, rhizobia differentiate into nitrogen-fixing bacteroids. This terminal differentiation is driven by host nodule-specific cysteine-rich (NCR) peptides that orchestrate the adaptation of free-living bacteria into intracellular residents. Medicago truncatula encodes a family of >700 NCR peptides that have conserved cysteine motifs. NCR247 is a cationic peptide with four cysteines that can form two intramolecular disulfide bonds in the oxidized forms. This peptide affects Sinorhizobium meliloti transcription, translation, and cell division at low concentrations and is antimicrobial at higher concentrations. By preparing the three possible disulfide-cross-linked NCR247 regioisomers, the reduced peptide, and a variant lacking cysteines, we performed a systematic study of the effects of intramolecular disulfide cross-linking and cysteines on the activities of an NCR peptide. The relative activities of the five NCR247 variants differed strikingly among the various bioassays, suggesting that the NCR peptide-based language used by plants to control the development of their bacterial partners during symbiosis is even greater than previously recognized. These patterns indicate that certain NCR bioactivities require cysteines whereas others do not. The results also suggest that NCR247 may exert some of its effects within the cell envelope whereas other activities occur in the cytoplasm. BacA, a membrane protein that is critical for symbiosis, provides protection against all bactericidal forms of NCR247. Oxidative folding protects NCR247 from degradation by the symbiotically relevant metalloprotease HrrP (host range restriction peptidase), suggesting that disulfide bond formation may additionally stabilize NCR peptides during symbiosis.

  5. Disulfide cross-linking influences symbiotic activities of nodule peptide NCR247

    PubMed Central

    Shabab, Mohammed; Arnold, Markus F. F.; Penterman, Jon; Wommack, Andrew J.; Bocker, Hartmut T.; Price, Paul A.; Griffitts, Joel S.; Nolan, Elizabeth M.; Walker, Graham C.

    2016-01-01

    Interactions of rhizobia with legumes establish the chronic intracellular infection that underlies symbiosis. Within nodules of inverted repeat-lacking clade (IRLC) legumes, rhizobia differentiate into nitrogen-fixing bacteroids. This terminal differentiation is driven by host nodule-specific cysteine-rich (NCR) peptides that orchestrate the adaptation of free-living bacteria into intracellular residents. Medicago truncatula encodes a family of >700 NCR peptides that have conserved cysteine motifs. NCR247 is a cationic peptide with four cysteines that can form two intramolecular disulfide bonds in the oxidized forms. This peptide affects Sinorhizobium meliloti transcription, translation, and cell division at low concentrations and is antimicrobial at higher concentrations. By preparing the three possible disulfide–cross-linked NCR247 regioisomers, the reduced peptide, and a variant lacking cysteines, we performed a systematic study of the effects of intramolecular disulfide cross-linking and cysteines on the activities of an NCR peptide. The relative activities of the five NCR247 variants differed strikingly among the various bioassays, suggesting that the NCR peptide-based language used by plants to control the development of their bacterial partners during symbiosis is even greater than previously recognized. These patterns indicate that certain NCR bioactivities require cysteines whereas others do not. The results also suggest that NCR247 may exert some of its effects within the cell envelope whereas other activities occur in the cytoplasm. BacA, a membrane protein that is critical for symbiosis, provides protection against all bactericidal forms of NCR247. Oxidative folding protects NCR247 from degradation by the symbiotically relevant metalloprotease HrrP (host range restriction peptidase), suggesting that disulfide bond formation may additionally stabilize NCR peptides during symbiosis. PMID:27551097

  6. Resveratrol cross-linked chitosan loaded with phospholipid for controlled release and antioxidant activity.

    PubMed

    Jeong, Hun; Samdani, Kunda J; Yoo, Dong Hyuck; Lee, Dong Won; Kim, Nam Hoon; Yoo, Il-Soo; Lee, Joong Hee

    2016-12-01

    Despite the therapeutic effects of resveratrol, its clinical application is restricted by its poor oral bioavailability and low water solubility. To overcome these physicochemical and pharmacokinetic limitations, encapsulation of resveratrol (RV) into nanodevices has been explored. Resveratrol cross-linked chitosan nanoparticles modified with phospholipids (RVC-lipid) were synthesized using a double emulsion technique. The surface morphology of RVC-lipid nanoparticles was evaluated with field emission scanning electron microscopy (FESEM) and transmission electron microscopy (TEM). Particle size was measured using dynamic light scattering technique (DLS), X-ray diffraction (XRD) was performed to identify the crystallographic nature and Fourier transform infrared spectroscopy (FTIR) was used to measure changes in the chemical structures of the resveratrol and RVC-lipid nanoparticles. Results showed RVC-lipid nanoparticle had a characteristic amorphous structure, a mean particle sizes of 570nm in DI water and 950nm in ethanol, and an encapsulation efficiency of 63.82% in aqueous medium and 85.59% in ethanol medium. In-vitro release studies demonstrated a slow and sustained release of resveratrol governed by diffusion. Based on assays of antioxidant activity the scavenging activity of RVC-lipid nanoparticles was inferior to that of resveratrol due to its prolonged release. We concluded that phospholipids are the potential carriers for resveratrol.

  7. Properties of collagen gels cross-linked by N-hydroxysuccinimide activated adipic acid deriviate.

    PubMed

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

    2014-08-01

    In order to improve the properties of collagen gel, N-hydroxysuccinimide activated adipic acid derivative (NHS-AA) was introduced into the formation of collagen fibrils. NHS-AA with different [NHS-AA]/[NH2] ratios (0.1-1.5, calculated by [ester group] of NHS-AA and [NH2] of lysine and hydroxylysine residues of collagen) was added after, simultaneously with or before the formation of collagen fibrils (abbreviated CAF, CSF and CBF, respectively) to obtain different collagen gels. With the same dose of NHS-AA, the cross-linking degree for CAF was lower than those for CSF and CBF. The formation of collagen fibrils was restrained by NHS-AA for CSF and CBF while that for CAF was unaffected. When the dose of NHS-AA increased from 0.1 to 1.5, the water contents of CSF and CBF increased while that of CAF had no obvious change. With lower dose of NHS-AA (0.1), CAF possessed higher value of G' (87.3Pa) and the best thermal stability (47.6°C). As the ratio of [NHS-AA]/[NH2] increased to 1.5, CSF had the maximum value of G' (288.8Pa) and CAF had the best thermal stability (52.9°C). These results showed collagen gels with different properties could be prepared by adding NHS-AA with different adding sequence and dose.

  8. FKBP65-dependent peptidyl-prolyl isomerase activity potentiates the lysyl hydroxylase 2-driven collagen cross-link switch

    PubMed Central

    Chen, Yulong; Terajima, Masahiko; Banerjee, Priyam; Guo, Houfu; Liu, Xin; Yu, Jiang; Yamauchi, Mitsuo; Kurie, Jonathan M.

    2017-01-01

    Bruck Syndrome is a connective tissue disease associated with inactivating mutations in lysyl hydroxylase 2 (LH2/PLOD2) or FK506 binding protein 65 (FKBP65/FKBP10). However, the functional relationship between LH2 and FKBP65 remains unclear. Here, we postulated that peptidyl prolyl isomerase (PPIase) activity of FKBP65 positively modulates LH2 enzymatic activity and is critical for the formation of hydroxylysine-aldehyde derived intermolecular collagen cross-links (HLCCs). To test this hypothesis, we analyzed collagen cross-links in Fkbp10-null and –wild-type murine embryonic fibroblasts. Although LH2 protein levels did not change, FKBP65 deficiency significantly diminished HLCCs and increased the non-hydroxylated lysine-aldehyde–derived collagen cross-links (LCCs), a pattern consistent with loss of LH2 enzymatic activity. The HLCC-to-LCC ratio was rescued in FKBP65-deficient murine embryonic fibroblasts by reconstitution with wild-type but not mutant FKBP65 that lacks intact PPIase domains. Findings from co-immunoprecipitation, protein-fragment complementation, and co-immunofluorescence assays showed that LH2 and FKBP65 are part of a common protein complex. We conclude that FKBP65 regulates LH2-mediated collagen cross-linking. Because LH2 promotes fibrosis and cancer metastasis, our findings suggest that pharmacologic strategies to target FKBP65 and LH2 may have complementary therapeutic activities. PMID:28378777

  9. Preparation, stability and antimicrobial activity of cationic cross-linked starch-iodine complexes.

    PubMed

    Klimaviciute, Rima; Bendoraitiene, Joana; Rutkaite, Ramune; Siugzdaite, Jurate; Zemaitaitis, Algirdas

    2012-12-01

    Cationic cross-linked starch (CCS)-iodine complexes containing different amounts of quaternary ammonium groups (different degrees of substitution (DS)) and iodine have been obtained by iodine adsorption on CCS from aqueous iodine potassium iodide solution. Equilibrium adsorption studies showed that with an increase of DS the amount of iodine adsorbed on CCS and the affinity of iodine to CCS increased linearly. The influences of the DS of CCS and the amount of adsorbed iodine on the stability of CCS-iodine complexes in a solution of 0.02M sodium acetate and reactivity toward l-tyrosine have been investigated. At the same DS, the stability of CCS-iodine complexes decreased with an increase of the amount of adsorbed iodine. With increasing the DS, the stability of CCS-iodine complexes increased. The iodine consumption in the reaction with l-tyrosine increased significantly with an increase of the amount of adsorbed iodine. The influence of DS on iodine consumption was lower and depended on the amount of adsorbed iodine. The antibacterial activity of CCS-iodine complexes against Bacillus cereus, Staphylococcus aureus, Pseudomonas aeruginosa and Escherichia coli was determined by the broth-dilution and spread-plate methods. The obtained results have demonstrated that an appropriate selection of the CCS-iodine complex composition (the DS of CCS and the amount of adsorbed iodine) could ensure good antimicrobial properties by keeping a low concentration of free iodine in the system. The main advantage of using CCS-iodine complexes as antimicrobial agents is the biodegradability of the polymeric matrix.

  10. Characterization of a redox active cross-linked complex between cyanobacterial photosystem I and soluble ferredoxin.

    PubMed Central

    Lelong, C; Boekema, E J; Kruip, J; Bottin, H; Rögner, M; Sétif, P

    1996-01-01

    A covalent stoichiometric complex between photosystem I (PSI) and ferredoxin from the cyanobacterium Synechocystis sp. PCC 6803 was generated by chemical cross-linking. The photoreduction of ferredoxin, studied by laser flash absorption spectroscopy between 460 and 600 nm, is a fast process in 60% of the covalent complexes, which exhibit spectral and kinetic properties very similar to those observed with the free partners. Two major phases with t(1/2) <1 micros and approximately 10-14 micros are observed at two different pH values (5.8 and 8.0). The remaining complexes do not undergo fast ferredoxin reduction and 20-25% of the complexes are still able to reduce free ferredoxin or flavodoxin efficiently, thus indicating that ferredoxin is not bound properly in this proportion of covalent complexes. The docking site of ferredoxin on PSI was determined by electron microscopy in combination with image analysis. Ferredoxin binds to the cytoplasmic side of PSI, with its mass center 77 angstroms distant from the center of the trimer and in close contact with a ridge formed by the subunits PsaC, PsaD and PsaE. This docking site corresponds to a close proximity between the [2Fe- 2S] center of ferredoxin and the terminal [4Fe-4S] acceptor FII of PSI and is very similar in position to the docking site of flavodoxin, an alternative electron acceptor of PSI. Images PMID:8641281

  11. 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

  12. Cross-linked enzyme aggregates (CLEAs) of selected lipases: a procedure for the proper calculation of their recovered activity

    PubMed Central

    2013-01-01

    In the last few years, synthesis of carrier-free immobilized biocatalysts by cross-linking of enzyme aggregates has appeared as a promising technique. Cross-linked enzyme aggregates (CLEAs) present several interesting advantages over carrier-bound immobilized enzymes, such as highly concentrated enzymatic activity, high stability of the produced superstructure, important production costs savings by the absence of a support, and the fact that no previous purification of the enzyme is needed. However, the published literature evidences that a) much specific non-systematic exploratory work is being done and, b) recovered activity calculations in CLEAs still need to be optimized. In this context, this contribution presents results of an optimized procedure for the calculation of the activity retained by CLEAs, based on the comparison of their specific activity relative to their free enzyme counterparts. The protocol implies determination of precipitable protein content in commercial enzyme preparations through precipitation with ammonium sulphate and a protein co-feeder. The identification of linear ranges of activity versus concentration/amount of protein in the test reaction is also required for proper specific activity determinations. By use of mass balances that involve the protein initially added to the synthesis medium, and the protein remaining in the supernatant and washing solutions (these last derived from activity measurements), the precipitable protein present in CLEAs is obtained, and their specific activity can be calculated. In the current contribution the described protocol was applied to CLEAs of Thermomyces lanuginosa lipase, which showed a recovered specific activity of 11.1% relative to native lipase. The approach described is simple and can easily be extended to other CLEAs and also to carrier-bound immobilized enzymes for accurate determination of their retained activity. PMID:23663379

  13. 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.

  14. Wear evaluation of a cross-linked medical grade polyethylene by ultra thin layer activation compared to gravimetry

    NASA Astrophysics Data System (ADS)

    Stroosnijder, Marinus F.; Hoffmann, Michael; Sauvage, Thierry; Blondiaux, Gilbert; Vincent, Laetitia

    2005-01-01

    Most of today's artificial joints rely on an articulating couple consisting of a CoCrMo alloy and a medical grade polyethylene. The wear of the polyethylene component is the major cause for long-term failure of these prostheses since the wear debris leads to adverse biological reactions. The polyethylene wear is usually measured by gravimetric methods, which are limited due to a low sensitivity and accuracy. To demonstrate the reliability of ultra thin layer activation (UTLA) as an alternative technique, wear tests on a cross-linked ultra-high-molecular weight polyethylene (XLPE) sliding against CoCrMo were performed on a wear tester featuring multi-directional sliding motion. The amount of polyethylene wear was evaluated by both UTLA and gravimetry. The particular TLA method used in this work employed the implantation of 7Be radioactive recoils into the polyethylene surface by means of a light mass particle beam. The results indicate that apart from its relatively high sensitivity, UTLA also offers the possibility for on-line measurements of polyethylene wear. This makes it a viable and complementary technique in wear test studies for medical implant purposes especially for those involving wear resistant materials and for rapid wear screening.

  15. Impact of estrogenic compounds on DNA integrity in human spermatozoa: evidence for cross-linking and redox cycling activities.

    PubMed

    Bennetts, L E; De Iuliis, G N; Nixon, B; Kime, M; Zelski, K; McVicar, C M; Lewis, S E; Aitken, R J

    2008-05-10

    A great deal of circumstantial evidence has linked DNA damage in human spermatozoa with adverse reproductive outcomes including reduced fertility and high rates of miscarriage. Although oxidative stress is thought to make a significant contribution to DNA damage in the male germ line, the factors responsible for creating this stress have not been elucidated. One group of compounds that are thought to be active in this context are the estrogens, either generated as a result of the endogenous metabolism of androgens within the male reproductive tract or gaining access to the latter as a consequence of environmental exposure. In this study, a wide variety of estrogenic compounds were assessed for their direct effects on human spermatozoa in vitro. DNA integrity was assessed using the Comet and TUNEL assays, lesion frequencies were quantified by QPCR using targets within the mitochondrial and nuclear (beta-globin) genomes, DNA adducts were characterized by mass spectrometry and redox activity was monitored using dihydroethidium (DHE) as the probe. Of the estrogenic and estrogen analogue compounds evaluated, catechol estrogens, quercetin, diethylstilbestrol and pyrocatechol stimulated intense redox activity while genistein was only active at the highest doses tested. Other estrogens and estrogen analogues, such as 17beta-estradiol, nonylphenol, bisphenol A and 2,3-dihydroxynaphthalene were inactive. Estrogen-induced redox activity was associated with a dramatic loss of motility and, in the case of 2-hydroxyestradiol, the induction of significant DNA fragmentation. Mass spectrometry also indicated that catechol estrogens were capable of forming dimers that can cross-link the densely packed DNA strands in sperm chromatin, impairing nuclear decondensation. These results highlight the potential importance of estrogenic compounds in creating oxidative stress and DNA damage in the male germ line and suggest that further exploration of these compounds in the aetiology of male

  16. Use of Activated Carbon in Packaging to Attenuate Formaldehyde-Induced and Formic Acid-Induced Degradation and Reduce Gelatin Cross-Linking in Solid Dosage Forms.

    PubMed

    Colgan, Stephen T; Zelesky, Todd C; Chen, Raymond; Likar, Michael D; MacDonald, Bruce C; Hawkins, Joel M; Carroll, Sophia C; Johnson, Gail M; Space, J Sean; Jensen, James F; DeMatteo, Vincent A

    2016-07-01

    Formaldehyde and formic acid are reactive impurities found in commonly used excipients and can be responsible for limiting drug product shelf-life. Described here is the use of activated carbon in drug product packaging to attenuate formaldehyde-induced and formic acid-induced drug degradation in tablets and cross-linking in hard gelatin capsules. Several pharmaceutical products with known or potential vulnerabilities to formaldehyde-induced or formic acid-induced degradation or gelatin cross-linking were subjected to accelerated stability challenges in the presence and absence of activated carbon. The effects of time and storage conditions were determined. For all of the products studied, activated carbon attenuated drug degradation or gelatin cross-linking. This novel use of activated carbon in pharmaceutical packaging may be useful for enhancing the chemical stability of drug products or the dissolution stability of gelatin-containing dosage forms and may allow for the 1) extension of a drug product's shelf-life when the limiting attribute is a degradation product induced by a reactive impurity, 2) marketing of a drug product in hotter and more humid climatic zones than currently supported without the use of activated carbon, and 3) enhanced dissolution stability of products that are vulnerable to gelatin cross-linking.

  17. Adjuvant Activity Enhanced by Cross-Linked CpG-Oligonucleotides in β-Glucan Nanogel and Its Antitumor Effect.

    PubMed

    Miyamoto, Noriko; Mochizuki, Shinichi; Fujii, Shota; Yoshida, Kenta; Sakurai, Kazuo

    2017-02-15

    Cancer vaccine has the ability to directly eradicate tumor cells by creating and activating cytotoxic T lymphocytes (CTLs). To achieve efficient CTL activity and to induce Th1 responses, it is essential to administer an appropriate adjuvant as well as an antigen. CpG-ODN is known as a ligand of Toll-like receptor 9 (TLR9) and strongly induces Th1 responses. In our previous study, we developed a CpG-ODN delivery system by use of the formation of complexes between ODN and a β-glucan SPG, denoted as CpG/SPG, and demonstrated that CpG/SPG induces high Th1 responses. In this study, we created a nanogel made from CpG/SPG complexes through DNA-DNA hybridization (cross-linked (CL)-CpG). Immunization with CL-CpG induced much stronger antigen-specific Th1 responses in combination with the antigenic protein ovalbumin (OVA) than that with CpG/SPG. Mice preimmunized with CL-CpG and OVA exhibited a long delay in tumor growth and an improved survival rate after tumor inoculation. These immune inductions can be attributed to the improvement of cellular uptake by the combination of increased size and the cluster effect of the β-glucan recognition site in the nanogel structure. In other words, the particle nature of CL-CpG, instead of the semiflexible rod conformation of CpG/SPG, enhanced the efficacy of a cancer vaccine. The present results indicate that CL-CpG can be used as a potent vaccine adjuvant for the treatment of cancers and infectious diseases.

  18. Biocatalytic methanolysis activities of cross-linked protein-coated microcrystalline lipase toward esterification/transesterification of relevant palm products.

    PubMed

    Raita, Marisa; Laosiripojana, Navadol; Champreda, Verawat

    2015-03-01

    Biocatalysis by immobilized lipase is an efficient alternative process for conversion of crude vegetable oil with high free fatty acid content to biodiesel, which is the limit of the conventional alkaline-catalyzed reaction. In this study, influences of solid-state organic and inorganic buffer core matrices with different pKa on catalytic performance of cross-linked protein coated microcrystalline biocatalysts prepared from Thermomyces lanuginosus lipase (CL-PCMC-LIP) toward esterification of palmitic acid (PA), transesterification of refined palm oil (RPO), and co-ester/transesterification of crude palm oil (CPO) to fatty acid methyl ester (FAME) was studied. Glycine, CAPSO (3-(cyclohexylamino)-2-hydroxy-1-propanesulfonic acid), and TAPS ([(2-hydroxy-1,1-bis(hydroxymethyl)ethyl)amino]-1-propanesulfonic acid) were shown to be potent core matrices for these reactions. The optimal reaction contained 4:1 [methanol]/[fatty acid] molar equivalence ratio with 20% (w/w) CL-PCMC-LIP on glycine in the presence of tert-butanol as a co-solvent. Deactivation effect of glycerol on the biocatalyst reactive surface was shown by FTIR, which could be alleviated by increasing co-solvent content. The maximal FAME yields from PA, RPO, and CPO reached 97.6, 94.9, and 95.5%, respectively on a molar basis under the optimum conditions after incubation at 50°C for 6h. The biocatalyst retained >80% activity after recycling in five consecutive batches. The work demonstrates the potential of CL-PCMC-LIP on one-step conversion of inexpensive crude fatty acid-rich feedstock to biodiesel.

  19. Selective activation of mitomycin A by thiols to form DNA cross-links and monoadducts: biochemical basis for the modulation of mitomycin cytotoxicity by the quinone redox potential.

    PubMed

    Paz, M M; Das, A; Palom, Y; He, Q Y; Tomasz, M

    2001-08-16

    Mitomycin A (MA) but not mitomycin C (MC) cross-linked linearized (32)P-pBR322 DNA in the presence of dithiothreitol (DTT) or glutathione (GSH), as shown by a sensitive DNA cross-link assay. Incubation of calf-thymus DNA with MA and DTT or mercaptoethanol (MER) resulted in the formation of MA-DNA adducts, which were isolated from nuclease digests of the drug-DNA complexes by HPLC. The adducts were characterized by their UV absorption spectra, electrospray ionization mass spectrometry (ESIMS), and facile conversion from 7-methoxy- to 7-amino-substituted mitosene type adducts upon 10% NH(4)OH treatment, which were identical with known adducts of MC. Both DNA interstrand and intrastrand cross-link adducts, linking two deoxyguanosine residues at N(2), as well as several deoxyguanosine-N(2) monoadducts of MA, were identified. No DNA adducts were formed with MC under the same conditions. A specificity of DNA cross-link formation for the CpG sequence was observed using 12-mer synthetic oligodeoxyribonucleotides as substrates and as DNA sequence models, in analogy to the known CpG sequence specificity of MC-induced DNA cross-links. MA is known to be more cytotoxic by 2-3 orders of magnitude than MC, and this property correlates with redox potentials of MA (-0.19 V) and MA analogues that are higher than those of MC (-0.40 V) and its analogues. It is suggested that the biochemical basis for the higher cytotoxic potency of MA is MA's propensity to be reductively activated by cellular thiols while MC is resistant to thiol activation. This distinction is probably derived from the large difference between the quinone redox potentials of the two drugs.

  20. Kinetics of sorption of polyaromatic hydrocarbons onto granular activated carbon and Macronet hyper-cross-linked polymers (MN200).

    PubMed

    Valderrama, C; Cortina, J L; Farran, A; Gamisans, X; Lao, C

    2007-06-01

    Polymeric supports are presented as an alternative to granular activated carbon (GAC) for organic contaminant removal from groundwater using permeable reactive barriers (PRB). The search for suitable polymeric sorbents for hydrocarbon extraction from aqueous streams has prompted the synthesis of new resins incorporating new functionalities or modifying the polymer network properties that solve many of the existing problems. Between them, the new type of polymeric sorbents Macronet Hypersol containing a styrene-divinylbenzene macroporous hyperreticulated network has been evaluated. Because of their potential sorptive properties, tests were conducted to determine the feasibility of using them as a low-cost reactive material for groundwater applications. The present work describes the sorption of six polycyclic hydrocarbons (PAHs) from aqueous solution onto both Macronet polymeric sorbent MN200 and granular activated carbon. Batch experiments were performed to determine loading rates of a family of PAHs (naphthalene, fluorene, anthracene, acenaphthene, pyrene, and fluoranthene), from a simple two-rings PAH (naphthalene) up to a four-ring PAH (pyrene). The behavior of a non-functionalized Macronet support (MN200) was compared with the behavior of a recognized material, granular activated carbon (GAC). Analyses of the respective rate data with three theoretical models (pseudo-first- and pseudo-second-order reaction models and the Elovich model) were used to describe the PAH sorption kinetics. Sorption rate constants were determined by graphical analysis of the proposed models. The study showed that sorption systems followed a pseudo-first-order reaction model, although the pseudo-second-order reaction model provides an acceptable description of the sorption process. Graphical analysis showed that the sorption process with activated carbon is a more complex process than the one observed for hyper-cross-linked polymers (MN200). A simulation of the barrier thickness needed

  1. Fc gamma receptor cross-linking activates p42, p38, and JNK/SAPK mitogen-activated protein kinases in murine macrophages: role for p42MAPK in Fc gamma receptor-stimulated TNF-alpha synthesis.

    PubMed

    Rose, D M; Winston, B W; Chan, E D; Riches, D W; Gerwins, P; Johnson, G L; Henson, P M

    1997-04-01

    Fc gamma R cross-linking on murine macrophages resulted in the activation of mitogen-activated protein kinase (MAPK) family members p42MAPK, p38, and c-Jun NH2-terminal kinase (JNK)/stress-activated protein kinase (SAPK). The temporal pattern of activation was distinct for each kinase. p42MAPK activation peaked at 5 min after receptor cross-linking, while peak p38 activity occurred 5 to 10 min later. Maximal JNK/SAPK activation occurred 20 min after Fc gamma R cross-linking. The selective MAPK/extracellular signal-regulated kinase-1 (MEK-1) inhibitor PD 098059 inhibited activation of p42MAPK induced by Fc gamma R cross-linking, but not p38 or JNK/SAPK activation. PD 098059 also inhibited the synthesis of TNF-alpha induced by Fc gamma R cross-linking (IC50 approximately 0.1 microM). Together, these results suggest that 1) the activation of MAPKs may play a role in Fc gammaR signal transduction, and 2) the activation of p42MAPK is necessary for Fc gamma R cross-linking-induced TNF-alpha synthesis.

  2. Anti-diabetic activity of cassava cross-linked octenyl succinic maltodextrin in STZ-induced diabetic mice.

    PubMed

    Wang, Li; Zheng, Maoqiang; Wang, Yingyao; Zhang, Ying; Qian, Haifeng; Zhang, Hui; Qi, Xiguang

    2014-03-01

    The effect of cassava cross-linked octenyl succinic maltodextrin (CCOMD) on diabetic mice was investigated in this study. For CCOMD-L (low dose) and CCOMD-H (high dose) groups, the body weights were recovered by 14.9% and 18.5%, respectively, which were significantly higher than that of model control group. It was also found that the blood glucose and insulin levels were ameliorated in the diabetic mice by the CCOMD diet. Moreover, the CCOMD diet decreased the plasma total cholesterol level (8.1-9.1%) and LDL cholesterol level (28.9-39.4%), and improved the plasma HDL cholesterol level (13.8-15.3%) and intestine short chain fatty acid content. The results indicated that CCOMD administration may be helpful for treating and preventing hyperlipidemia and hyperglycemia in diabetes.

  3. Regulation of Sodium Channel Activity by Capping of Actin Filaments

    PubMed Central

    Shumilina, Ekaterina V.; Negulyaev, Yuri A.; Morachevskaya, Elena A.; Hinssen, Horst; Khaitlina, Sofia Yu

    2003-01-01

    Ion transport in various tissues can be regulated by the cortical actin cytoskeleton. Specifically, involvement of actin dynamics in the regulation of nonvoltage-gated sodium channels has been shown. Herein, inside-out patch clamp experiments were performed to study the effect of the heterodimeric actin capping protein CapZ on sodium channel regulation in leukemia K562 cells. The channels were activated by cytochalasin-induced disruption of actin filaments and inactivated by G-actin under ionic conditions promoting rapid actin polymerization. CapZ had no direct effect on channel activity. However, being added together with G-actin, CapZ prevented actin-induced channel inactivation, and this effect occurred at CapZ/actin molar ratios from 1:5 to 1:100. When actin was allowed to polymerize at the plasma membrane to induce partial channel inactivation, subsequent addition of CapZ restored the channel activity. These results can be explained by CapZ-induced inhibition of further assembly of actin filaments at the plasma membrane due to the modification of actin dynamics by CapZ. No effect on the channel activity was observed in response to F-actin, confirming that the mechanism of channel inactivation does not involve interaction of the channel with preformed filaments. Our data show that actin-capping protein can participate in the cytoskeleton-associated regulation of sodium transport in nonexcitable cells. PMID:12686620

  4. Nanocomposites of C3N4 with Layers of MoS2 and Nitrogenated RGO, Obtained by Covalent Cross-linking: Synthesis, Characterization and HER Activity.

    PubMed

    Pramoda, K; Gupta, Uttam; Chhetri, Manjeet; Bandyopadhyay, Arkamita; Pati, Swapan K; Rao, Chintamani Nagesa Ramachandra

    2017-03-07

    Generation of hydrogen by photochemical, electrochemical and other means is a vital area of research today and a variety of materials has been explored as catalysts for this purpose. C3N4, MoS2 and nitrogenated RGO (NRGO) are some of the important catalytic materials investigated for the HER reaction but the observed catalytic activities are somewhat marginal. Prompted by preliminary reports that covalent cross-linking of 2D materials to generate hetero assemblies or nanocomposites may have beneficial effect on the catalytic activity, we have synthesized nanocomposites wherein C3N4 is covalently bonded to MoS2 or NRGO nanosheets. The photochemical HER activity of the C3N4-MoS2 nanocomposite is found to be remarkable with a activity of 12778 µmoles h-1g-1 and a TOF of 2.35 h-1. The physical mixture of C3N4 and MoS2, on the other hand, does not exhibit notable catalytic activity. Encouraged by this result, we have studied electrochemical HER activity of these composites as well. C3N4-MoS2 shows superior activity relative to a physical mixture of MoS2 and C3N4. DFT calculations have been carried out to understand the HER activity of the nanocomposites. Charge-transfer between the components and greater planarity of cross-linked layers are important causes of the superior catalytic activity of the nanocomposites. Covalent linking of such 2D materials appears to be a worthwhile strategy for catalysis and other applications.

  5. Hydrogel microspheres for stabilization of an antioxidant enzyme: effect of emulsion cross-linking of a dual polysaccharide system on the protection of enzyme activity.

    PubMed

    Tang, Deh-Wei; Yu, Shu-Huei; Wu, Wen-Shin; Hsieh, Hao-Ying; Tsai, Yi-Chin; Mi, Fwu-Long

    2014-01-01

    Catalase is an antioxidant enzyme abundant in natural resources. However, the enzyme is usually inactivated by gastric acid and digestive enzymes after oral ingestion. In this study, carboxymethyl chitosan (CM-chitosan) and hyaluronic acid (HA) conjugate hydrogel microspheres have been prepared by an emulsion cross-linking technique to retain the activity of catalase in simulated gastrointestinal (GI) fluids. Cross-linking reduced the swelling capability and increased the resistance toward hyaluronidase digestion of prepared HA-CM-chitosan hydrogel microspheres. Catalase entrapped in the hydrogel microspheres exhibited superior stability over a wide pH range (pH 2.0 and 6.0-8.0) as compared to the native enzyme. The entrapped catalase was also protected against degradation by digestive enzymes. Following the treatments, the catalase-loaded microspheres, in contrast to native catalase, could effectively decrease the intracellular H2O2 level and protect HT-29 colonic epithelial cells against H2O2-induced oxidative damage to preserve cell viability. These results suggested that the HA-CM-chitosan hydrogel microspheres can be used for entrapment, protection and intestinal delivery of catalase for H2O2 scavenging.

  6. Actin network architecture can determine myosin motor activity.

    PubMed

    Reymann, Anne-Cécile; Boujemaa-Paterski, Rajaa; Martiel, Jean-Louis; Guérin, Christophe; Cao, Wenxiang; Chin, Harvey F; De La Cruz, Enrique M; Théry, Manuel; Blanchoin, Laurent

    2012-06-08

    The organization of actin filaments into higher-ordered structures governs eukaryotic cell shape and movement. Global actin network size and architecture are maintained in a dynamic steady state through regulated assembly and disassembly. Here, we used experimentally defined actin structures in vitro to investigate how the activity of myosin motors depends on network architecture. Direct visualization of filaments revealed myosin-induced actin network deformation. During this reorganization, myosins selectively contracted and disassembled antiparallel actin structures, while parallel actin bundles remained unaffected. The local distribution of nucleation sites and the resulting orientation of actin filaments appeared to regulate the scalability of the contraction process. This "orientation selection" mechanism for selective contraction and disassembly suggests how the dynamics of the cellular actin cytoskeleton can be spatially controlled by actomyosin contractility.

  7. Evidence that Plasmodium falciparum chromosome end clusters are cross-linked by protein and are the sites of both virulence gene silencing and activation.

    PubMed

    Marty, Allison J; Thompson, Jennifer K; Duffy, Michael F; Voss, Till S; Cowman, Alan F; Crabb, Brendan S

    2006-10-01

    The malaria parasite Plasmodium falciparum undergoes antigenic variation through allelic exclusion and variant expression of surface proteins encoded by the var gene family. Regulation of var genes is under epigenetic control and involves reversible silencing and activation that requires the physical repositioning of a var locus into a transcriptionally permissive zone of the nuclear periphery. P. falciparum chromosome ends appear to aggregate into large perinuclear clusters which house both subtelomeric and chromosome central var genes. In this study we further define the composition of telomeric clusters using fluorescent in situ hybridization, and provide evidence that chromosome end clusters are formed by cross-linking protein. In addition, we demonstrate that a subtelomeric reporter gene and a var gene remain within clusters regardless of their transcriptional status. Our findings support a model whereby a highly localized structure dedicated to the activation of a single var gene can be housed within a gene dense chromosome end cluster that is otherwise transcriptionally silent.

  8. 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.

  9. Influence of unmodified and β-glycerophosphate cross-linked chitosan on anti-Candida activity of clotrimazole in semi-solid delivery systems.

    PubMed

    Szymańska, Emilia; Winnicka, Katarzyna; Wieczorek, Piotr; Sacha, Paweł Tomasz; Tryniszewska, Elżbieta Anna

    2014-09-30

    The combination of an antifungal agent and drug carrier with adjunctive antimicrobial properties represents novel strategy of complex therapy in pharmaceutical technology. The goal of this study was to investigate the unmodified and ion cross-linked chitosan's influence on anti-Candida activity of clotrimazole used as a model drug in hydrogels. It was particularly crucial to explore whether the chitosans' structure modification by β-glycerophosphate altered its antifungal properties. Antifungal studies (performed by plate diffusion method according to CLSI reference protocol) revealed that hydrogels obtained with chitosan/β-glycerophosphate displayed lower anti-Candida effect, probably as a result of weakened polycationic properties of chitosan in the presence of ion cross-linker. Designed chitosan hydrogels with clotrimazole were found to be more efficient against tested Candida strains and showed more favorable drug release profile compared to commercially available product. These observations indicate that novel chitosan formulations may be considered as promising semi-solid delivery system of clotrimazole.

  10. Active Chemical Thermodynamics promoted by activity of cortical actin

    NASA Astrophysics Data System (ADS)

    Bhattacharya, Bhaswati; Chaudhuri, Abhishek; Gowrishankar, Kripa; Rao, Madan

    2011-03-01

    The spatial distribution and dynamics of formation and breakup of the nanoclusters of cell surface proteins is controlled by the active remodeling dynamics of the underlying cortical actin. To explain these observations, we have proposed a novel mechanism of nanoclustering, involving the transient binding to and advection along constitutively occuring ``asters'' of cortical actin. We study the consequences of such active actin-based clustering, in the context of chemical reactions involving conformational changes of cell surface proteins. We find that the active remodeling of cortical actin, can give rise to a dramatic increase in efficiency and extent of conformational spread, even at low levels of expression at the cell surface. We define a activity temperature (τa) arising due to actin activities which can be used to describe chemical thermodynamics of the system. We plot TTT (time-temparature-transformation) curves and compute the Arrhenius factors which depend on τa . With this, the active asters can be treated as enzymes whose enzymatic reaction rate can be related to the activity.

  11. 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.

  12. 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.

  13. 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.

  14. Buckling-induced F-actin fragmentation modulates the contraction of active cytoskeletal networks.

    PubMed

    Li, Jing; Biel, Thomas; Lomada, Pranith; Yu, Qilin; Kim, Taeyoon

    2017-04-11

    Actomyosin contractility originating from interactions between F-actin and myosin facilitates various structural reorganizations of the actin cytoskeleton. Cross-linked actomyosin networks show a tendency to contract to single or multiple foci, which has been investigated extensively in numerous studies. Recently, it was suggested that suppression of F-actin buckling via an increase in bending rigidity significantly reduces network contraction. In this study, we demonstrate that networks may show the largest contraction at intermediate bending rigidity, not at the lowest rigidity, if filaments are severed by buckling arising from myosin activity as demonstrated in recent experiments; if filaments are very flexible, frequent severing events can severely deteriorate network connectivity, leading to the formation of multiple small foci and low network contraction. By contrast, if filaments are too stiff, the networks exhibit minimal contraction due to the inhibition of filament buckling. This study reveals that buckling-induced filament severing can modulate the contraction of active cytoskeletal networks, which has been neglected to date.

  15. BO-1055, a novel DNA cross-linking agent with remarkable low myelotoxicity shows potent activity in sarcoma models

    PubMed Central

    Ambati, Srikanth R.; Shieh, Jae-Hung; Pera, Benet; Lopes, Eloisi Caldas; Chaudhry, Anisha; Wong, Elissa W.P.; Saxena, Ashish; Su, Tsann-Long; Moore, Malcolm A.S.

    2016-01-01

    DNA damaging agents cause rapid shrinkage of tumors and form the basis of chemotherapy for sarcomas despite significant toxicities. Drugs having superior efficacy and wider therapeutic windows are needed to improve patient outcomes. We used cell proliferation and apoptosis assays in sarcoma cell lines and benign cells; γ-H2AX expression, comet assay, immunoblot analyses and drug combination studies in vitro and in patient derived xenograft (PDX) models. BO-1055 caused apoptosis and cell death in a concentration and time dependent manner in sarcoma cell lines. BO-1055 had potent activity (submicromolar IC50) against Ewing sarcoma and rhabdomyosarcoma, intermediate activity in DSRCT (IC50 = 2-3μM) and very weak activity in osteosarcoma (IC50 >10μM) cell lines. BO-1055 exhibited a wide therapeutic window compared to other DNA damaging drugs. BO-1055 induced more DNA double strand breaks and γH2AX expression in cancer cells compared to benign cells. BO-1055 showed inhibition of tumor growth in A673 xenografts and caused tumor regression in cyclophosphamide resistant patient-derived Ewing sarcoma xenografts and A204 xenografts. Combination of BO-1055 and irinotecan demonstrated synergism in Ewing sarcoma PDX models. Potent activity on sarcoma cells and its relative lack of toxicity presents a strong rationale for further development of BO-1055 as a therapeutic agent. PMID:27248664

  16. 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.

  17. AIE-Active Tetraphenylethylene Cross-Linked N-Isopropylacrylamide Polymer: A Long-Term Fluorescent Cellular Tracker.

    PubMed

    Ma, Hengchang; Qi, Chunxuan; Cheng, Chao; Yang, Zengming; Cao, Haiying; Yang, Zhiwang; Tong, Jinhui; Yao, Xiaoqiang; Lei, Ziqiang

    2016-04-06

    There is a great demand to understand cell transplantation, migration, division, fusion, and lysis. Correspondingly, illuminant object-labeled bioprobes have been employed as long-term cellular tracers, which could provide valuable insights into detecting these biological processes. In this work, we designed and synthesized a fluorescent polymer, which was comprised of hydrophilic N-isopropylacrylamide polymers as matrix and a hydrophobic tetraphenylethene (TPE) unit as AIE-active cross-linkers (DDBV). It was found that when the feed molar ratio of N-isopropylacrylamides to cross-linkers was 22:1, the produced polymers demonstrated the desirable LCST at 37.5 °C. And also, the temperature sensitivity of polymers could induce phase transfer within a narrow window (32-38 °C). Meanwhile, phase transfer was able to lead the florescent response. And thus, we concluded that two responses occur when one stimulus is input. Therefore, the new cross-linker of DDBV rendered a new performance from PNIPAm and a new chance to create new materials. Moreover, the resulted polymers demonstrated very good biocompatibility with living A549 human lung adenocarcinoma cells and L929 mouse fibroblast cells, respectively. Both of these cells retained very active viabilities in the concentration range of 7.8-125 μL/mg of polymers. Notably, P[(NIPAm)22-(DDBV)1] (P6) could be readily internalized by living cells with a noninvasive manner. The cellular staining by the fluorescent polymer is so indelible that it enables cell tracing for at least 10 passages.

  18. Structural Analysis of Guanylyl Cyclase-Activating Protein-2 (GCAP-2) Homodimer by Stable Isotope-Labeling, Chemical Cross-Linking, and Mass Spectrometry

    NASA Astrophysics Data System (ADS)

    Pettelkau, Jens; Thondorf, Iris; Theisgen, Stephan; Lilie, Hauke; Schröder, Thomas; Arlt, Christian; Ihling, Christian H.; Sinz, Andrea

    2013-12-01

    The topology of the GCAP-2 homodimer was investigated by chemical cross-linking and high resolution mass spectrometry. Complementary conducted size-exclusion chromatography and analytical ultracentrifugation studies indicated that GCAP-2 forms a homodimer both in the absence and in the presence of Ca2+. In-depth MS and MS/MS analysis of the cross-linked products was aided by 15 N-labeled GCAP-2. The use of isotope-labeled protein delivered reliable structural information on the GCAP-2 homodimer, enabling an unambiguous discrimination between cross-links within one monomer (intramolecular) or between two subunits (intermolecular). The limited number of cross-links obtained in the Ca2+-bound state allowed us to deduce a defined homodimeric GCAP-2 structure by a docking and molecular dynamics approach. In the Ca2+-free state, GCAP-2 is more flexible as indicated by the higher number of cross-links. We consider stable isotope-labeling to be indispensable for deriving reliable structural information from chemical cross-linking data of multi-subunit protein assemblies.

  19. A redox-active, compact molecule for cross-linking amyloidogenic peptides into nontoxic, off-pathway aggregates: In vitro and in vivo efficacy and molecular mechanisms

    SciTech Connect

    Derrick, Jeffrey S.; Kerr, Richard A.; Nam, Younwoo; Oh, Shin Bi; Lee, Hyuck Jin; Earnest, Kaylin G.; Suh, Nayoung; Peck, Kristy L.; Ozbil, Mehmet; Korshavn, Kyle J.; Ramamoorthy, Ayyalusamy; Prabhakar, Rajeev; Merino, Edward J.; Shearer, Jason; Lee, Joo -Yong; Ruotolo, Brandon T.; Lim, Mi Hee

    2015-11-17

    Chemical reagents targeting and controlling amyloidogenic peptides have received much attention for helping identify their roles in the pathogenesis of protein-misfolding disorders. In this paper, we report a novel strategy for redirecting amyloidogenic peptides into nontoxic, off-pathway aggregates, which utilizes redox properties of a small molecule (DMPD, N,N-dimethyl-p-phenylenediamine) to trigger covalent adduct formation with the peptide. In addition, for the first time, biochemical, biophysical, and molecular dynamics simulation studies have been performed to demonstrate a mechanistic understanding for such an interaction between a small molecule (DMPD) and amyloid-β (Aβ) and its subsequent anti-amyloidogenic activity, which, upon its transformation, generates ligand–peptide adducts via primary amine-dependent intramolecular cross-linking correlated with structural compaction. Furthermore, in vivo efficacy of DMPD toward amyloid pathology and cognitive impairment was evaluated employing 5xFAD mice of Alzheimer’s disease (AD). Such a small molecule (DMPD) is indicated to noticeably reduce the overall cerebral amyloid load of soluble Aβ forms and amyloid deposits as well as significantly improve cognitive defects in the AD mouse model. Altogether our in vitro and in vivo studies of DMPD toward Aβ with the first molecular-level mechanistic investigations present the feasibility of developing new, innovative approaches that employ redox-active compounds without the structural complexity as next-generation chemical tools for amyloid management.

  20. A redox-active, compact molecule for cross-linking amyloidogenic peptides into nontoxic, off-pathway aggregates: In vitro and in vivo efficacy and molecular mechanisms

    DOE PAGES

    Derrick, Jeffrey S.; Kerr, Richard A.; Nam, Younwoo; ...

    2015-11-17

    Chemical reagents targeting and controlling amyloidogenic peptides have received much attention for helping identify their roles in the pathogenesis of protein-misfolding disorders. In this paper, we report a novel strategy for redirecting amyloidogenic peptides into nontoxic, off-pathway aggregates, which utilizes redox properties of a small molecule (DMPD, N,N-dimethyl-p-phenylenediamine) to trigger covalent adduct formation with the peptide. In addition, for the first time, biochemical, biophysical, and molecular dynamics simulation studies have been performed to demonstrate a mechanistic understanding for such an interaction between a small molecule (DMPD) and amyloid-β (Aβ) and its subsequent anti-amyloidogenic activity, which, upon its transformation, generates ligand–peptidemore » adducts via primary amine-dependent intramolecular cross-linking correlated with structural compaction. Furthermore, in vivo efficacy of DMPD toward amyloid pathology and cognitive impairment was evaluated employing 5xFAD mice of Alzheimer’s disease (AD). Such a small molecule (DMPD) is indicated to noticeably reduce the overall cerebral amyloid load of soluble Aβ forms and amyloid deposits as well as significantly improve cognitive defects in the AD mouse model. Altogether our in vitro and in vivo studies of DMPD toward Aβ with the first molecular-level mechanistic investigations present the feasibility of developing new, innovative approaches that employ redox-active compounds without the structural complexity as next-generation chemical tools for amyloid management.« less

  1. 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

  2. Self-assembled complexes of non-cross-linked amphiphilic polymeric ligands with inorganic species: highly active and reusable solid-phase polymeric catalysts.

    PubMed

    Yamada, Yoichi M A

    2005-07-01

    I present herein the development of highly active and reusable polymeric catalysts produced by self-assembly process of non-cross-linked amphiphilic polymeric ligands with inorganic species. Thus, PWAA 1 prepared from H3PW12O40 and poly[(N-isopropylacrylamide)-co-(acrylamide with ammonium salt)] is suitable for oxidation of alcohols, amines, and sulfides in aqueous hydrogen peroxide. PdAS 2 produced by self-organization of (NH4)2PdCl4 and poly[(N-isopropylacrylamide)10-co-diphenylphosphinostyrene] is an excellent recyclable catalyst for Suzuki-Miyaura reaction in water, water-organic solvent, and organic solvent. It is commercially available from Tokyo Kasei Kogyo (TCI). PdAS-V 3 assembled from (NH4)2PdCl4 and poly[(N-isopropylacrylamide)5-co-diphenylphosphinostyrene] provides recycling system of itself for Mizorogi-Heck reaction. TiSS 4 made from Ti(O-i-Pr)4 and poly(styryl-linked binaphtholate-co-styrene) promotes an enantioselective carbonyl-ene reaction as a recyclable catalyst.

  3. Villin severing activity enhances actin-based motility in vivo.

    PubMed

    Revenu, Céline; Courtois, Matthieu; Michelot, Alphée; Sykes, Cécile; Louvard, Daniel; Robine, Sylvie

    2007-03-01

    Villin, an actin-binding protein associated with the actin bundles that support microvilli, bundles, caps, nucleates, and severs actin in a calcium-dependant manner in vitro. We hypothesized that the severing activity of villin is responsible for its reported role in enhancing cell plasticity and motility. To test this hypothesis, we chose a loss of function strategy and introduced mutations in villin based on sequence comparison with CapG. By pyrene-actin assays, we demonstrate that this mutant has a strongly reduced severing activity, whereas nucleation and capping remain unaffected. The bundling activity and the morphogenic effects of villin in cells are also preserved in this mutant. We thus succeeded in dissociating the severing from the three other activities of villin. The contribution of villin severing to actin dynamics is analyzed in vivo through the actin-based movement of the intracellular bacteria Shigella flexneri in cells expressing villin and its severing variant. The severing mutations abolish the gain of velocity induced by villin. To further analyze this effect, we reconstituted an in vitro actin-based bead movement in which the usual capping protein is replaced by either the wild type or the severing mutant of villin. Confirming the in vivo results, villin-severing activity enhances the velocity of beads by more than two-fold and reduces the density of actin in the comets. We propose a model in which, by severing actin filaments and capping their barbed ends, villin increases the concentration of actin monomers available for polymerization, a mechanism that might be paralleled in vivo when an enterocyte undergoes an epithelio-mesenchymal transition.

  4. Villin Severing Activity Enhances Actin-based Motility In Vivo

    PubMed Central

    Revenu, Céline; Courtois, Matthieu; Michelot, Alphée; Sykes, Cécile; Louvard, Daniel

    2007-01-01

    Villin, an actin-binding protein associated with the actin bundles that support microvilli, bundles, caps, nucleates, and severs actin in a calcium-dependant manner in vitro. We hypothesized that the severing activity of villin is responsible for its reported role in enhancing cell plasticity and motility. To test this hypothesis, we chose a loss of function strategy and introduced mutations in villin based on sequence comparison with CapG. By pyrene-actin assays, we demonstrate that this mutant has a strongly reduced severing activity, whereas nucleation and capping remain unaffected. The bundling activity and the morphogenic effects of villin in cells are also preserved in this mutant. We thus succeeded in dissociating the severing from the three other activities of villin. The contribution of villin severing to actin dynamics is analyzed in vivo through the actin-based movement of the intracellular bacteria Shigella flexneri in cells expressing villin and its severing variant. The severing mutations abolish the gain of velocity induced by villin. To further analyze this effect, we reconstituted an in vitro actin-based bead movement in which the usual capping protein is replaced by either the wild type or the severing mutant of villin. Confirming the in vivo results, villin-severing activity enhances the velocity of beads by more than two-fold and reduces the density of actin in the comets. We propose a model in which, by severing actin filaments and capping their barbed ends, villin increases the concentration of actin monomers available for polymerization, a mechanism that might be paralleled in vivo when an enterocyte undergoes an epithelio-mesenchymal transition. PMID:17182858

  5. Subcellular distribution and dynamics of active proteasome complexes unraveled by a workflow combining in vivo complex cross-linking and quantitative proteomics.

    PubMed

    Fabre, Bertrand; Lambour, Thomas; Delobel, Julien; Amalric, François; Monsarrat, Bernard; Burlet-Schiltz, Odile; Bousquet-Dubouch, Marie-Pierre

    2013-03-01

    Through protein degradation, the proteasome plays fundamental roles in different cell compartments. Although the composition of the 20S catalytic core particle (CP) has been well documented, little is known about the composition and dynamics of the regulatory complexes that play a crucial role in its activity, or about how they associate with the CP in different cell compartments, different cell lines, and in response to external stimuli. Because of difficulties performing acceptable cell fractionation while maintaining complex integrity, it has been challenging to characterize proteasome complexes by proteomic approaches. Here, we report an integrated protocol, combining a cross-linking procedure on intact cells with cell fractionation, proteasome immuno-purification, and robust label-free quantitative proteomic analysis by mass spectrometry to determine the distribution and dynamics of cellular proteasome complexes in leukemic cells. Activity profiles of proteasomes were correlated fully with the composition of protein complexes and stoichiometry. Moreover, our results suggest that, at the subcellular level, proteasome function is regulated by dynamic interactions between the 20S CP and its regulatory proteins-which modulate proteasome activity, stability, localization, or substrate uptake-rather than by profound changes in 20S CP composition. Proteasome plasticity was observed both in the 20S CP and in its network of interactions following IFNγ stimulation. The fractionation protocol also revealed specific proteolytic activities and structural features of low-abundance microsomal proteasomes from U937 and KG1a cells. These could be linked to their important roles in the endoplasmic reticulum associated degradation pathway in leukemic cells.

  6. 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

  7. 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.

  8. 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...

  9. 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.

  10. 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.

  11. Molecular motions involved in Na-K-Cl cotransporter-mediated ion transport and transporter activation revealed by internal cross-linking between transmembrane domains 10 and 11/12.

    PubMed

    Monette, Michelle Y; Somasekharan, Suma; Forbush, Biff

    2014-03-14

    We examined the relationship between transmembrane domain (TM) 10 and TM11/12 in NKCC1, testing homology models based on the structure of AdiC in the same transporter superfamily. We hypothesized that introduced cysteine pairs would be close enough for disulfide formation and would alter transport function: indeed, evidence for cross-link formation with low micromolar concentrations of copper phenanthroline or iodine was found in 3 of 8 initially tested pairs and in 1 of 26 additionally tested pairs. Inhibition of transport was observed with copper phenanthroline and iodine treatment of P676C/A734C and I677C/A734C, consistent with the proximity of these residues and with movement of TM10 during the occlusion step of ion transport. We also found Cu(2+) inhibition of the single-cysteine mutant A675C, suggesting that this residue and Met(382) of TM3 are involved in a Cu(2+)-binding site. Surprisingly, cross-linking of P676C/I730C was found to prevent rapid deactivation of the transporter while not affecting the dephosphorylation rate, thus uncoupling the phosphorylation and activation steps. Consistent with this, (a) cross-linking of P676C/I730C was dependent on activation state, and (b) mutants lacking the phosphoregulatory domain could still be activated by cross-linking. These results suggest a model of NKCC activation that involves movement of TM12 relative to TM10, which is likely tied to movement of the large C terminus, a process somehow triggered by phosphorylation of the regulatory domain in the N terminus.

  12. 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.

  13. 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

  14. 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.

  15. 3D actin network centerline extraction with multiple active contours.

    PubMed

    Xu, Ting; Vavylonis, Dimitrios; Huang, Xiaolei

    2014-02-01

    Fluorescence microscopy is frequently used to study two and three dimensional network structures formed by cytoskeletal polymer fibers such as actin filaments and actin cables. While these cytoskeletal structures are often dilute enough to allow imaging of individual filaments or bundles of them, quantitative analysis of these images is challenging. To facilitate quantitative, reproducible and objective analysis of the image data, we propose a semi-automated method to extract actin networks and retrieve their topology in 3D. Our method uses multiple Stretching Open Active Contours (SOACs) that are automatically initialized at image intensity ridges and then evolve along the centerlines of filaments in the network. SOACs can merge, stop at junctions, and reconfigure with others to allow smooth crossing at junctions of filaments. The proposed approach is generally applicable to images of curvilinear networks with low SNR. We demonstrate its potential by extracting the centerlines of synthetic meshwork images, actin networks in 2D Total Internal Reflection Fluorescence Microscopy images, and 3D actin cable meshworks of live fission yeast cells imaged by spinning disk confocal microscopy. Quantitative evaluation of the method using synthetic images shows that for images with SNR above 5.0, the average vertex error measured by the distance between our result and ground truth is 1 voxel, and the average Hausdorff distance is below 10 voxels.

  16. Myosin II filament assemblies in the active lamella of fibroblasts: their morphogenesis and role in the formation of actin filament bundles

    PubMed Central

    1995-01-01

    The morphogenesis of myosin II structures in active lamella undergoing net protrusion was analyzed by correlative fluorescence and electron microscopy. In rat embryo fibroblasts (REF 52) microinjected with tetramethylrhodamine-myosin II, nascent myosin spots formed close to the active edge during periods of retraction and then elongated into wavy ribbons of uniform width. The spots and ribbons initially behaved as distinct structural entities but subsequently aligned with each other in a sarcomeric-like pattern. Electron microscopy established that the spots and ribbons consisted of bipolar minifilaments associated with each other at their head-containing ends and arranged in a single row in an "open" zig-zag conformation or as a "closed" parallel stack. Ribbons also contacted each other in a nonsarcomeric, network-like arrangement as described previously (Verkhovsky and Borisy, 1993. J. Cell Biol. 123:637-652). Myosin ribbons were particularly pronounced in REF 52 cells, but small ribbons and networks were found also in a range of other mammalian cells. At the edge of the cell, individual spots and open ribbons were associated with relatively disordered actin filaments. Further from the edge, myosin filament alignment increased in parallel with the development of actin bundles. In actin bundles, the actin cross-linking protein, alpha-actinin, was excluded from sites of myosin localization but concentrated in paired sites flanking each myosin ribbon, suggesting that myosin filament association may initiate a pathway for the formation of actin filament bundles. We propose that zig-zag assemblies of myosin II filaments induce the formation of actin bundles by pulling on an actin filament network and that co-alignment of actin and myosin filaments proceeds via folding of myosin II filament assemblies in an accordion-like fashion. PMID:7490299

  17. 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.

  18. Actin interaction and regulation of cyclin-dependent kinase 5/p35 complex activity.

    PubMed

    Xu, Jiqing; Tsutsumi, Koji; Tokuraku, Kiyotaka; Estes, Katherine A; Hisanaga, Shin-ichi; Ikezu, Tsuneya

    2011-01-01

    Cyclin-dependent kinase 5 (Cdk5) plays a critical role during neurodevelopment, synaptic plasticity, and neurodegeneration. Cdk5 activity depends on association with neuronal proteins p35 and p25, a proteolytic product of p35. Cdk5 regulates the actin cytoskeletal dynamics that are essential for neuronal migration, neuritic growth, and synaptogenesis. However, little is known about the interaction of actin and Cdk5 and its effect on neuronal Cdk5 activity. In a previous study, we observed that Cdk5/p35 activity is negatively correlated with co-immunoprecipitated F-actin (filamentous actin) amounts in the mouse brain, and suggested that F-actin inhibits the formation of the Cdk5/p35 complex [Journal of Neuroscience (2008) vol. 28, p. 14511]. The experiments reported here were undertaken to elucidate the relationship between actin and the formation of the Cdk5/p35 complex and its activity. Instead of an F-actin-mediated inhibition, we propose that G-actin (globular actin) in the F-actin preparations is responsible for inhibiting Cdk5/p35 and Cdk5/p25 kinase activity. We found that F-actin binds to p35 but not p25 or Cdk5. We have shown that G-actin binds directly to Cdk5 without disrupting the formation of the Cdk5/p35 or Cdk5/p25 complexes. G-actin potently suppressed Cdk5/p35 and Cdk5/p25 activity when either histone H1 or purified human tau protein were used as substrates, indicating a substrate-independent inhibitory effect of G-actin on Cdk5 activity. Finally, G-actin suppressed the activity of Cdk5 immunoprecipitated from wild type and p35-deficient mouse brain, suggesting that G-actin suppresses endogenous Cdk5 activity in a p35-independent manner. Together, these results suggest a novel mechanism of actin cytoskeletal regulation of Cdk5/p35 activity.

  19. 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.

  20. Contact dependent suppression of CD4 T cell activation and proliferation by B cells activated through IgD cross-linking.

    PubMed

    Preciado-Llanes, Lorena; Wing, James B; Foster, Rachel A; Carlring, Jennifer; Lees, Andrew; Read, Robert C; Heath, Andrew W

    2014-09-20

    Although the co-stimulatory interaction between B and T cells is well defined, recent evidence suggests that B cells also have a regulatory role. Here, we show that B cells activated using anti-IgD conjugated to dextran (α-δ-dex) directly inhibit TCR-induced CD4 T cell activation, proliferation and cytokine production. This effect was observed in CD4 T cells activated both with and without CD28 co-stimulation. T cell viability was unaffected, and the T cell suppressive effect was mediated by contact with IgD activated purified B cells and not by IL-10 or other soluble factors. This is the first evidence of IgD activated B cells mediating inhibition of activation and proliferation of CD4 T cells in humans. This article is protected by copyright. All rights reserved.

  1. 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.

  2. 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.

  3. 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.

  4. 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.

  5. 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.

  6. 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.

  7. 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.

  8. 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.

  9. 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.

  10. Cross-linked beads of activated oil palm ash zeolite/chitosan composite as a bio-adsorbent for the removal of methylene blue and acid blue 29 dyes.

    PubMed

    Khanday, W A; Asif, M; Hameed, B H

    2017-02-01

    Cross-linked beads of activated oil palm ash zeolite/chitosan (Z-AC/C) composite were prepared through the hydrothermal treatment of NaOH activated oil palm ash followed by beading with chitosan. The effects of initial dye concentration (50-400mg/L), temperature (30°C-50°C) and pH (3-13) on batch adsorption of methylene blue (MB) and acid blue 29 (AB29) were studied. Adsorption of both dyes was better described by Pseudo-second-order kinetics and Freundlich isotherm model. The maximum adsorption capacities of Z-AC/C were 151.51, 169.49, and 199.20mg/g for MB and 212.76, 238.09, and 270.27mg/g for AB29 at 30°C, 40°C, and 50°C, respectively.

  11. 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

  12. 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.

  13. 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.

  14. 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.

  15. 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.

  16. 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.

  17. Criticalities in crosslinked actin networks due to myosin activity

    NASA Astrophysics Data System (ADS)

    Sheinman, Michael

    2013-03-01

    Many essential processes in cells and tissues, like motility and morphogenesis, are orchestrated by molecular motors applying internal, active stresses on crosslinked networks of actin filaments. Using scaling analysis, mean-field calculation, numerical modelling and in vitro experiments of such active networks we predict and observe different mechanical regimes exhibiting interesting critical behaviours with non-trivial power-law dependencies. Firstly, we find that the presence of active stresses can dramatically increase the stiffness of a floppy network, as was observed in reconstituted intracellular F-actin networks with myosin motors and extracellular gels with contractile cells. Uniform internal stress results in an anomalous, critical mechanical regime only in the vicinity of the rigidity percolation points of the network. However, taking into account heterogeneity of motors, we demonstrate that the motors, stiffening any floppy network, induce large non-affine fluctuations, giving rise to a critical mechanical regime. Secondly, upon increasing motor concentration, the resulting large internal stress is able to significantly enhance unbinding of the network's crosslinks and, therefore, disconnect the initially well-connected network to isolated clusters. However, during this process, when the network approaches marginal connectivity the internal stresses are expected to drop drastically such that the connectivity stabilizes. This general argument and detailed numerical simulations show that motors should drive a well connected network to a close vicinity of a critical point of marginal connectivity. Experiments clearly confirm this conclusion and demonstrate robust critical connectivity of initially well-connected networks, ruptured by the motor activity for a wide range of parameters. M. Sheinman, C.P. Broedersz and F.C. MacKintosh, Phys. Rev. Lett, in press. J. Alvarado, M. Sheinman, A. Sharma, F.C. MacKintosh and G. Koenderink, in preparation.

  18. A defence-related Olea europaea β-glucosidase hydrolyses and activates oleuropein into a potent protein cross-linking agent.

    PubMed

    Koudounas, Konstantinos; Banilas, Georgios; Michaelidis, Christos; Demoliou, Catherine; Rigas, Stamatis; Hatzopoulos, Polydefkis

    2015-04-01

    Oleuropein, the major secoiridoid compound in olive, is involved in a sophisticated two-component defence system comprising a β-glucosidase enzyme that activates oleuropein into a toxic glutaraldehyde-like structure. Although oleuropein deglycosylation studies have been monitored extensively, an oleuropein β-glucosidase gene has not been characterized as yet. Here, we report the isolation of OeGLU cDNA from olive encoding a β-glucosidase belonging to the defence-related group of terpenoid-specific glucosidases. In planta recombinant protein expression assays showed that OeGLU deglycosylated and activated oleuropein into a strong protein cross-linker. Homology and docking modelling predicted that OeGLU has a characteristic (β/α)8 TIM barrel conformation and a typical construction of a pocket-shaped substrate recognition domain composed of conserved amino acids supporting the β-glucosidase activity and non-conserved residues associated with aglycon specificity. Transcriptional analysis in various olive organs revealed that the gene was developmentally regulated, with its transcript levels coinciding well with the spatiotemporal patterns of oleuropein degradation and aglycon accumulation in drupes. OeGLU upregulation in young organs reflects its prominent role in oleuropein-mediated defence system. High gene expression during drupe maturation implies an additional role in olive secondary metabolism, through the degradation of oleuropein and reutilization of hydrolysis products.

  19. 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.

  20. Cross-linked polyvinyl polymers versus polyureas as designed supports for catalytically active M(0) nanoclusters. Part III. Nanometer scale structure of the cross-linked polyurea support EnCat 30 and of the Pd(II)/EnCat 30 and Pd(0)/EnCat 30NP catalysts.

    PubMed

    Centomo, P; Zecca, M; Zoleo, A; Maniero, A L; Canton, P; Jerábek, K; Corain, B

    2009-05-28

    The cross-linked polyurea support EnCat 30, its related macromolecular complex Pd(II)/EnCat 30 and its related Pd(0)/EnCat 30NP nanocomposite are thoroughly investigated with SEM, TEM, ISEC and ESR in the solid state (SEM and TEM) and swollen state in THF (ISEC and ESR). Pd(II)/EnCat 30 and its related Pd(0)/EnCat 30NP are obtained by microencapsulation of palladium acetate in a polyurea framework, which is formed upon hydrolysis/condensation of mixtures of multi-functional oligo-arylisocyanates in dichloroethane. Most remarkably, both Pd(II)/EnCat and Pd(0)/EnCat 30NP turn out to be far more (nano)porous and swellable materials than the blank polyurea matrix (EnCat 30). It is proposed that there is a strong nanostructural effect exerted by Pd(II) species due to its interaction with functional groups (amines stemming from the hydrolysis of the isocyanato groups or ureido groups belonging to the polymer chains) during the growth of the cross-linked polymer framework. As a consequence, the catalytic species in both Pd(II)/EnCat 30 and Pd(0)/EnCat 30NP are much more accessible to molecules diffusing from liquid phases in contact with the materials and, hence, are better catalysts than expected from the morphology of blank polyurea EnCat 30.

  1. 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.

  2. Cross-linking of B7-H1 on EBV-transformed B cells induces apoptosis through reactive oxygen species production, JNK signaling activation, and fasL expression.

    PubMed

    Kim, Yeong Seok; Park, Ga Bin; Lee, Hyun-Kyung; Song, Hyunkeun; Choi, In-Hak; Lee, Wang Jae; Hur, Dae Young

    2008-11-01

    B7-H1 is a newly identified member of the B7 family with important regulatory functions in cell-mediated immune responses, and it is expressed in human immune cells and several tumors. We first observed that expression of surface B7-H1 on B cells was increased during the immortalization process by EBV, which is strongly related to both inflammation and tumorigenesis. Cross-linking of B7-H1 on EBV-transformed B cells using anti-B7-H1 Ab (clone 130002) induced reactive oxygen species (ROS) generation, mitochondrial disruption, release of apoptotic proteins from mitochondria, and subsequent apoptosis. Inhibition of caspases and ROS generation recovered B7-H1-mediated apoptosis and proteolytic activities of caspase-8, -9, and -3. We observed that B7-H1 stimulation induced both transcription and translation of fasL. ZB4, an antagonistic anti-fas Ab, and NOK-1, an antagonistic anti-fasL Ab, effectively blocked apoptosis without exerting any influence on ROS generation. N-acetylcysteine (NAC) completely blocked the induction of fasL mRNA and protein. We found that B7-H1 stimulation activated the phosphorylation of JNK and c-jun and down-regulated ERK1/2 and p-Akt. NAC blocked the activation of JNK and down-regulation of ERK, but both z-VAD-fmk (N-benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone) and ZB4 did not inhibit JNK activation of B7-H1 stimulation. SP600125 blocked fasL induction and apoptosis but did not affect ROS generation after B7-H1 stimulation. Taken together, we concluded that B7-H1-mediated apoptosis on EBV-transformed B cells may be involved in the induction of fasL, which is evoked by ROS generation and JNK activation after cross-linking of B7-H1. These results provide a new concept for understanding reverse signaling through B7-H1 and another mechanism of tumor immunotherapy using anti-B7-H1.

  3. 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.

  4. Novel binding interactions of the DNA fragment d(pGpG) cross-linked by the antitumor active compound tetrakis(mu-carboxylato)dirhodium(II,II).

    PubMed

    Chifotides, Helen T; Koshlap, Karl M; Pérez, Lisa M; Dunbar, Kim R

    2003-09-03

    Insight into the N7/O6 equatorial binding interactions of the antitumor active complex Rh(2)(OAc)(4)(H(2)O)(2) (OAc(-) = CH(3)CO(2)(-)) with the nucleotide 5'-GMP and the DNA fragment d(pGpG) has been obtained by one- (1D) and two-dimensional (2D) NMR spectroscopy. The lack of N7 protonation at low pH values and the significant increase in the acidity of N1-H (pK(a) approximately 5.6 as compared to 8.5 for N7 only bound platinum adducts), indicated by the pH dependence study of the H8 (1)H NMR resonance for the HT (head-to-tail) isomer of Rh(2)(OAc)(2)(5'-GMP)(2), are consistent with bidentate N7/O6 binding of the guanine. The H8 (1)H NMR resonance of the HH (head-to-head) Rh(2)(OAc)(2)(5'-GMP)(2) isomer, as well as the 5'-G and 3'-G H8 resonances of the Rh(2)(OAc)(2) [d(pGpG)] adduct exhibit pH-independent titration curves, attributable to the added effect of the 5'-phosphate group deprotonation at a pH value similar to that of the N1 site. The enhancement in the acidity of N1-H, with respect to N7 only bound metal adducts, afforded by the O6 binding of the bases to the rhodium centers, has been corroborated by monitoring the pH dependence of the purine C6 and C2 (13)C NMR resonances for Rh(2)(OAc)(2)(5'-GMP)(2) and Rh(2)(OAc)(2) [d(pGpG)]. The latter studies resulted in pK(a) values in good agreement with those derived from the pH-dependent (1)H NMR titrations of the H8 resonances. Comparison of the (13)C NMR resonances of C6 and C2 for the dirhodium adducts Rh(2)(OAc)(2)(5'-GMP)(2) and Rh(2)(OAc)(2) [d(pGpG)] with the corresponding resonances of the unbound ligands at pH 8.0, showed substantial downfield shifts of Deltadelta approximately 11.0 and 6.0 ppm, respectively. The HH arrangement of the bases in the Rh(2)(OAc)(2) [d(pGpG)] adduct is evidenced by intense H8/H8 ROE cross-peaks in the 2D ROESY NMR spectrum. The presence of the terminal 5'-phosphate group in d(pGpG) results in stabilization of one left-handed Rh(2)(OAc)(2) [d(pGpG)] HH1 L conformer, due to

  5. Plasma Membrane Calcium ATPase Activity Is Regulated by Actin Oligomers through Direct Interaction*

    PubMed Central

    Dalghi, Marianela G.; Fernández, Marisa M.; Ferreira-Gomes, Mariela; Mangialavori, Irene C.; Malchiodi, Emilio L.; Strehler, Emanuel E.; Rossi, Juan Pablo F. C.

    2013-01-01

    As recently described by our group, plasma membrane calcium ATPase (PMCA) activity can be regulated by the actin cytoskeleton. In this study, we characterize the interaction of purified G-actin with isolated PMCA and examine the effect of G-actin during the first polymerization steps. As measured by surface plasmon resonance, G-actin directly interacts with PMCA with an apparent 1:1 stoichiometry in the presence of Ca2+ with an apparent affinity in the micromolar range. As assessed by the photoactivatable probe 1-O-hexadecanoyl-2-O-[9-[[[2-[125I]iodo-4-(trifluoromethyl-3H-diazirin-3-yl)benzyl]oxy]carbonyl]nonanoyl]-sn-glycero-3-phosphocholine, the association of PMCA to actin produced a shift in the distribution of the conformers of the pump toward a calmodulin-activated conformation. G-actin stimulates Ca2+-ATPase activity of the enzyme when incubated under polymerizing conditions, displaying a cooperative behavior. The increase in the Ca2+-ATPase activity was related to an increase in the apparent affinity for Ca2+ and an increase in the phosphoenzyme levels at steady state. Although surface plasmon resonance experiments revealed only one binding site for G-actin, results clearly indicate that more than one molecule of G-actin was needed for a regulatory effect on the pump. Polymerization studies showed that the experimental conditions are compatible with the presence of actin in the first stages of assembly. Altogether, these observations suggest that the stimulatory effect is exerted by short oligomers of actin. The functional interaction between actin oligomers and PMCA represents a novel regulatory pathway by which the cortical actin cytoskeleton participates in the regulation of cytosolic Ca2+ homeostasis. PMID:23803603

  6. Plasma membrane calcium ATPase activity is regulated by actin oligomers through direct interaction.

    PubMed

    Dalghi, Marianela G; Fernández, Marisa M; Ferreira-Gomes, Mariela; Mangialavori, Irene C; Malchiodi, Emilio L; Strehler, Emanuel E; Rossi, Juan Pablo F C

    2013-08-09

    As recently described by our group, plasma membrane calcium ATPase (PMCA) activity can be regulated by the actin cytoskeleton. In this study, we characterize the interaction of purified G-actin with isolated PMCA and examine the effect of G-actin during the first polymerization steps. As measured by surface plasmon resonance, G-actin directly interacts with PMCA with an apparent 1:1 stoichiometry in the presence of Ca(2+) with an apparent affinity in the micromolar range. As assessed by the photoactivatable probe 1-O-hexadecanoyl-2-O-[9-[[[2-[(125)I]iodo-4-(trifluoromethyl-3H-diazirin-3-yl)benzyl]oxy]carbonyl]nonanoyl]-sn-glycero-3-phosphocholine, the association of PMCA to actin produced a shift in the distribution of the conformers of the pump toward a calmodulin-activated conformation. G-actin stimulates Ca(2+)-ATPase activity of the enzyme when incubated under polymerizing conditions, displaying a cooperative behavior. The increase in the Ca(2+)-ATPase activity was related to an increase in the apparent affinity for Ca(2+) and an increase in the phosphoenzyme levels at steady state. Although surface plasmon resonance experiments revealed only one binding site for G-actin, results clearly indicate that more than one molecule of G-actin was needed for a regulatory effect on the pump. Polymerization studies showed that the experimental conditions are compatible with the presence of actin in the first stages of assembly. Altogether, these observations suggest that the stimulatory effect is exerted by short oligomers of actin. The functional interaction between actin oligomers and PMCA represents a novel regulatory pathway by which the cortical actin cytoskeleton participates in the regulation of cytosolic Ca(2+) homeostasis.

  7. Ca2+ regulation of gelsolin activity: binding and severing of F-actin.

    PubMed Central

    Kinosian, H J; Newman, J; Lincoln, B; Selden, L A; Gershman, L C; Estes, J E

    1998-01-01

    Regulation of the F-actin severing activity of gelsolin by Ca2+ has been investigated under physiologic ionic conditions. Tryptophan fluorescence intensity measurements indicate that gelsolin contains at least two Ca2+ binding sites with affinities of 2.5 x 10(7) M-1 and 1.5 x 10(5) M-1. At F-actin and gelsolin concentrations in the range of those found intracellularly, gelsolin is able to bind F-actin with half-maximum binding at 0.14 microM free Ca2+ concentration. Steady-state measurements of gelsolin-induced actin depolymerization suggest that half-maximum depolymerization occurs at approximately 0.4 microM free Ca2+ concentration. Dynamic light scattering measurements of the translational diffusion coefficient for actin filaments and nucleated polymerization assays for number concentration of actin filaments both indicate that severing of F-actin occurs slowly at micromolar free Ca2+ concentrations. The data suggest that binding of Ca2+ to the gelsolin-F-actin complex is the rate-limiting step for F-actin severing by gelsolin; this Ca2+ binding event is a committed step that results in a Ca2+ ion bound at a high-affinity, EGTA-resistant site. The very high affinity of gelsolin for the barbed end of an actin filament drives the binding reaction equilibrium toward completion under conditions where the reaction rate is slow. PMID:9826630

  8. 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

  9. Regulation of myosin II activity by actin architecture

    NASA Astrophysics Data System (ADS)

    Weirich, Kimberly; Stam, Samantha; McCall, Patrick; Munro, Edwin; Gardel, Margaret

    2015-03-01

    Networks of actin filaments containing myosin II motors generate forces and motions that promote biological processes such as cell division, motility, and cargo transport. In cells, actin filaments are arranged in various structures from disordered meshworks to tight bundles. Clusters of myosin II motors, known as myosin filaments, crosslink and generate force on neighboring actin filaments. We hypothesized that the local actin architecture controls the magnitude and duration of force generated by myosin II motors. We used fluorescence imaging to directly measure the mobility of myosin II filaments on actin networks and bundles with varying actin filament polarity, orientation, spacing, and length. On unipolar bundles, myosin exhibits fast, unidirectional motion consistent with their unloaded gliding speed. On mixed polarity bundles, myosin speed is reduced by one order of magnitude and marked by direction switching and trapping. Increasing filament spacing and bundle flexibility reduces the duration of trapping and enhances the mobility of motors. Simulations indicate that stable trapping is a signature of large generated forces while increased mobility indicates force release. Our data underscore that the efficiency of force generation by myosin motors in an actin network depends sensitively on its architecture and suggests actin crosslinking proteins are tuned to optimize actomyosin contractility.

  10. 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

  11. Diffusion of GPI-anchored proteins is influenced by the activity of dynamic cortical actin.

    PubMed

    Saha, Suvrajit; Lee, Il-Hyung; Polley, Anirban; Groves, Jay T; Rao, Madan; Mayor, Satyajit

    2015-11-05

    Molecular diffusion at the surface of living cells is believed to be predominantly driven by thermal kicks. However, there is growing evidence that certain cell surface molecules are driven by the fluctuating dynamics of cortical cytoskeleton. Using fluorescence correlation spectroscopy, we measure the diffusion coefficient of a variety of cell surface molecules over a temperature range of 24-37 °C. Exogenously incorporated fluorescent lipids with short acyl chains exhibit the expected increase of diffusion coefficient over this temperature range. In contrast, we find that GPI-anchored proteins exhibit temperature-independent diffusion over this range and revert to temperature-dependent diffusion on cell membrane blebs, in cells depleted of cholesterol, and upon acute perturbation of actin dynamics and myosin activity. A model transmembrane protein with a cytosolic actin-binding domain also exhibits the temperature-independent behavior, directly implicating the role of cortical actin. We show that diffusion of GPI-anchored proteins also becomes temperature dependent when the filamentous dynamic actin nucleator formin is inhibited. However, changes in cortical actin mesh size or perturbation of branched actin nucleator Arp2/3 do not affect this behavior. Thus cell surface diffusion of GPI-anchored proteins and transmembrane proteins that associate with actin is driven by active fluctuations of dynamic cortical actin filaments in addition to thermal fluctuations, consistent with expectations from an "active actin-membrane composite" cell surface.

  12. Diffusion of GPI-anchored proteins is influenced by the activity of dynamic cortical actin

    PubMed Central

    Saha, Suvrajit; Lee, Il-Hyung; Polley, Anirban; Groves, Jay T.; Rao, Madan; Mayor, Satyajit

    2015-01-01

    Molecular diffusion at the surface of living cells is believed to be predominantly driven by thermal kicks. However, there is growing evidence that certain cell surface molecules are driven by the fluctuating dynamics of cortical cytoskeleton. Using fluorescence correlation spectroscopy, we measure the diffusion coefficient of a variety of cell surface molecules over a temperature range of 24–37°C. Exogenously incorporated fluorescent lipids with short acyl chains exhibit the expected increase of diffusion coefficient over this temperature range. In contrast, we find that GPI-anchored proteins exhibit temperature-independent diffusion over this range and revert to temperature-dependent diffusion on cell membrane blebs, in cells depleted of cholesterol, and upon acute perturbation of actin dynamics and myosin activity. A model transmembrane protein with a cytosolic actin-binding domain also exhibits the temperature-independent behavior, directly implicating the role of cortical actin. We show that diffusion of GPI-anchored proteins also becomes temperature dependent when the filamentous dynamic actin nucleator formin is inhibited. However, changes in cortical actin mesh size or perturbation of branched actin nucleator Arp2/3 do not affect this behavior. Thus cell surface diffusion of GPI-anchored proteins and transmembrane proteins that associate with actin is driven by active fluctuations of dynamic cortical actin filaments in addition to thermal fluctuations, consistent with expectations from an “active actin-membrane composite” cell surface. PMID:26378258

  13. How Actin Initiates the Motor Activity of Myosin

    PubMed Central

    Llinas, Paola; Isabet, Tatiana; Song, Lin; Ropars, Virginie; Zong, Bin; Benisty, Hannah; Sirigu, Serena; Morris, Carl; Kikuti, Carlos; Safer, Dan; Sweeney, H. Lee; Houdusse, Anne

    2015-01-01

    SUMMARY Fundamental to cellular processes are directional movements driven by molecular motors. A common theme for these and other molecular machines driven by ATP is that controlled release of hydrolysis products is essential to use the chemical energy efficiently. Mechanochemical transduction by myosin motors on actin is coupled to unknown structural changes that result in the sequential release of inorganic phosphate (Pi) and MgADP. We present here a myosin structure possessing an actin-binding interface and a tunnel (back door) that creates an escape route for Pi with a minimal rotation of the myosin lever arm that drives movements. We propose that this state represents the beginning of the powerstroke on actin, and that Pi translocation from the nucleotide pocket triggered by actin binding initiates myosin force generation. This elucidates how actin initiates force generation and movement, and may represent a strategy common to many molecular machines. PMID:25936506

  14. How actin initiates the motor activity of Myosin.

    PubMed

    Llinas, Paola; Isabet, Tatiana; Song, Lin; Ropars, Virginie; Zong, Bin; Benisty, Hannah; Sirigu, Serena; Morris, Carl; Kikuti, Carlos; Safer, Dan; Sweeney, H Lee; Houdusse, Anne

    2015-05-26

    Fundamental to cellular processes are directional movements driven by molecular motors. A common theme for these and other molecular machines driven by ATP is that controlled release of hydrolysis products is essential for using the chemical energy efficiently. Mechanochemical transduction by myosin motors on actin is coupled to unknown structural changes that result in the sequential release of inorganic phosphate (Pi) and MgADP. We present here a myosin structure possessing an actin-binding interface and a tunnel (back door) that creates an escape route for Pi with a minimal rotation of the myosin lever arm that drives movements. We propose that this state represents the beginning of the powerstroke on actin and that Pi translocation from the nucleotide pocket triggered by actin binding initiates myosin force generation. This elucidates how actin initiates force generation and movement and may represent a strategy common to many molecular machines.

  15. Coordinated integrin activation by actin-dependent force during T-cell migration.

    PubMed

    Nordenfelt, Pontus; Elliott, Hunter L; Springer, Timothy A

    2016-10-10

    For a cell to move forward it must convert chemical energy into mechanical propulsion. Force produced by actin polymerization can generate traction across the plasma membrane by transmission through integrins to their ligands. However, the role this force plays in integrin activation is unknown. Here we show that integrin activity and cytoskeletal dynamics are reciprocally linked, where actin-dependent force itself appears to regulate integrin activity. We generated fluorescent tension-sensing constructs of integrin αLβ2 (LFA-1) to visualize intramolecular tension during cell migration. Using quantitative imaging of migrating T cells, we correlate tension in the αL or β2 subunit with cell and actin dynamics. We find that actin engagement produces tension within the β2 subunit to induce and stabilize an active integrin conformational state and that this requires intact talin and kindlin motifs. This supports a general mechanism where localized actin polymerization can coordinate activation of the complex machinery required for cell migration.

  16. 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.

  17. Differential Effects of G- and F-Actin on the Plasma Membrane Calcium Pump Activity

    PubMed Central

    Vanagas, Laura; de La Fuente, María Candelaria; Dalghi, Marianela; Ferreira-Gomes, Mariela; Rossi, Rolando C.; Strehler, Emanuel E.; Rossi, Juan P. F. C.

    2014-01-01

    We have previously shown that plasma membrane calcium ATPase (PMCA) pump activity is affected by the membrane protein concentration (Vanagas et al., Biochim Biophys Acta 1768:1641–1644, 2007). Results show evidences for the involvement of the actin cytoskeleton. In this study, we explored the relationship between the polymerization state of actin and its effects on purified PMCA activity. Our results show that PMCA associates with the actin cytoskeleton and this interaction causes a modulation of the catalytic activity involving the phosphorylated intermediate of the pump. The state of actin polymerization determines whether it acts as an activator or an inhibitor of the pump: G-actin and/or short oligomers activate the pump, while F-actin inhibits it. The effects of actin on PMCA are the consequence of direct interaction as demonstrated by immunoblotting and cosedimentation experiments. Taken together, these findings suggest that interactions with actin play a dynamic role in the regulation of PMCA-mediated Ca2+ extrusion through the membrane. Our results provide further evidence of the activation–inhibition phenomenon as a property of many cytoskeleton-associated membrane proteins where the cytoskeleton is no longer restricted to a mechanical function but is dynamically involved in modulating the activity of integral proteins with which it interacts. PMID:23152090

  18. 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.

  19. Efficient synthesis of spacer-N-linked double-headed glycosides carrying N-acetylglucosamine and N,N'-diacetylchitobiose and their cross-linking activities with wheat germ agglutinin.

    PubMed

    Misawa, Yoshinori; Masaka, Ryuichi; Maeda, Kayo; Yano, Megumi; Murata, Takeomi; Kawagishi, Hirokazu; Usui, Taichi

    2008-02-25

    We describe here an efficient synthetic route to spacer-N-linked double-headed glycosides via a simple two-step procedure. N-Acetylglucosamine (GlcNAc) and N,N'-diacetylchitobiose [(GlcNAc)(2)] were treated with ammonia and the resulting N-beta-glycosylamines were coupled to a series of dicarboxylic acids. Condensation with each dicarboxylic acid proceeded stereoselectively to give the corresponding beta-N-linked double-headed glycoside without the need for any protection/deprotection steps. Interaction of the resulting N-linked double-headed glycosides with wheat germ agglutinin (WGA) were then investigated using a precipitation assay and an optical biosensor based on surface plasmon resonance (SPR). Spacer-N-linked double-headed glycosides bearing GlcNAc and (GlcNAc)(2) were found to be capable of binding and precipitating WGA as divalent ligands. However, the length of the spacer groups between the two terminal sugar residues was found to greatly influence the cross-linking activities with the lectin.

  20. 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.

  1. Spatiotemporal regulation of chemical reaction kinetics of cell surface molecules by active remodeling of cortical actin

    NASA Astrophysics Data System (ADS)

    Bhattacharyya, Bhaswati; Chaudhuri, Abhishek; Gowrishankar, Kripa; Mayor, Satyajit; Rao, Madan

    2010-03-01

    Cell surface proteins such as lipid tethered GPI-anchored proteins and Ras-proteins are distributed as monomers and nanoclusters on the surface of living cells. Recent work from our laboratory suggests that the spatial distribution and dynamics of formation and breakup of these nanoclusters is controlled by the active remodeling dynamics of the underlying cortical actin. To explain these observations, we propose a novel mechanism of nanoclustering, involving the transient binding to and advection along constitutively occuring ``asters'' of cortical actin. Here we study the consequences of such active actin based clustering, in the context of chemical reactions involving conformational changes of cell surface proteins. We find that active remodeling of cortical actin, can give rise to a dramatic increase in the reaction efficiency and output levels. In general, such actin driven clustering of membrane proteins could be a cellular mechanism to spatiotemporally regulate and amplify local chemical reaction rates, in the context of signalling and endocytosis.

  2. 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.

  3. Maleimidobenzoyl-G-actin: Structural properties and interaction with skeletal myosin subfragment-1

    SciTech Connect

    Bettache, N.; Bertrand, R.; Kassab, R. )

    1990-09-25

    The authors have investigated various structural and interaction properties of maleimidobenzoyl-G-actin (MBS-actin), a new, internally cross-linked G-actin derivative that does not exhibit, at moderate protein concentration, the salt-and myosin subfragment 1 (S-1)--induced polymerizations of G-actin and reacts reversibly and covalently in solution with S-1 at or near the F-actin binding region of the heavy chain. The far-ultraviolet CD spectrum and {alpha}-helix content of the MBS-actin were identical with those displayed by native G-actin. {sup 45}Ca{sup 2+} measurements showed the same content of tightly bound Ca{sup 2+} in MBS-actin as in G-actin and the EDTA treatment of the modified protein promoted the same red shift of the intrinsic fluorescence spectrum as observed with native G-actin. Incubation of concentrated MBS-actin solutions with 100 mM KCl+5 mM MgCl{sub 2} led to the polymerization of the actin derivative when the critical monomer concentration reached 1.6mg/mL, at 25{degree}C, pH 8.0. The MBS-F-actin formed activated the Mg{sup 2+}-ATPase of S-1 to the same extent as native F-actin. The MBS-G-actin exhibited a DNase I inhibitor activity very close to that found with native G-actin and was to be at all affected by its specific covalent conjugation to S-1. This finding led them to isolate, for the first time, by gel filtration, a ternary complex comprising DNase I tightly bound to MBS-actin cross-linked to the S-1 heavy chain, demonstrating that S-1 and DNase I bind at distinct sites on G-actin. Collectively, the data illustrate further the nativeness of the MBS-G-actin and its potential use in solution studies of the actin-myosin head interactions.

  4. 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.

  5. 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

  6. 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

  7. Lamellipodial actin mechanically links myosin activity with adhesion site formation

    PubMed Central

    Giannone, Gregory; Dubin-Thaler, Benjamin; Rossier, Olivier; Cai, Yunfei; Chaga, Oleg; Jiang, Guoying; Beaver, William; Döbereiner, Hans-Günther; Freund, Yoav; Borisy, Gary; Sheetz, Michael P.

    2013-01-01

    Summary Cell motility proceeds by cycles of edge protrusion, adhesion and retraction. Whether these functions are coordinated by biochemical or biomechanical processes is unknown. We find that myosin II pulls the rear of the lamellipodial actin network, causing upward bending, edge retraction and initiation of new adhesion sites. The network then separates from the edge and condenses over the myosin. Protrusion resumes as lamellipodial actin regenerates from the front and extends rearward until it reaches newly assembled myosin, initiating the next cycle. Upward bending, observed by evanescence and electron microscopy, results in ruffle formation when adhesion strength is low. Correlative fluorescence and electron microscopy shows that the regenerating lamellipodium forms a cohesive, separable layer of actin above the lamellum. Thus, actin polymerization periodically builds a mechanical link, the lamellipodium, connecting myosin motors with the initiation of adhesion sites, suggesting that the major functions driving motility are coordinated by a biomechanical process. PMID:17289574

  8. 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

  9. 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.

  10. 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.

  11. Endoplasmic reticulum stress-mediated apoptosis of EBV-transformed B cells by cross-linking of CD70 is dependent upon generation of reactive oxygen species and activation of p38 MAPK and JNK pathway.

    PubMed

    Park, Ga Bin; Kim, Yeong Seok; Lee, Hyun-Kyung; Song, Hyunkeun; Cho, Dae-Ho; Lee, Wang Jae; Hur, Dae Young

    2010-12-15

    CD70 is expressed in normal activated immune cells as well as in several types of tumors. It has been established that anti-CD70 mAb induces complement-dependent death of CD70(+) tumor cells, but how anti-CD70 mAb affects the intrinsic signaling is poorly defined. In this report, we show that ligation of CD70 expressed on EBV-transformed B cells using anti-CD70 mAb induced production of reactive oxygen species (ROS) and subsequent apoptosis. We observed an early expression of endoplasmic reticulum (ER) stress response genes that preceded the release of apoptotic molecules from the mitochondria and the cleavage of caspases. CD70-induced apoptosis was inhibited by pretreatment with the ER stress inhibitor salubrinal, ROS quencher N-acetylcysteine, and Ca(2+) chelator BAPTA. We supposed that ROS generation might be the first event of CD70-induced apoptosis because N-acetylcysteine blocked increases of ROS and Ca(2+), but BAPTA did not block ROS generation. We also found that CD70 stimulation activated JNK and p38 MAPK. JNK inhibitor SP600125 and p38 inhibitor SB203580 effectively blocked upregulation of ER stress-related genes and cleavage of caspases. Inhibition of ROS generation completely blocked phosphorylation of JNK and p38 MAPK and induction of ER stress-related genes. Taken together, we concluded that cross-linking of CD70 on EBV-transformed B cells triggered ER stress-mediated apoptosis via ROS generation and JNK and p38 MAPK pathway activation. Our report reveals alternate mechanisms of direct apoptosis through CD70 signaling and provides data supporting CD70 as a viable target for an Ab-based therapy against EBV-related tumors.

  12. 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

  13. 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

  14. 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

  15. Tropomyosin movement on F-actin during muscle activation explained by energy landscapes.

    PubMed

    Orzechowski, Marek; Moore, Jeffrey R; Fischer, Stefan; Lehman, William

    2014-03-01

    Muscle contraction is regulated by tropomyosin movement across the thin filament surface, which exposes or blocks myosin-binding sites on actin. Recent atomic structures of F-actin-tropomyosin have yielded the positions of tropomyosin on myosin-free and myosin-decorated actin. Here, the repositioning of α-tropomyosin between these locations on F-actin was systematically examined by optimizing the energy of the complex for a wide range of tropomyosin positions on F-actin. The resulting energy landscape provides a full-map of the F-actin surface preferred by tropomyosin, revealing a broad energy basin associated with the tropomyosin position that blocks myosin-binding. This is consistent with previously proposed low-energy oscillations of semi-rigid tropomyosin, necessary for shifting of tropomyosin following troponin-binding. In contrast, the landscape shows much less favorable energies when tropomyosin locates near its myosin-induced "open-state" position. This indicates that spontaneous movement of tropomyosin away from its energetic "ground-state" to the open-state is unlikely in absence of myosin. Instead, myosin-binding must drive tropomyosin toward the open-state to activate the thin filament. Additional energy landscapes were computed for disease-causing actin mutants that distort the topology of the actin-tropomyosin energy landscape, explaining their phenotypes. Thus, the computation of such energy landscapes offers a sensitive way to estimate the impact of mutations.

  16. The interplay between neuronal activity and actin dynamics mimic the setting of an LTD synaptic tag

    PubMed Central

    Szabó, Eszter C.; Manguinhas, Rita; Fonseca, Rosalina

    2016-01-01

    Persistent forms of plasticity, such as long-term depression (LTD), are dependent on the interplay between activity-dependent synaptic tags and the capture of plasticity-related proteins. We propose that the synaptic tag represents a structural alteration that turns synapses permissive to change. We found that modulation of actin dynamics has different roles in the induction and maintenance of LTD. Inhibition of either actin depolymerisation or polymerization blocks LTD induction whereas only the inhibition of actin depolymerisation blocks LTD maintenance. Interestingly, we found that actin depolymerisation and CaMKII activation are involved in LTD synaptic-tagging and capture. Moreover, inhibition of actin polymerisation mimics the setting of a synaptic tag, in an activity-dependent manner, allowing the expression of LTD in non-stimulated synapses. Suspending synaptic activation also restricts the time window of synaptic capture, which can be restored by inhibiting actin polymerization. Our results support our hypothesis that modulation of the actin cytoskeleton provides an input-specific signal for synaptic protein capture. PMID:27650071

  17. 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

  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. Formin 1 Regulates Ectoplasmic Specialization in the Rat Testis Through Its Actin Nucleation and Bundling Activity

    PubMed Central

    Li, Nan; Mruk, Dolores D.; Wong, Chris K. C.; Han, Daishu; Lee, Will M.

    2015-01-01

    During spermatogenesis, developing spermatids and preleptotene spermatocytes are transported across the adluminal compartment and the blood-testis barrier (BTB), respectively, so that spermatids line up near the luminal edge to prepare for spermiation, whereas preleptotene spermatocytes enter the adluminal compartment to differentiate into late spermatocytes to prepare for meiosis I/II. These cellular events involve actin microfilament reorganization at the testis-specific, actin-rich Sertoli-spermatid and Sertoli-Sertoli cell junction called apical and basal ectoplasmic specialization (ES). Formin 1, an actin nucleation protein known to promote actin microfilament elongation and bundling, was expressed at the apical ES but limited to stage VII of the epithelial cycle, whereas its expression at the basal ES/BTB stretched from stage III to stage VI, diminished in stage VII, and was undetectable in stage VIII tubules. Using an in vitro model of studying Sertoli cell BTB function by RNA interference and biochemical assays to monitor actin bundling and polymerization activity, a knockdown of formin 1 in Sertoli cells by approximately 70% impeded the tight junction-permeability function. This disruptive effect on the tight junction barrier was mediated by a loss of actin microfilament bundling and actin polymerization capability mediated by changes in the localization of branched actin-inducing protein Arp3 (actin-related protein 3), and actin bundling proteins Eps8 (epidermal growth factor receptor pathway substrate 8) and palladin, thereby disrupting cell adhesion. Formin 1 knockdown in vivo was found to impede spermatid adhesion, transport, and polarity, causing defects in spermiation in which elongated spermatids remained embedded into the epithelium in stage IX tubules, mediated by changes in the spatiotemporal expression of Arp3, Eps8, and palladin. In summary, formin 1 is a regulator of ES dynamics. PMID:25901598

  20. Plant villin, lily P-135-ABP, possesses G-actin binding activity and accelerates the polymerization and depolymerization of actin in a Ca2+-sensitive manner.

    PubMed

    Yokota, Etsuo; Tominaga, Motoki; Mabuchi, Issei; Tsuji, Yasunori; Staiger, Christopher J; Oiwa, Kazuhiro; Shimmen, Teruo

    2005-10-01

    From germinating pollen of lily, two types of villins, P-115-ABP and P-135-ABP, have been identified biochemically. Ca(2+)-CaM-dependent actin-filament binding and bundling activities have been demonstrated for both villins previously. Here, we examined the effects of lily villins on the polymerization and depolymerization of actin. P-115-ABP and P-135-ABP present in a crude protein extract prepared from germinating pollen bound to a DNase I affinity column in a Ca(2+)-dependent manner. Purified P-135-ABP reduced the lag period that precedes actin filament polymerization from monomers in the presence of either Ca(2+) or Ca(2+)-CaM. These results indicated that P-135-ABP can form a complex with G-actin in the presence of Ca(2+) and this complex acts as a nucleus for polymerization of actin filaments. However, the nucleation activity of P-135-ABP is probably not relevant in vivo because the assembly of G-actin saturated with profilin, a situation that mimics conditions found in pollen, was not accelerated in the presence of P-135-ABP. P-135-ABP also enhanced the depolymerization of actin filaments during dilution-mediated disassembly. Growth from filament barbed ends in the presence of Ca(2+)-CaM was also prevented, consistent with filament capping activity. These results suggested that lily villin is involved not only in the arrangement of actin filaments into bundles in the basal and shank region of the pollen tube, but also in regulating and modulating actin dynamics through its capping and depolymerization (or fragmentation) activities in the apical region of the pollen tube, where there is a relatively high concentration of Ca(2+).

  1. 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

  2. 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

  3. 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.

  4. 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.

  5. 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

  6. S-NO-actin: S-nitrosylation kinetics and the effect on isolated vascular smooth muscle.

    PubMed

    Dalle-Donne, I; Milzani, A; Giustarini, D; Di Simplicio, P; Colombo, R; Rossi, R

    2000-02-01

    We describe the modification of reactive actin sulfhydryls by S-nitrosoglutathione. Kinetics of S-nitrosylation and denitrosylation suggest that only one cysteine of actin is involved in the reactions. By using the bifunctional sulfhydryl cross-linking reagent N,N'-1,4-phenylenebismaleimide and the monofunctional reagent N-iodoacetyl-N'-(5-sulpho-1-naphthyl)ethylenediamine, we identified this residue as Cys374. The time course of filament formation followed by high-shear viscosity changes revealed that S-nitrosylated G-actin polymerizes less efficiently than native monomers. The observed decrease in specific viscosity at steady state is due mainly to a marked inhibition of filament end-to-end annealing and, partially, to a reduction in F-actin concentration. Finally, S-nitrosylated actin acts as nitric oxide donor showing a fast, potent vasodilating activity at unusually low concentrations, being comparable with that of low molecular weight nitrosothiols.

  7. 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.

  8. Bypass of a psoralen DNA interstrand cross-link by DNA polymerases β, ι, and κ in vitro.

    PubMed

    Smith, Leigh A; Makarova, Alena V; Samson, Laura; Thiesen, Katherine E; Dhar, Alok; Bessho, Tadayoshi

    2012-11-06

    Repair of DNA interstrand cross-links in mammalian cells involves several biochemically distinctive processes, including the release of one of the cross-linked strands and translesion DNA synthesis (TLS). In this report, we investigated the in vitro TLS activity of a psoralen DNA interstrand cross-link by three DNA repair polymerases, DNA polymerases β, κ, and ι. DNA polymerase β is capable of bypassing a psoralen cross-link with a low efficiency. Cell extracts prepared from DNA polymerase β knockout mouse embryonic fibroblasts showed a reduced bypass activity of the psoralen cross-link, and purified DNA polymerase β restored the bypass activity. In addition, DNA polymerase ι misincorporated thymine across the psoralen cross-link and DNA polymerase κ extended these mispaired primer ends, suggesting that DNA polymerase ι may serve as an inserter and DNA polymerase κ may play a role as an extender in the repair of psoralen DNA interstrand cross-links. The results demonstrated here indicate that multiple DNA polymerases could participate in TLS steps in mammalian DNA interstrand cross-link repair.

  9. F-actin polymerization and retrograde flow drive sustained PLCγ1 signaling during T cell activation

    PubMed Central

    Babich, Alexander; Li, Shuixing; O'Connor, Roddy S.; Milone, Michael C.; Freedman, Bruce D.

    2012-01-01

    Activation of T cells by antigen-presenting cells involves assembly of signaling molecules into dynamic microclusters (MCs) within a specialized membrane domain termed the immunological synapse (IS). Actin and myosin IIA localize to the IS, and depletion of F-actin abrogates MC movement and T cell activation. However, the mechanisms that coordinate actomyosin dynamics and T cell receptor signaling are poorly understood. Using pharmacological inhibitors that perturb individual aspects of actomyosin dynamics without disassembling the network, we demonstrate that F-actin polymerization is the primary driver of actin retrograde flow, whereas myosin IIA promotes long-term integrity of the IS. Disruption of F-actin retrograde flow, but not myosin IIA contraction, arrested MC centralization and inhibited sustained Ca2+ signaling at the level of endoplasmic reticulum store release. Furthermore, perturbation of retrograde flow inhibited PLCγ1 phosphorylation within MCs but left Zap70 activity intact. These studies highlight the importance of ongoing actin polymerization as a central driver of actomyosin retrograde flow, MC centralization, and sustained Ca2+ signaling. PMID:22665519

  10. 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.

  11. Actin depolymerization affects stress-induced translational activity of potato tuber tissue

    PubMed

    Morelli; Zhou; Yu; Lu; Vayda

    1998-04-01

    Changes in polymerized actin during stress conditions were correlated with potato (Solanum tuberosum L.) tuber protein synthesis. Fluorescence microscopy and immunoblot analyses indicated that filamentous actin was nearly undetectable in mature, quiescent aerobic tubers. Mechanical wounding of postharvest tubers resulted in a localized increase of polymerized actin, and microfilament bundles were visible in cells of the wounded periderm within 12 h after wounding. During this same period translational activity increased 8-fold. By contrast, low-oxygen stress caused rapid reduction of polymerized actin coincident with acute inhibition of protein synthesis. Treatment of aerobic tubers with cytochalasin D, an agent that disrupts actin filaments, reduced wound-induced protein synthesis in vivo. This effect was not observed when colchicine, an agent that depolymerizes microtubules, was used. Neither of these drugs had a significant effect in vitro on run-off translation of isolated polysomes. However, cytochalasin D did reduce translational competence in vitro of a crude cellular fraction containing both polysomes and cytoskeletal elements. These results demonstrate the dependence of wound-induced protein synthesis on the integrity of microfilaments and suggest that the dynamics of the actin cytoskeleton may affect translational activity during stress conditions.

  12. Gestalt-binding of tropomyosin on actin during thin filament activation.

    PubMed

    Lehman, William; Orzechowski, Marek; Li, Xiaochuan Edward; Fischer, Stefan; Raunser, Stefan

    2013-08-01

    Our thesis is that thin filament function can only be fully understood and muscle regulation then elucidated if atomic structures of the thin filament are available to reveal the positions of tropomyosin on actin in all physiological states. After all, it is tropomyosin influenced by troponin that regulates myosin-crossbridge cycling on actin and therefore controls contraction in all muscles. In addition, we maintain that a complete appreciation of thin filament activation also requires that the mechanical properties of tropomyosin itself are recognized and then related to the effect of myosin-association on actin. Taking the Gestalt-binding of tropomyosin into account, coupled with our electron microscopy structures and computational chemistry, we propose a comprehensive mechanism for tropomyosin regulatory movement over the actin filament surface that explains the cooperative muscle activation process. In fact, well-known point mutations of critical amino acids on the actin-tropomyosin binding interface disrupt Gestalt-binding and are associated with a number of inherited myopathies. Moreover, dysregulation of tropomyosin may also be a factor that interferes with the gatekeeping operation of non-muscle tropomyosin in the controlling interactions of a wide variety of cellular actin-binding proteins. The clinical relevance of Gestalt-binding is discussed in articles by the Marston and the Gunning groups in this special journal issue devoted to the impact of tropomyosin on biological systems.

  13. Postsynaptic actin regulates active zone spacing and glutamate receptor apposition at the Drosophila neuromuscular junction.

    PubMed

    Blunk, Aline D; Akbergenova, Yulia; Cho, Richard W; Lee, Jihye; Walldorf, Uwe; Xu, Ke; Zhong, Guisheng; Zhuang, Xiaowei; Littleton, J Troy

    2014-07-01

    Synaptic communication requires precise alignment of presynaptic active zones with postsynaptic receptors to enable rapid and efficient neurotransmitter release. How transsynaptic signaling between connected partners organizes this synaptic apparatus is poorly understood. To further define the mechanisms that mediate synapse assembly, we carried out a chemical mutagenesis screen in Drosophila to identify mutants defective in the alignment of active zones with postsynaptic glutamate receptor fields at the larval neuromuscular junction. From this screen we identified a mutation in Actin 57B that disrupted synaptic morphology and presynaptic active zone organization. Actin 57B, one of six actin genes in Drosophila, is expressed within the postsynaptic bodywall musculature. The isolated allele, act(E84K), harbors a point mutation in a highly conserved glutamate residue in subdomain 1 that binds members of the Calponin Homology protein family, including spectrin. Homozygous act(E84K) mutants show impaired alignment and spacing of presynaptic active zones, as well as defects in apposition of active zones to postsynaptic glutamate receptor fields. act(E84K) mutants have disrupted postsynaptic actin networks surrounding presynaptic boutons, with the formation of aberrant actin swirls previously observed following disruption of postsynaptic spectrin. Consistent with a disruption of the postsynaptic actin cytoskeleton, spectrin, adducin and the PSD-95 homolog Discs-Large are all mislocalized in act(E84K) mutants. Genetic interactions between act(E84K) and neurexin mutants suggest that the postsynaptic actin cytoskeleton may function together with the Neurexin-Neuroligin transsynaptic signaling complex to mediate normal synapse development and presynaptic active zone organization.

  14. Postsynaptic actin regulates active zone spacing and glutamate receptor apposition at the Drosophila neuromuscular junction

    PubMed Central

    Blunk, Aline D.; Akbergenova, Yulia; Cho, Richard W.; Lee, Jihye; Walldorf, Uwe; Xu, Ke; Zhong, Guisheng; Zhuang, Xiaowei; Littleton, J. Troy

    2014-01-01

    Synaptic communication requires precise alignment of presynaptic active zones with postsynaptic receptors to enable rapid and efficient neurotransmitter release. How transsynaptic signaling between connected partners organizes this synaptic apparatus is poorly understood. To further define the mechanisms that mediate synapse assembly, we carried out a chemical mutagenesis screen in Drosophila to identify mutants defective in the alignment of active zones with postsynaptic glutamate receptor fields at the larval neuromuscular junction. From this screen we identified a mutation in actin 57B that disrupted synaptic morphology and presynaptic active zone organization. Actin 57B, one of six actin genes in Drosophila, is expressed within the postsynaptic bodywall musculature. The isolated allele, actE84K, harbors a point mutation in a highly conserved glutamate residue in subdomain 1 that binds members of the Calponin Homology protein family, including spectrin. Homozygous actE84K mutants show impaired alignment and spacing of presynaptic active zones, as well as defects in apposition of active zones to postsynaptic glutamate receptor fields. actE84K mutants have disrupted postsynaptic actin networks surrounding presynaptic boutons, with the formation of aberrant actin swirls previously observed following disruption of postsynaptic spectrin. Consistent with a disruption of the postsynaptic actin cytoskeleton, spectrin, adducin and the PSD-95 homolog Disc-Large are all mislocalized in actE84K mutants. Genetic interactions between actE84K and neurexin mutants suggest that the postsynaptic actin cytoskeleton may function together with the Neurexin-Neuroligin transsynaptic signaling complex to mediate normal synapse development and presynaptic active zone organization. PMID:25066865

  15. 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.

  16. Propagating cell-membrane waves driven by curved activators of actin polymerization.

    PubMed

    Peleg, Barak; Disanza, Andrea; Scita, Giorgio; Gov, Nir

    2011-04-21

    Cells exhibit propagating membrane waves which involve the actin cytoskeleton. One type of such membranal waves are Circular Dorsal Ruffles (CDR) which are related to endocytosis and receptor internalization. Experimentally, CDRs have been associated with membrane bound activators of actin polymerization of concave shape. We present experimental evidence for the localization of convex membrane proteins in these structures, and their insensitivity to inhibition of myosin II contractility in immortalized mouse embryo fibroblasts cell cultures. These observations lead us to propose a theoretical model which explains the formation of these waves due to the interplay between complexes that contain activators of actin polymerization and membrane-bound curved proteins of both types of curvature (concave and convex). Our model predicts that the activity of both types of curved proteins is essential for sustaining propagating waves, which are abolished when one type of curved activator is removed. Within this model waves are initiated when the level of actin polymerization induced by the curved activators is higher than some threshold value, which allows the cell to control CDR formation. We demonstrate that the model can explain many features of CDRs, and give several testable predictions. This work demonstrates the importance of curved membrane proteins in organizing the actin cytoskeleton and cell shape.

  17. Coordinated integrin activation by actin-dependent force during T-cell migration

    PubMed Central

    Nordenfelt, Pontus; Elliott, Hunter L.; Springer, Timothy A.

    2016-01-01

    For a cell to move forward it must convert chemical energy into mechanical propulsion. Force produced by actin polymerization can generate traction across the plasma membrane by transmission through integrins to their ligands. However, the role this force plays in integrin activation is unknown. Here we show that integrin activity and cytoskeletal dynamics are reciprocally linked, where actin-dependent force itself appears to regulate integrin activity. We generated fluorescent tension-sensing constructs of integrin αLβ2 (LFA-1) to visualize intramolecular tension during cell migration. Using quantitative imaging of migrating T cells, we correlate tension in the αL or β2 subunit with cell and actin dynamics. We find that actin engagement produces tension within the β2 subunit to induce and stabilize an active integrin conformational state and that this requires intact talin and kindlin motifs. This supports a general mechanism where localized actin polymerization can coordinate activation of the complex machinery required for cell migration. PMID:27721490

  18. 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.

  19. 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.

  20. 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.

  1. 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.

  2. 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

  3. 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

  4. 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.

  5. 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.

  6. A Dichotomy in Cortical Actin and Chemotactic Actin Activity between Human Memory and Naive T Cells Contributes to Their Differential Susceptibility to HIV-1 Infection*

    PubMed Central

    Wang, Weifeng; Guo, Jia; Yu, Dongyang; Vorster, Paul J.; Chen, WanJun; Wu, Yuntao

    2012-01-01

    Human memory and naive CD4 T cells can mainly be identified by the reciprocal expression of the CD45RO or CD45RA isoforms. In HIV-1 infection, blood CD45RO memory CD4 T cells are preferentially infected and serve as a major viral reservoir. The molecular mechanism dictating this differential susceptibility to HIV-1 remains largely obscure. Here, we report that the different susceptibility of memory and naive T cells to HIV is not determined by restriction factors such as Apobec3G or BST2. However, we observed a phenotypic distinction between human CD45RO and CD45RA resting CD4 T cells in their cortical actin density and actin dynamics. CD45RO CD4 T cells possess a higher cortical actin density and can be distinguished as CD45RO+Actinhigh. In contrast, CD45RA T cells are phenotypically CD45RA+Actinlow. In addition, the cortical actin in CD45RO memory CD4 T cells is more dynamic and can respond to low dosages of chemotactic induction by SDF-1, whereas that of naive cells cannot, despite a similar level of the chemokine receptor CXCR4 present on both cells. We further demonstrate that this difference in the cortical actin contributes to their differential susceptibility to HIV-1; resting memory but not naive T cells are highly responsive to HIV-mediated actin dynamics that promote higher levels of viral entry and early DNA synthesis in resting memory CD4 T cells. Furthermore, transient induction of actin dynamics in resting naive T cells rescues HIV latent infection following CD3/CD28 stimulation. These results suggest a key role of chemotactic actin activity in facilitating HIV-1 latent infection of these T cell subsets. PMID:22879601

  7. 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.

  8. 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.

  9. 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

  10. 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.

  11. 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.

  12. Self-organization of actin networks by a monomeric myosin

    PubMed Central

    Saczko-Brack, Dario; Warchol, Ewa; Rogez, Benoit; Kröss, Markus; Heissler, Sarah M.; Sellers, James R.; Batters, Christopher; Veigel, Claudia

    2016-01-01

    The organization of actomyosin networks lies at the center of many types of cellular motility, including cell polarization and collective cell migration during development and morphogenesis. Myosin-IXa is critically involved in these processes. Using total internal reflection fluorescence microscopy, we resolved actin bundles assembled by myosin-IXa. Electron microscopic data revealed that the bundles consisted of highly ordered lattices with parallel actin polarity. The myosin-IXa motor domains aligned across the network, forming cross-links at a repeat distance of precisely 36 nm, matching the helical repeat of actin. Single-particle image processing resolved three distinct conformations of myosin-IXa in the absence of nucleotide. Using cross-correlation of a modeled actomyosin crystal structure, we identified sites of additional mass, which can only be accounted for by the large insert in loop 2 exclusively found in the motor domain of class IX myosins. We show that the large insert in loop 2 binds calmodulin and creates two coordinated actin-binding sites that constrain the actomyosin interactions generating the actin lattices. The actin lattices introduce orientated tracks at specific sites in the cell, which might install platforms allowing Rho-GTPase–activating protein (RhoGAP) activity to be focused at a definite locus. In addition, the lattices might introduce a myosin-related, force-sensing mechanism into the cytoskeleton in cell polarization and collective cell migration. PMID:27956608

  13. Actin polymerization-dependent activation of Cas-L promotes immunological synapse stability

    PubMed Central

    Santos, Luís C; Blair, David A; Kumari, Sudha; Cammer, Michael; Iskratsch, Thomas; Herbin, Olivier; Alexandropoulos, Konstantina; Dustin, Michael L; Sheetz, Michael P

    2016-01-01

    The immunological synapse formed between a T-cell and an antigen-presenting cell is important for cell–cell communication during T-cell-mediated immune responses. Immunological synapse formation begins with stimulation of the T-cell receptor (TCR). TCR microclusters are assembled and transported to the center of the immunological synapse in an actin polymerization-dependent process. However, the physical link between TCR and actin remains elusive. Here we show that lymphocyte-specific Crk-associated substrate (Cas-L), a member of a force sensing protein family, is required for transport of TCR microclusters and for establishing synapse stability. We found that Cas-L is phosphorylated at TCR microclusters in an actin polymerization-dependent fashion. Furthermore, Cas-L participates in a positive feedback loop leading to amplification of Ca2+ signaling, inside–out integrin activation, and actomyosin contraction. We propose a new role for Cas-L in T-cell activation as a mechanical transducer linking TCR microclusters to the underlying actin network and coordinating multiple actin-dependent structures in the immunological synapse. Our studies highlight the importance of mechanotransduction processes in T-cell-mediated immune responses. PMID:27359298

  14. PLEKHG3 enhances polarized cell migration by activating actin filaments at the cell front

    PubMed Central

    Nguyen, Trang Thi Thu; Park, Wei Sun; Park, Byung Ouk; Kim, Cha Yeon; Oh, Yohan; Kim, Jin Man; Choi, Hana; Kyung, Taeyoon; Kim, Cheol-Hee; Lee, Gabsang; Hahn, Klaus M.; Meyer, Tobias; Heo, Won Do

    2016-01-01

    Cells migrate by directing Ras-related C3 botulinum toxin substrate 1 (Rac1) and cell division control protein 42 (Cdc42) activities and by polymerizing actin toward the leading edge of the cell. Previous studies have proposed that this polarization process requires a local positive feedback in the leading edge involving Rac small GTPase and actin polymerization with PI3K likely playing a coordinating role. Here, we show that the pleckstrin homology and RhoGEF domain containing G3 (PLEKHG3) is a PI3K-regulated Rho guanine nucleotide exchange factor (RhoGEF) for Rac1 and Cdc42 that selectively binds to newly polymerized actin at the leading edge of migrating fibroblasts. Optogenetic inactivation of PLEKHG3 showed that PLEKHG3 is indispensable both for inducing and for maintaining cell polarity. By selectively binding to newly polymerized actin, PLEKHG3 promotes local Rac1/Cdc42 activation to induce more local actin polymerization, which in turn promotes the recruitment of more PLEKHG3 to induce and maintain cell front. Thus, autocatalytic reinforcement of PLEKHG3 localization to the leading edge of the cell provides a molecular basis for the proposed positive feedback loop that is required for cell polarization and directed migration. PMID:27555588

  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. Actin activates Pseudomonas aeruginosa ExoY nucleotidyl cyclase toxin and ExoY-like effector domains from MARTX toxins

    PubMed Central

    Belyy, Alexander; Raoux-Barbot, Dorothée; Saveanu, Cosmin; Namane, Abdelkader; Ogryzko, Vasily; Worpenberg, Lina; David, Violaine; Henriot, Veronique; Fellous, Souad; Merrifield, Christien; Assayag, Elodie; Ladant, Daniel; Renault, Louis; Mechold, Undine

    2016-01-01

    The nucleotidyl cyclase toxin ExoY is one of the virulence factors injected by the Pseudomonas aeruginosa type III secretion system into host cells. Inside cells, it is activated by an unknown eukaryotic cofactor to synthesize various cyclic nucleotide monophosphates. ExoY-like adenylate cyclases are also found in Multifunctional-Autoprocessing Repeats-in-ToXin (MARTX) toxins produced by various Gram-negative pathogens. Here we demonstrate that filamentous actin (F-actin) is the hitherto unknown cofactor of ExoY. Association with F-actin stimulates ExoY activity more than 10,000 fold in vitro and results in stabilization of actin filaments. ExoY is recruited to actin filaments in transfected cells and alters F-actin turnover. Actin also activates an ExoY-like adenylate cyclase MARTX effector domain from Vibrio nigripulchritudo. Finally, using a yeast genetic screen, we identify actin mutants that no longer activate ExoY. Our results thus reveal a new sub-group within the class II adenylyl cyclase family, namely actin-activated nucleotidyl cyclase (AA-NC) toxins. PMID:27917880

  18. 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

  19. Amyloid-β and proinflammatory cytokines utilize a prion protein-dependent pathway to activate NADPH oxidase and induce cofilin-actin rods in hippocampal neurons.

    PubMed

    Walsh, Keifer P; Minamide, Laurie S; Kane, Sarah J; Shaw, Alisa E; Brown, David R; Pulford, Bruce; Zabel, Mark D; Lambeth, J David; Kuhn, Thomas B; Bamburg, James R

    2014-01-01

    Neurites of neurons under acute or chronic stress form bundles of filaments (rods) containing 1∶1 cofilin∶actin, which impair transport and synaptic function. Rods contain disulfide cross-linked cofilin and are induced by treatments resulting in oxidative stress. Rods form rapidly (5-30 min) in >80% of cultured hippocampal or cortical neurons treated with excitotoxic levels of glutamate or energy depleted (hypoxia/ischemia or mitochondrial inhibitors). In contrast, slow rod formation (50% of maximum response in ∼6 h) occurs in a subpopulation (∼20%) of hippocampal neurons upon exposure to soluble human amyloid-β dimer/trimer (Aβd/t) at subnanomolar concentrations. Here we show that proinflammatory cytokines (TNFα, IL-1β, IL-6) also induce rods at the same rate and within the same neuronal population as Aβd/t. Neurons from prion (PrP(C))-null mice form rods in response to glutamate or antimycin A, but not in response to proinflammatory cytokines or Aβd/t. Two pathways inducing rod formation were confirmed by demonstrating that NADPH-oxidase (NOX) activity is required for prion-dependent rod formation, but not for rods induced by glutamate or energy depletion. Surprisingly, overexpression of PrP(C) is by itself sufficient to induce rods in over 40% of hippocampal neurons through the NOX-dependent pathway. Persistence of PrP(C)-dependent rods requires the continuous activity of NOX. Removing inducers or inhibiting NOX activity in cells containing PrP(C)-dependent rods causes rod disappearance with a half-life of about 36 min. Cofilin-actin rods provide a mechanism for synapse loss bridging the amyloid and cytokine hypotheses for Alzheimer disease, and may explain how functionally diverse Aβ-binding membrane proteins induce synaptic dysfunction.

  20. LIM Kinase 1 Modulates Cortical Actin and CXCR4 Cycling and Is Activated by HIV-1 to Initiate Viral Infection*

    PubMed Central

    Vorster, Paul J.; Guo, Jia; Yoder, Alyson; Wang, Weifeng; Zheng, Yanfang; Xu, Xuehua; Yu, Dongyang; Spear, Mark; Wu, Yuntao

    2011-01-01

    Almost all viral pathogens utilize a cytoskeleton for their entry and intracellular transport. In HIV-1 infection, binding of the virus to blood resting CD4 T cells initiates a temporal course of cortical actin polymerization and depolymerization, a process mimicking the chemotactic response initiated from chemokine receptors. The actin depolymerization has been suggested to promote viral intracellular migration through cofilin-mediated actin treadmilling. However, the role of the virus-mediated actin polymerization in HIV infection is unknown, and the signaling molecules involved remain unidentified. Here we describe a pathogenic mechanism for triggering early actin polymerization through HIV-1 envelope-mediated transient activation of the LIM domain kinase (LIMK), a protein that phosphorylates cofilin. We demonstrate that HIV-mediated LIMK activation is through gp120-triggered transient activation of the Rack-PAK-LIMK pathway, and that knockdown of LIMK through siRNA decreases filamentous actin, increases CXCR4 trafficking, and diminishes viral DNA synthesis. These results suggest that HIV-mediated early actin polymerization may directly regulate the CXCR4 receptor during viral entry and is involved in viral DNA synthesis. Furthermore, we also demonstrate that in resting CD4 T cells, actin polymerization can be triggered through transient treatment with a pharmacological agent, okadaic acid, that activates LIMK and promotes HIV latent infection of resting CD4 T cells. Taken together, our results suggest that HIV hijacks LIMK to control the cortical actin dynamics for the initiation of viral infection of CD4 T cells. PMID:21321123

  1. 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.

  2. 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.

  3. 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.

  4. 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

  5. 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

  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. Cross-Linking of the Fingers Subdomain of Human Immunodeficiency Virus Type 1 Reverse Transcriptase to Template-Primer

    PubMed Central

    Peletskaya, Elena N.; Boyer, Paul L.; Kogon, Alex A.; Clark, Patrick; Kroth, Heiko; Sayer, Jane M.; Jerina, Donald M.; Hughes, Stephen H.

    2001-01-01

    Cross-linking experiments were performed with human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) mutants with unique cysteine residues at several positions (positions 65, 67, 70, and 74) in the fingers subdomain of the p66 subunit. Two approaches were used—photoaffinity cross-linking and disulfide chemical cross-linking (using an oligonucleotide that contained an N2-modified dG with a reactive thiol group). In the former case, cross-linking can occur to any nucleotide in either DNA strand, and in the latter case, a specific cross-link is produced between the template and the enzyme. Neither the introduction of the unique cysteine residues into the fingers nor the modification of these residues with photocross-linking reagents caused a significant decrease in the enzymatic activities of RT. We were able to use this model system to investigate interactions between specific points on the fingers domain of RT and double-stranded DNA (dsDNA). Photoaffinity cross-linking of the template to the modified RTs with Cys residues in positions 65, 67, 70, and 74 of the fingers domain of the p66 subunit was relatively efficient. Azide-modified Cys residues produced 10 to 25% cross-linking, whereas diazirine modified residues produced 5 to 8% cross-linking. Disulfide cross-linking yields were up to 90%. All of the modified RTs preferentially photocross-linked to the 5′ extended template strand of the dsDNA template-primer substrate. The preferred sites of interactions were on the extended template, 5 to 7 bases beyond the polymerase active site. HIV-1 RT is quite flexible. There are conformational changes associated with substrate binding. Cross-linking was used to detect intramolecular movements associated with binding of the incoming deoxynucleoside triphosphate (dNTP). Binding an incoming dNTP at the polymerase active site decreases the efficiency of cross-linking, but causes only modest changes in the preferred positions of cross-linking. This suggests

  9. Drosophila protein kinase N (Pkn) is a negative regulator of actin-myosin activity during oogenesis.

    PubMed

    Ferreira, Tânia; Prudêncio, Pedro; Martinho, Rui Gonçalo

    2014-10-15

    Nurse cell dumping is an actin-myosin based process, where 15 nurse cells of a given egg chamber contract and transfer their cytoplasmic content through the ring canals into the growing oocyte. We isolated two mutant alleles of protein kinase N (pkn) and showed that Pkn negatively-regulates activation of the actin-myosin cytoskeleton during the onset of dumping. Using live-cell imaging analysis we observed that nurse cell dumping rates sharply increase during the onset of fast dumping. Such rate increase was severely impaired in pkn mutant nurse cells due to excessive nurse cell actin-myosin activity and/or loss of tissue integrity. Our work demonstrates that the transition between slow and fast dumping is a discrete event, with at least a five to six-fold dumping rate increase. We show that Pkn negatively regulates nurse cell actin-myosin activity. This is likely to be important for directional cytoplasmic flow. We propose Pkn provides a negative feedback loop to help avoid excessive contractility after local activation of Rho GTPase.

  10. 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.

  11. 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.

  12. Isolation and removal of proteolytic enzymes with magnetic cross-linked erythrocytes

    NASA Astrophysics Data System (ADS)

    Šafařík, Ivo; Šafaříková, Mirka

    2001-01-01

    New magnetic adsorbents for batch isolation and removal of various proteolytic enzymes were prepared by glutaraldehyde cross-linking of bovine, porcine and human erythrocytes in the presence of fine magnetic particles. Trypsin, chymotrypsin, alkaline bacterial protease and proteases present in various commercial enzyme preparations were efficiently adsorbed on these adsorbents; on the contrary, proteins without proteolytic activity were not adsorbed.

  13. The F-actin bundler α-actinin Ain1 is tailored for ring assembly and constriction during cytokinesis in fission yeast

    PubMed Central

    Li, Yujie; Christensen, Jenna R.; Homa, Kaitlin E.; Hocky, Glen M.; Fok, Alice; Sees, Jennifer A.; Voth, Gregory A.; Kovar, David R.

    2016-01-01

    The actomyosin contractile ring is a network of cross-linked actin filaments that facilitates cytokinesis in dividing cells. Contractile ring formation has been well characterized in Schizosaccharomyces pombe, in which the cross-linking protein α-actinin SpAin1 bundles the actin filament network. However, the specific biochemical properties of SpAin1 and whether they are tailored for cytokinesis are not known. Therefore we purified SpAin1 and quantified its ability to dynamically bind and bundle actin filaments in vitro using a combination of bulk sedimentation assays and direct visualization by two-color total internal reflection fluorescence microscopy. We found that, while SpAin1 bundles actin filaments of mixed polarity like other α-actinins, SpAin1 has lower bundling activity and is more dynamic than human α-actinin HsACTN4. To determine whether dynamic bundling is important for cytokinesis in fission yeast, we created the less dynamic bundling mutant SpAin1(R216E). We found that dynamic bundling is critical for cytokinesis, as cells expressing SpAin1(R216E) display disorganized ring material and delays in both ring formation and constriction. Furthermore, computer simulations of initial actin filament elongation and alignment revealed that an intermediate level of cross-linking best facilitates filament alignment. Together our results demonstrate that dynamic bundling by SpAin1 is important for proper contractile ring formation and constriction. PMID:27075176

  14. Calcium-induced movement of troponin-I relative to actin in skeletal muscle thin filaments.

    PubMed

    Tao, T; Gong, B J; Leavis, P C

    1990-03-16

    The role of troponin-I (the inhibitory subunit of troponin) in the regulation by Ca2+ of skeletal muscle contraction was investigated with resonance energy transfer and photo cross-linking techniques. The effect of Ca2+ on the proximity of troponin-I to actin in reconstituted rabbit skeletal thin filaments was determined. The distance between the cysteine residue at position 133 (Cys133) of troponin-I and Cys374 of actin increases by approximately 15 angstroms on binding of Ca2+ to troponin-C. Also, troponin-I labeled at Cys133 with benzophenone-4-maleimide could be photo cross-linked to actin in the absence of Ca2+, but not in its presence. These results suggest that troponin-I is attached to actin in the Ca2(+)-free or relaxed state of muscle, and that it detaches from actin on Ca2+ activation of contraction. Thus, troponin-I may function as a Ca2(+)-dependent molecular switch in regulation of skeletal muscle contraction.

  15. Plasma membrane restricted RhoGEF activity is sufficient for RhoA-mediated actin polymerization

    PubMed Central

    van Unen, Jakobus; Reinhard, Nathalie R.; Yin, Taofei; Wu, Yi I.; Postma, Marten; Gadella, Theodorus W.J.; Goedhart, Joachim

    2015-01-01

    The small GTPase RhoA is involved in cell morphology and migration. RhoA activity is tightly regulated in time and space and depends on guanine exchange factors (GEFs). However, the kinetics and subcellular localization of GEF activity towards RhoA are poorly defined. To study the mechanism underlying the spatiotemporal control of RhoA activity by GEFs, we performed single cell imaging with an improved FRET sensor reporting on the nucleotide loading state of RhoA. By employing the FRET sensor we show that a plasma membrane located RhoGEF, p63RhoGEF, can rapidly activate RhoA through endogenous GPCRs and that localized RhoA activity at the cell periphery correlates with actin polymerization. Moreover, synthetic recruitment of the catalytic domain derived from p63RhoGEF to the plasma membrane, but not to the Golgi apparatus, is sufficient to activate RhoA. The synthetic system enables local activation of endogenous RhoA and effectively induces actin polymerization and changes in cellular morphology. Together, our data demonstrate that GEF activity at the plasma membrane is sufficient for actin polymerization via local RhoA signaling. PMID:26435194

  16. Actin filament organization in activated mast cells is regulated by heterotrimeric and small GTP-binding proteins

    PubMed Central

    1994-01-01

    Rat peritoneal mast cells, both intact and permeabilized, have been used widely as model secretory cells. GTP-binding proteins and calcium play a major role in controlling their secretory response. Here we have examined changes in the organization of actin filaments in intact mast cells after activation by compound 48/80, and in permeabilized cells after direct activation of GTP-binding proteins by GTP-gamma-S. In both cases, a centripetal redistribution of cellular F-actin was observed: the content of F-actin was reduced in the cortical region and increased in the cell interior. The overall F-actin content was increased. Using permeabilized cells, we show that AIF4-, an activator of heterotrimeric G proteins, induces the disassembly of F-actin at the cortex, while the appearance of actin filaments in the interior of the cell is dependent on two small GTPases, rho and rac. Rho was found to be responsible for de novo actin polymerization, presumably from a membrane-bound monomeric pool, while rac was required for an entrapment of the released cortical filaments. Thus, a heterotrimeric G-protein and the small GTPases, rho and rac, participate in affecting the changes in the actin cytoskeleton observed after activation of mast cells. PMID:8051203

  17. An actin monomer binding activity localizes to the carboxyl-terminal half of the Saccharomyces cerevisiae cyclase-associated protein.

    PubMed

    Freeman, N L; Chen, Z; Horenstein, J; Weber, A; Field, J

    1995-03-10

    The Saccharomyces cerevisiae adenylyl cyclase complex contains at least two subunits, a 200-kDa catalytic subunit and a 70-kDa cyclase-associated protein, CAP (also called Srv2p). Genetic studies suggested two roles for CAP, one as a positive regulator of cAMP levels in yeast and a second role as a cytoskeletal regulator. We present evidence showing that CAP sequesters monomeric actin (Kd in the range of 0.5-5 microM), decreasing actin incorporation into actin filaments. Anti-CAP monoclonal antibodies co-immunoprecipitate a protein with a molecular size of about 46 kDa. When CAP was purified from yeast using an anti-CAP monoclonal antibody column, the 46-kDa protein co-purified with a stoichiometry of about 1:1 with CAP. Western blots identified the 46-kDa protein as yeast actin. CAP also bound to muscle actin in vitro in immunoprecipitation assays and falling ball viscometry assays. Experiments with pyrene-labeled actin demonstrated that CAP sequesters actin monomers. The actin monomer binding activity is localized to the carboxyl-terminal half of CAP. Together, these data suggest that yeast CAP regulates the yeast cytoskeleton by sequestering actin monomers.

  18. 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.

  19. 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.

  20. 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

  1. 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.

  2. ACTINIC MASK INSPECTION AT THE ALS: RISK REDUCTION ACTIVITIES FOR 2003

    SciTech Connect

    Barty, A; Levesque, R; Ayers, J; Liu, Y; Gullikson, E; Barale, P

    2004-01-05

    This document reports on risk reduction activities performed at the VNL during CY2003 as a part of the Lith-343 actinic inspection project funded by International SEMATECH. The risk reduction activities described in this document comprise deliverable items 3.1.3, 3.1.4, 3.1.5 and 3.1.6 of Amendment 6 to the VNL EUV mask blank technology transfer contract.

  3. Structure of the 34 kDa F-actin-bundling protein ABP34 from Dictyostelium discoideum.

    PubMed

    Kim, Min-Kyu; Kim, Ji-Hye; Kim, Ji-Sun; Kang, Sa-Ouk

    2015-09-01

    The crystal structure of the 34 kDa F-actin-bundling protein ABP34 from Dictyostelium discoideum was solved by Ca(2+)/S-SAD phasing and refined at 1.89 Å resolution. ABP34 is a calcium-regulated actin-binding protein that cross-links actin filaments into bundles. Its in vitro F-actin-binding and F-actin-bundling activities were confirmed by a co-sedimentation assay and transmission electron microscopy. The co-localization of ABP34 with actin in cells was also verified. ABP34 adopts a two-domain structure with an EF-hand-containing N-domain and an actin-binding C-domain, but has no reported overall structural homologues. The EF-hand is occupied by a calcium ion with a pentagonal bipyramidal coordination as in the canonical EF-hand. The C-domain structure resembles a three-helical bundle and superposes well onto the rod-shaped helical structures of some cytoskeletal proteins. Residues 216-244 in the C-domain form part of the strongest actin-binding sites (193-254) and exhibit a conserved sequence with the actin-binding region of α-actinin and ABP120. Furthermore, the second helical region of the C-domain is kinked by a proline break, offering a convex surface towards the solvent area which is implicated in actin binding. The F-actin-binding model suggests that ABP34 binds to the side of the actin filament and residues 216-244 fit into a pocket between actin subdomains -1 and -2 through hydrophobic interactions. These studies provide insights into the calcium coordination in the EF-hand and F-actin-binding site in the C-domain of ABP34, which are associated through interdomain interactions.

  4. 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.

  5. Myosin II ATPase activity mediates the long-term potentiation-induced exodus of stable F-actin bound by drebrin A from dendritic spines.

    PubMed

    Mizui, Toshiyuki; Sekino, Yuko; Yamazaki, Hiroyuki; Ishizuka, Yuta; Takahashi, Hideto; Kojima, Nobuhiko; Kojima, Masami; Shirao, Tomoaki

    2014-01-01

    The neuronal actin-binding protein drebrin A forms a stable structure with F-actin in dendritic spines. NMDA receptor activation causes an exodus of F-actin bound by drebrin A (DA-actin) from dendritic spines, suggesting a pivotal role for DA-actin exodus in synaptic plasticity. We quantitatively assessed the extent of DA-actin localization to spines using the spine-dendrite ratio of drebrin A in cultured hippocampal neurons, and found that (1) chemical long-term potentiation (LTP) stimulation induces rapid DA-actin exodus and subsequent DA-actin re-entry in dendritic spines, (2) Ca(2+) influx through NMDA receptors regulates the exodus and the basal accumulation of DA-actin, and (3) the DA-actin exodus is blocked by myosin II ATPase inhibitor, but is not blocked by myosin light chain kinase (MLCK) or Rho-associated kinase (ROCK) inhibitors. These results indicate that myosin II mediates the interaction between NMDA receptor activation and DA-actin exodus in LTP induction. Furthermore, myosin II seems to be activated by a rapid actin-linked mechanism rather than slow MLC phosphorylation. Thus the myosin-II mediated DA-actin exodus might be an initial event in LTP induction, triggering actin polymerization and spine enlargement.

  6. 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.

  7. 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

  8. 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.

  9. 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

  10. 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 reinforced by 5 wt % CNFs showed superior properties as a promising material for the development of chitosan-based biomaterials.

  11. ROMP-based thermosetting polymers from modified castor oil with various cross-linking agents

    NASA Astrophysics Data System (ADS)

    Ding, Rui

    Polymers derived from bio-renewable resources are finding an increase in global demand. In addition, polymers with distinctive functionalities are required in certain advanced fields, such as aerospace and civil engineering. In an attempt to meet both these needs, the goal of this work aims to develop a range of bio-based thermosetting matrix polymers for potential applications in multifunctional composites. Ring-opening metathesis polymerization (ROMP), which recently has been explored as a powerful method in polymer chemistry, was employed as a unique pathway to polymerize agricultural oil-based reactants. Specifically, a novel norbornyl-functionalized castor oil alcohol (NCA) was investigated to polymerize different cross-linking agents using ROMP. The effects of incorporating dicyclopentadiene (DCPD) and a norbornene-based crosslinker (CL) were systematically evaluated with respect to curing behavior and thermal mechanical properties of the polymers. Isothermal differential scanning calorimetry (DSC) was used to investigate the conversion during cure. Dynamic DSC scans at multiple heating rates revealed conversion-dependent activation energy by Ozawa-Flynn-Wall analysis. The glass transition temperature, storage modulus, and loss modulus for NCA/DCPD and NCA/CL copolymers with different cross-linking agent loading were compared using dynamic mechanical analysis. Cross-link density was examined to explain the very different dynamic mechanical behavior. Mechanical stress-strain curves were developed through tensile test, and thermal stability of the cross-linked polymers was evaluated by thermogravimetric analysis to further investigate the structure-property relationships in these systems.

  12. 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.

  13. AMP-activated protein kinase induces actin cytoskeleton reorganization in epithelial cells.

    PubMed

    Miranda, Lisa; Carpentier, Sarah; Platek, Anna; Hussain, Nusrat; Gueuning, Marie-Agnès; Vertommen, Didier; Ozkan, Yurda; Sid, Brice; Hue, Louis; Courtoy, Pierre J; Rider, Mark H; Horman, Sandrine

    2010-06-04

    AMP-activated protein kinase (AMPK), a known regulator of cellular and systemic energy balance, is now recognized to control cell division, cell polarity and cell migration, all of which depend on the actin cytoskeleton. Here we report the effects of A769662, a pharmacological activator of AMPK, on cytoskeletal organization and signalling in epithelial Madin-Darby canine kidney (MDCK) cells. We show that AMPK activation induced shortening or radiation of stress fibers, uncoupling from paxillin and predominance of cortical F-actin. In parallel, Rho-kinase downstream targets, namely myosin regulatory light chain and cofilin, were phosphorylated. These effects resembled the morphological changes in MDCK cells exposed to hyperosmotic shock, which led to Ca(2+)-dependent AMPK activation via calmodulin-dependent protein kinase kinase-beta(CaMKKbeta), a known upstream kinase of AMPK. Indeed, hypertonicity-induced AMPK activation was markedly reduced by the STO-609 CaMKKbeta inhibitor, as was the increase in MLC and cofilin phosphorylation. We suggest that AMPK links osmotic stress to the reorganization of the actin cytoskeleton.

  14. 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.

  15. Quantitative assessment of fibrinogen cross-linking by epsilon aminocaproic acid in patients with end-stage liver disease.

    PubMed

    Quach, Thien; Tippens, Melissa; Szlam, Fania; Van Dyke, Rebecca; Levy, Jerrold H; Csete, Marie

    2004-01-01

    Analysis of the effectiveness of antifibrinolytic therapy for liver transplant recipients is hampered by lack of quantitative assays for assessing drug effects. We adapted chemical engineering tools used in polymerization studies to quantify fibrinogen cross-linking by plasma from liver transplant patients obtained before and after epsilon aminocaproic acid (EACA) therapy. A target fluorescein isothiocyanate-fibrinogen (FITC-fibrinogen) molecule was constructed; it fluoresces in a quantifiable pattern when in solution, and undergoes cross-linking in the presence of plasmin inhibitors. Cross-linking quenches the fluorescent signal, and the quenching is a quantifiable endpoint. Thus fluorescence from this reporter molecule can be used to assess functional improvement in fibrinogen cross-linking as a result of antifibrinolytic therapies, and it is sensitive to picomolar amounts of plasmin inhibitors and activators. Cross-linking of FITC-fibrinogen by patient plasma, before and after EACA therapy, was assessed using fluorescence spectrometry. Fluorescence patterns from FITC-fibrinogen indicated no significant cross-linking of the target fibrinogen as a consequence of EACA in posttreatment plasma. When the fibrinogen-FITC target was assayed without plasma in the presence of EACA at concentrations that bracket therapeutic levels (100 and 400 microg/ml), significant fluorescence quenching (target FITC-fibrinogen cross-linking) was achieved. These results suggest that fibrinogen-FITC fluorescence is sensitive enough to detect EACA activity in clinically relevant ranges, but that EACA given in usual doses is insufficient to promote fibrinogen cross-linking in patients with end-stage liver disease.

  16. Wear measurement of highly cross-linked UHMWPE using a 7Be tracer implantation technique.

    PubMed

    Wimmer, Markus A; Laurent, Michel P; Dwiwedi, Yasha; Gallardo, Luis A; Chipps, Kelly A; Blackmon, Jeffery C; Kozub, Raymond L; Bardayan, Daniel W; Gross, Carl J; Stracener, Daniel W; Smith, Michael S; Nesaraja, Caroline D; Erikson, Luke; Patel, Nidhi; Rehm, Karl E; Ahmad, Irshad; Greene, John P; Greife, Uwe

    2013-04-01

    The very low wear rates achieved with the current highly cross-linked ultrahigh molecular weight polyethylenes (UHMWPE) used in joint prostheses have proven to be difficult to measure accurately by gravimetry. Tracer methods are therefore being explored. The purpose of this study was to perform a proof-of-concept experiment on the use of the radioactive tracer beryllium-7 ((7)Be) for the determination of in vitro wear in a highly cross-linked orthopedic UHMWPE. Three cross-linked and four conventional UHMWPE pins made from compression-molded GUR 1050, were activated with 10(9) to 10(10) (7)Be nuclei using a new implantation setup that produced a homogenous distribution of implanted nuclei up to 8.5 μm below the surface. The pins were tested for wear in a six-station pin-on-flat apparatus for up to 7.1 million cycles (178 km). A Germanium gamma detector was employed to determine activity loss of the UHMWPE pins at preset intervals during the wear test. The wear of the cross-linked UHMWPE pins was readily detected and estimated to be 17 ± 3 μg per million cycles. The conventional-to-cross-linked ratio of the wear rates was 13.1 ± 0.8, in the expected range for these materials. Oxidative degradation damage from implantation was negligible; however, a weak dependence of wear on implantation dose was observed limiting the number of radioactive tracer atoms that can be introduced. Future applications of this tracer technology may include the analysis of location-specific wear, such as loss of material in the post or backside of a tibial insert.

  17. 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.

  18. 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.

  19. 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

  20. Yap/Taz transcriptional activity is essential for vascular regression via Ctgf expression and actin polymerization

    PubMed Central

    Nagasawa-Masuda, Ayumi; Terai, Kenta

    2017-01-01

    Vascular regression is essential to remove redundant vessels during the formation of an efficient vascular network that can transport oxygen and nutrient to every corner of the body. However, no mechanism is known to explain how major blood vessels regress during development. Here we use the dorsal part of the caudal vein plexus (dCVP) in Zebrafish to investigate the mechanism of regression and discover a new role of Yap/Taz in vascular regression. During regression, Yap/Taz is activated by blood circulation in the endothelial cells. This leads to induction of Ctgf and actin polymerization. Interference with Yap/Taz activation decreased Ctgf production, which decreased actin polymerization and vascular regression. These results implicate a novel role of Yap/Taz in vascular regression. PMID:28369143

  1. Cortactin Adopts a Globular Conformation and Bundles Actin into Sheets

    SciTech Connect

    Cowieson, Nathan P.; King, Gordon; Cookson, David; Ross, Ian; Huber, Thomas; Hume, David A.; Kobe, Bostjan; Martin, Jennifer L.

    2008-08-21

    Cortactin is a filamentous actin-binding protein that plays a pivotal role in translating environmental signals into coordinated rearrangement of the cytoskeleton. The dynamic reorganization of actin in the cytoskeleton drives processes including changes in cell morphology, cell migration, and phagocytosis. In general, structural proteins of the cytoskeleton bind in the N-terminal region of cortactin and regulatory proteins in the C-terminal region. Previous structural studies have reported an extended conformation for cortactin. It is therefore unclear how cortactin facilitates cross-talk between structural proteins and their regulators. In the study presented here, circular dichroism, chemical cross-linking, and small angle x-ray scattering are used to demonstrate that cortactin adopts a globular conformation, thereby bringing distant parts of the molecule into close proximity. In addition, the actin bundling activity of cortactin is characterized, showing that fully polymerized actin filaments are bundled into sheet-like structures. We present a low resolution structure that suggests how the various domains of cortactin interact to coordinate its array of binding partners at sites of actin branching.

  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. Cancer-associated Fibroblasts Induce a Collagen Cross-link Switch in Tumor Stroma

    PubMed Central

    Pankova, Daniela; Chen, Yulong; Terajima, Masahiko; Schliekelman, Mark J.; Baird, Brandi N.; Fahrenholtz, Monica; Sun, Li; Gill, Bartley J.; Vadakkan, Tegy J.; Kim, Min P.; Ahn, Young-Ho; Roybal, Jonathon D.; Liu, Xin; Parra Cuentas, Edwin Roger; Rodriguez, Jaime; Wistuba, Ignacio I.; Creighton, Chad J.; Gibbons, Don L.; Hicks, John M.; Dickinson, Mary E.; West, Jennifer L.; Grande-Allen, K. Jane; Hanash, Samir M.; Yamauchi, Mitsuo; Kurie, Jonathan M.

    2015-01-01

    Intratumoral collagen cross-links heighten stromal stiffness and stimulate tumor cell invasion, but it is unclear how collagen cross-linking is regulated in epithelial tumors. To address this question, we used KrasLA1 mice, which develop lung adenocarcinomas from somatic activation of a KrasG12D allele. The lung tumors in KrasLA1 mice were highly fibrotic and contained cancer-associated fibroblasts (CAFs) that produced collagen and generated stiffness in collagen gels. In xenograft tumors generated by injection of wild-type mice with lung adenocarcinoma cells alone or in combination with CAFs, the total concentration of collagen cross-links was the same in tumors generated with or without CAFs, but co-injected tumors had higher hydroxylysine aldehyde-derived collagen cross-links (HLCCs) and lower lysine-aldehyde-derived collagen cross-links (LCCs). Therefore, we postulated that an LCC-to-HLCC switch induced by CAFs promotes the migratory and invasive properties of lung adenocarcinoma cells. To test this hypothesis, we created co-culture models in which CAFs are positioned interstitially or peripherally in tumor cell aggregates, mimicking distinct spatial orientations of CAFs in human lung cancer. In both contexts, CAFs enhanced the invasive properties of tumor cells in 3-dimensional (3D) collagen gels. Tumor cell aggregates that attached to CAF networks on a Matrigel surface dissociated and migrated on the networks. Lysyl hydroxylase 2 (PLOD2/LH2), which drives HLCC formation, was expressed in CAFs, and LH2 depletion abrogated the ability of CAFs to promote tumor cell invasion and migration. PMID:26631572

  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. 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.

  6. Activator-inhibitor coupling between Rho signaling and actin assembly make the cell cortex an excitable medium

    PubMed Central

    Bement, William M.; Leda, Marcin; Moe, Alison M.; Kita, Angela M.; Larson, Matthew E.; Golding, Adriana E.; Pfeuti, Courtney; Su, Kuan-Chung; Miller, Ann L.; Goryachev, Andrew B.; von Dassow, George

    2016-01-01

    Animal cell cytokinesis results from patterned activation of the small GTPase Rho, which directs assembly of actomyosin in the equatorial cortex. Cytokinesis is restricted to a portion of the cell cycle following anaphase onset in which the cortex is responsive to signals from the spindle. We show that shortly after anaphase onset oocytes and embryonic cells of frogs and echinoderms exhibit cortical waves of Rho activity and F-actin polymerization. The waves are modulated by cyclin-dependent kinase 1 (Cdk1) activity and require the Rho GEF (guanine nucleotide exchange factor), Ect2. Surprisingly, during wave propagation, while Rho activity elicits F-actin assembly, F-actin subsequently inactivates Rho. Experimental and modeling results show that waves represent excitable dynamics of a reaction diffusion system with Rho as the activator and F-actin the inhibitor. We propose that cortical excitability explains fundamental features of cytokinesis including its cell cycle regulation. PMID:26479320

  7. Both cross-links and monoadducts induced in DNA by psoralens can lead to sister chromatid exchange formation

    SciTech Connect

    Cortes, F. Facultad de Biologia, Sevilla ); Morgan, W.F.; Varcarcel, E.R.; Cleaver, J.E.; Wolff, S. )

    1991-09-01

    The relative importance of DNA-DNA cross-links and bulky monoadducts in sister chromatid exchange (SCE) formation was investigated in three human fibroblast cell lines with different repair capabilities. These cell lines included normal cells, which can repair both classes of lesion; xeroderma pigmentosum (XP) cells, which cannot repair either psoralen-induced cross-links or monoadducts; and an XP revertant that repairs only cross-links and not monoadducts. SCEs were induced by two psoralen derivatives. After activation with long-wave ultraviolet light, HMT produces cross-links and monoadducts in DNA, whereas 5-MIP produces only monoadducts. In normal human cells both psoralens induced SCEs, but if cells were allowed to repair for 18 h before bromodeoxyuridine (BrdUrd) was added for SCE analysis, the SCE frequency was significantly reduced. XP cells showed an SCE frequency that remained high regardless of whether SCEs were analyzed immediately after psoralen exposure of 18 h later. In the XP revertant that repairs only cross-links, both psoralens induced a high yield of SCEs when BrdUrd was added immediately after psoralen treatment. These observations indicate that both cross-links and monoadducts are lesions in DNA that can lead to SCE formation.

  8. Cross-linking staphylococcal enterotoxin A bound to major histocompatibility complex class I is required for TNF-alpha secretion

    NASA Technical Reports Server (NTRS)

    Wright, A. D.; Chapes, S. K.

    1999-01-01

    The mechanism of how superantigens function to activate cells has been linked to their ability to bind and cross-link the major histocompatibility complex class II (MHCII) molecule. Cells that lack the MHCII molecule also respond to superantigens, however, with much less efficiency. Therefore, the purpose of this study was to confirm that staphylococcal enterotoxin A (SEA) could bind the MHCI molecule and to test the hypothesis that cross-linking SEA bound to MHCII-deficient macrophages would induce a more robust cytokine response than without cross-linking. We used a capture enzyme-linked immunosorbent assay and an immunprecipitation assay to directly demonstrate that MHCI molecules bind SEA. Directly cross-linking MHCI using monoclonal antibodies or cross-linking bound SEA with an anti-SEA antibody or biotinylated SEA with avidin increased TNF-alpha and IL-6 secretion by MHCII(-/-) macrophages. The induction of a vigorous macrophage cytokine response by SEA/anti-SEA cross-linking of MHCI offers a mechanism to explain how MHCI could play an important role in superantigen-mediated pathogenesis. Copyright 1999 Academic Press.

  9. Molecular Basis for the Dual Function of Eps8 on Actin Dynamics: Bundling and Capping

    PubMed Central

    Hazelwood, Larnele; Disanza, Andrea; Liu, HongJun; Perlade, Emilie; Malabarba, Maria Grazia; Pasqualato, Sebastiano; Maiolica, Alessio; Confalonieri, Stefano; Le Clainche, Christophe; Offenhauser, Nina; Block, Jennifer; Rottner, Klemens; Di Fiore, Pier Paolo; Carlier, Marie-France; Volkmann, Niels; Hanein, Dorit; Scita, Giorgio

    2010-01-01

    Actin capping and cross-linking proteins regulate the dynamics and architectures of different cellular protrusions. Eps8 is the founding member of a unique family of capping proteins capable of side-binding and bundling actin filaments. However, the structural basis through which Eps8 exerts these functions remains elusive. Here, we combined biochemical, molecular, and genetic approaches with electron microscopy and image analysis to dissect the molecular mechanism responsible for the distinct activities of Eps8. We propose that bundling activity of Eps8 is mainly mediated by a compact four helix bundle, which is contacting three actin subunits along the filament. The capping activity is mainly mediated by a amphipathic helix that binds within the hydrophobic pocket at the barbed ends of actin blocking further addition of actin monomers. Single-point mutagenesis validated these modes of binding, permitting us to dissect Eps8 capping from bundling activity in vitro. We further showed that the capping and bundling activities of Eps8 can be fully dissected in vivo, demonstrating the physiological relevance of the identified Eps8 structural/functional modules. Eps8 controls actin-based motility through its capping activity, while, as a bundler, is essential for proper intestinal morphogenesis of developing Caenorhabditis elegans. PMID:20532239

  10. Activation of endothelial nitric oxide synthase is dependent on its interaction with globular actin in human umbilical vein endothelial cells.

    PubMed

    Mi, Qiongyu; Chen, Nan; Shaifta, Yasin; Xie, Liping; Lu, Hui; Liu, Zhen; Chen, Qi; Hamid, Colleen; Becker, Silke; Ji, Yong; Ferro, Albert

    2011-09-01

    Endothelial nitric oxide synthase (eNOS) has been reported to associate with globular actin, and this association increases eNOS activity. Adenosine, histamine, salbutamol and thrombin cause activation of eNOS through widely different mechanisms. Whether these eNOS agonists can regulate eNOS activity through affecting its association with actin is unknown. As previously reported, we confirmed in cultured human umbilical vein endothelial cells (HUVEC) that histamine and thrombin increased intracellular Ca(2+) whereas adenosine and salbutamol did not, and that these four agonists caused different effects on actin filament structure. Nevertheless, despite their divergent effects on intracellular Ca(2+) and on actin filament structure, we found by immunoprecipitation that adenosine, histamine, salbutamol and thrombin all caused an increase in association between eNOS and globular actin. This increase of association was inhibited by pre-treatment with phalloidin, an actin filament stabilizer. All of these agonists also increased phosphorylation of eNOS on serine residue 1177, eNOS activity, and cyclic guanosine-3', 5'-monophosphate, and these increases were all attenuated by phalloidin. Agonist-induced phosphorylation of eNOS on serine 1177 was attenuated by Akt inhibition, whereas association of eNOS with actin was not. We also found, in HEK-293 cells transfected with the eNOS mutants eNOS-S1177A or eNOS-S1177D, that the association between eNOS and globular actin was decreased as compared to cells transfected with wild-type eNOS. We conclude that association of globular actin with eNOS plays an essential and necessary role in agonist-induced eNOS activation, through enabling its phosphorylation by Akt at serine residue 1177.

  11. 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.

  12. 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.

  13. 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

  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. 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.

  2. 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.

  3. 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.

  4. 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

  5. A biosensor for the protease TACE reveals actin damage induced TACE activation

    PubMed Central

    Chapnick, Douglas A.; Bunker, Eric; Liu, Xuedong

    2016-01-01

    Ligand shedding has gained increased attention as a major posttranslational modification mechanism used by cells to respond to diverse environmental conditions. The TACEadam17 protease is a critical mediator of such ligand shedding, regulating the maturation and release of an impressive range of extracellular substrates that drive diverse cellular responses. Exactly how this protease is itself activated remains unclear, in part due to the lack of available tools to measure TACE activity with temporal and spatial resolution in live cells. We have developed a FRET based biosensor for TACE activity (TSen), which is capable of reporting TACE activation kinetics in live cells with a high degree of specificity. TSen was used in combination with chemical biology to probe the dependence of various means of TACE activation on p38 and Erk kinase activities, as well as to identify a novel connection between actin cytoskeletal disruption and TACE activation. Such cytoskeletal disruption leads to rapid and robust TACE activation in some cell types and accumulation of TACE at the plasma membrane, allowing for increased cleavage of endogenous substrates. Our study highlights both the versatility of TSen as a tool to understand the mechanisms of TACE activation in live cells and the importance of actin cytoskeletal integrity as a modulator of TACE activity. PMID:25714465

  6. 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

  7. Mitogen Activated Protein Kinase Activated Protein Kinase 2 Regulates Actin Polymerization and Vascular Leak in Ventilator Associated Lung Injury

    PubMed Central

    Damarla, Mahendra; Hasan, Emile; Boueiz, Adel; Le, Anne; Pae, Hyun Hae; Montouchet, Calypso; Kolb, Todd; Simms, Tiffany; Myers, Allen; Kayyali, Usamah S.; Gaestel, Matthias; Peng, Xinqi; Reddy, Sekhar P.; Damico, Rachel; Hassoun, Paul M.

    2009-01-01

    Mechanical ventilation, a fundamental therapy for acute lung injury, worsens pulmonary vascular permeability by exacting mechanical stress on various components of the respiratory system causing ventilator associated lung injury. We postulated that MK2 activation via p38 MAP kinase induced HSP25 phosphorylation, in response to mechanical stress, leading to actin stress fiber formation and endothelial barrier dysfunction. We sought to determine the role of p38 MAP kinase and its downstream effector MK2 on HSP25 phosphorylation and actin stress fiber formation in ventilator associated lung injury. Wild type and MK2−/− mice received mechanical ventilation with high (20 ml/kg) or low (7 ml/kg) tidal volumes up to 4 hrs, after which lungs were harvested for immunohistochemistry, immunoblotting and lung permeability assays. High tidal volume mechanical ventilation resulted in significant phosphorylation of p38 MAP kinase, MK2, HSP25, actin polymerization, and an increase in pulmonary vascular permeability in wild type mice as compared to spontaneous breathing or low tidal volume mechanical ventilation. However, pretreatment of wild type mice with specific p38 MAP kinase or MK2 inhibitors abrogated HSP25 phosphorylation and actin polymerization, and protected against increased lung permeability. Finally, MK2−/− mice were unable to phosphorylate HSP25 or increase actin polymerization from baseline, and were resistant to increases in lung permeability in response to HVT MV. Our results suggest that p38 MAP kinase and its downstream effector MK2 mediate lung permeability in ventilator associated lung injury by regulating HSP25 phosphorylation and actin cytoskeletal remodeling. PMID:19240800

  8. Macromolecular cross-linked enzyme aggregates (M-CLEAs) of α-amylase.

    PubMed

    Nadar, Shamraja S; Muley, Abhijeet B; Ladole, Mayur R; Joshi, Pranoti U

    2016-03-01

    Macromolecular cross-linked enzyme aggregates (M-CLEAs) of α-amylase were prepared by precipitation and subsequent cross-linking. The non-toxic, biodegradable, biocompatible, renewable polysaccharide based macromolecular cross-linkers viz. agar, chitosan, dextran, and gum arabic were used as a substitute for traditional glutaraldehyde to augment activity recovery toward macromolecular substrate. Macromolecular cross-linkers were prepared by periodate mediated controlled oxidation of polysaccharides. The effects of precipitating agent, concentration and different cross-linkers on activity recovery of α-amylase CLEAs were investigated. α-Amylase aggregated with ammonium sulphate and cross-linked by dextran showed 91% activity recovery, whereas glutaraldehyde CLEAs (G-CLEAs) exhibited 42% activity recovery. M-CLEAs exhibited higher thermal stability in correlation with α-amylase and G-CLEAs. Moreover, dextran and chitosan M-CLEAs showed same affinity for starch hydrolysis as of free α-amylase. The changes in secondary structures revealed the enhancements in structural and conformational rigidity attributed by cross-linkers. Finally, after five consecutive cycles dextran M-CLEAs retained 1.25 times higher initial activity than G-CLEAs.

  9. Catechol chemistry inspired approach to construct self-cross-linked polymer nanolayers as versatile biointerfaces.

    PubMed

    Liu, Xinyue; Deng, Jie; Ma, Lang; Cheng, Chong; Nie, Chuanxiong; He, Chao; Zhao, Changsheng

    2014-12-16

    In this study, we proposed a catechol chemistry inspired approach to construct surface self-cross-linked polymer nanolayers for the design of versatile biointerfaces. Several representative biofunctional polymers, P(SS-co-AA), P(SBMA-co-AA), P(EGMA-co-AA), P(VP-co-AA), and P(MTAC-co-AA), were first synthesized via reversible addition-fragmentation chain transfer (RAFT) polymerization, and then the catecholic molecules (dopamine, DA) were conjugated to the acrylic acid (AA) units by the facile carbodiimide chemistry. Then, the catechol (Cat) group conjugated biofunctional polymers, named PSS-Cat, PSBMA-Cat, PEGMA-Cat, PVP-Cat, and PMTAC-Cat, were applied for the construction of self-cross-linked nanolayers on polymeric substrates via the pH induced catechol cross-linking and immobilization. The XPS spectra, surface morphology, and wettability gave robust evidence that the catechol conjugated polymers were successfully coated, and the coated substrates possessed increased surface roughness and hydrophilicity. Furthermore, the systematic in vitro investigation of protein adsorption, platelet adhesion, activated partial thromboplastin time (APTT), thrombin time (TT), cell viability, and antibacterial ability confirmed that the coated nanolayers conferred the substrates with versatile biological performances. The PSS-Cat coated substrate had low blood component activation and excellent anticoagulant activity; while the PEGMA-Cat and PSBMA-Cat showed ideal resistance to protein fouling and inhibition of platelet activation. The PSS-Cat and PVP-Cat coated substrates exhibited promoted endothelial cell proliferation and viability. The PMTAC-Cat coated substrate showed an outstanding activity on bacterial inhibition. In conclusion, the catechol chemistry inspired approach allows the self-cross-linked nanolayers to be easily immobilized on polymeric substrates with the stable conformation and multiple biofunctionalities. It is expected that this low-cost and facile

  10. Why is Actin Patchy?

    NASA Astrophysics Data System (ADS)

    Carlsson, Anders

    2009-03-01

    The intracellular protein actin, by reversibly polymerizing into filaments, generates forces for motion and shape changes of many types of biological cells. Fluorescence imaging studies show that actin often occurs in the form of localized patches of size roughly one micrometer at the cell membrane. Patch formation is most prevalent when the free-actin concentration is low. I investigate possible mechanisms for the formation of actin patches by numerically simulating the ``dendritic nucleation'' model of actin network growth. The simulations include filament growth, capping, branching, severing, and debranching. The attachment of membrane-bound activators to actin filaments, and subsequent membrane diffusion of unattached activators, are also included. It is found that as the actin concentration increases from zero, the actin occurs in patches at lower actin concentrations, and the size of the patches increases with increasing actin concentration. At a critical value of the actin concentration, the system undergoes a transition to complete coverage. The results are interpreted within the framework of reaction-diffusion equations in two dimensions.

  11. Arf1 and Arf6 Promote Ventral Actin Structures formed by acute Activation of Protein Kinase C and Src

    PubMed Central

    Caviston, Juliane P.; Cohen, Lee Ann; Donaldson, Julie G.

    2016-01-01

    Arf proteins regulate membrane traffic and organelle structure. Although Arf6 is known to initiate actin-based changes in cell surface architecture, Arf1 may also function at the plasma membrane. Here we show that acute activation of protein kinase C (PKC) induced by the phorbol ester PMA led to the formation of motile actin structures on the ventral surface of Beas-2b cells, a lung bronchial epithelial cell line. Ventral actin structures also formed in PMA-treated HeLa cells that had elevated levels of Arf activation. For both cell types, formation of the ventral actin structures was enhanced by expression of active forms of either Arf1 or Arf6, and by the expression of guanine nucleotide exchange factors that activate these Arfs. By contrast, formation of these structures was blocked by inhibitors of PKC and Src, and required phosphatidylinositol 4, 5-bisphosphate, Rac, Arf6 and Arf1. Furthermore, expression of ASAP1, an Arf1 GTPase activating protein (GAP) was more effective at inhibiting the ventral actin structures than was ACAP1, an Arf6 GAP. This study adds to the expanding role for Arf1 in the periphery and identifies a requirement for Arf1, a “Golgi Arf”, in the reorganization of the cortical actin cytoskeleton on ventral surfaces, against the substratum. PMID:24916416

  12. Structural and viscoelastic properties of actin networks formed by espin or pathologically relevant espin mutants.

    PubMed

    Lieleg, Oliver; Schmoller, Kurt M; Purdy Drew, Kirstin R; Claessens, Mireille M A E; Semmrich, Christine; Zheng, Lili; Bartles, James R; Bausch, Andreas R

    2009-11-09

    The structural organization of the cytoskeleton determines its viscoelastic response which is crucial for the correct functionality of living cells. Both the mechanical response and microstructure of the cytoskeleton are regulated on a microscopic level by the local activation of different actin binding and/or bundling proteins (ABPs). Misregulations in the expression of these ABPs or mutations in their sequence can entail severe cellular dysfunctions and diseases. Here, we study the structural and viscoelastic properties of reconstituted actin networks cross-linked by the ABP espin and compare the obtained network properties to those of other bundled actin networks. Moreover, we quantify the impact of pathologically relevant espin mutations on the viscoelastic properties of these cytoskeletal networks.

  13. 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

  14. Effect of O6-methylguanine on DNA interstrand cross-link formation by chloroethylnitrosoureas and 2-chloroethyl(methylsulfonyl)methanesulfonate.

    PubMed

    Dolan, M E; Pegg, A E; Hora, N K; Erickson, L C

    1988-07-01

    Exposure of HT29 cells in culture to O6-methylguanine is known to result in a reduction in O6-alkylguanine-DNA alkyltransferase (AGT) activity and an enhancement of sensitivity to the cytotoxic effects of chloroethylating agents. Since cytotoxicity of these agents may be mediated by the formation of interstrand cross-links, alkaline elution analysis was performed on HT29 cells treated with 1-(2-chloroethyl)-1-nitrosourea, 1-(2-chloroethyl)-3-cyclohexyl-1-nitrosourea, and Clomesone [2-chloroethyl(methylsulfonyl)methanesulfonate] in the presence or absence of O6-methylguanine pretreatment to determine if the enhanced toxicity was due to an increase in the number of cross-links formed. Interstrand cross-linking by 1-(2-chloroethyl)-1-nitrosourea or 1-(2-chloroethyl)-3-cyclohexyl-1-nitrosourea was increased by pretreatment with 0.4 mM O6-methylguanine for 24 h. Cross-linking by Clomesone was observed only in cells exposed to 0.4 mM O6-methylguanine for 24 h prior to administration of the drug and for 12 h after administration, suggesting that the resynthesis of the AGT may prevent the cross-linking by Clomesone. Complete recovery of AGT activity after reduction to 20 to 30% of the basal level upon treatment with 0.4 mM O6-methylguanine required between 8 h and 15 h in both HT29 cells and in Raji cells which were also sensitized to 1-(2-chloro-ethyl)-3-cyclohexyl-1-nitrosourea by exposure to O6-methylguanine. These data suggest that the enhancement of chloroethylnitrosourea toxicity after treatment with O6-methylguanine may be related to an increase in the number of DNA cross-links and that the relatively rapid rate of AGT recovery plays a role in prevention of cross-links resulting from Clomesone.

  15. Actin depolymerization mediated loss of SNTA1 phosphorylation and Rac1 activity has implications on ROS production, cell migration and apoptosis.

    PubMed

    Bhat, Sehar Saleem; Parray, Arif Ali; Mushtaq, Umar; Fazili, Khalid Majid; Khanday, Firdous Ahmad

    2016-06-01

    Alpha-1-syntrophin (SNTA1) and Rac1 are part of a signaling pathway via the dystrophin glycoprotein complex (DGC). Both SNTA1 and Rac1 proteins are over-expressed in various carcinomas. It is through the DGC signaling pathway that SNTA1 has been shown to act as a link between the extra cellular matrix, the internal cell signaling apparatus and the actin cytoskeleton. SNTA1 is involved in the modulation of the actin cytoskeleton and actin reorganization. Rac1 also controls actin cytoskeletal organization in the cell. In this study, we present the interplay between f-actin, SNTA1 and Rac1. We analyzed the effect of actin depolymerization on SNTA1 tyrosine phosphorylation and Rac1 activity using actin depolymerizing drugs, cytochalasin D and latrunculin A. Our results indicate a marked decrease in the tyrosine phosphorylation of SNTA1 upon actin depolymerization. Results suggest that actin depolymerization mediated loss of SNTA1 phosphorylation leads to loss of interaction between SNTA1 and Rac1, with a concomitant loss of Rac1 activation. The loss of SNTA1tyrosine phosphorylation and Rac1 activity by actin depolymerization results in increased apoptosis, decreased cell migration and decreased reactive oxygen species (ROS) levels in breast carcinoma cells. Collectively, our results present a possible role of f-actin in the SNTA1-Rac1 signaling pathway and implications of actin depolymerization on cell migration, ROS production and apoptosis.

  16. Lenalidomide augments actin remodeling and lowers NK-cell activation thresholds

    PubMed Central

    Lagrue, Kathryn; Carisey, Alex; Morgan, David J.; Chopra, Rajesh

    2015-01-01

    As multiple myeloma (MM) progresses, natural killer (NK)-cell responses decline against malignant plasma cells. The immunomodulatory drug lenalidomide is widely used for treatment of MM but its influence on NK-cell biology is unclear. Here, we report that lenalidomide lowers the threshold for NK-cell activation, causing a 66% decrease in the 50% effective concentration (EC50) for activation through CD16, and a 38% decrease in EC50 for NK group 2 member D (NKG2D)–mediated activation, allowing NK cells to respond to lower doses of ligand. In addition, lenalidomide augments NK-cell responses, causing a twofold increase in the proportion of primary NK cells producing interferon-γ (IFN-γ), and a 20-fold increase in the amount of IFN-γ produced per cell. Importantly, lenalidomide did not trigger IFN-γ production in unstimulated NK cells. Thus, lenalidomide enhances the NK-cell arm of the immune response, without activating NK cells inappropriately. Of particular clinical importance, lenalidomide also allowed NK cells to be activated by lower doses of rituximab, an anti-CD20 monoclonal antibody (mAb) widely used to treat B-cell malignancies. This supports combined use of lenalidomide and rituximab in a clinical setting. Finally, superresolution microscopy revealed that lenalidomide increased the periodicity of cortical actin at immune synapses, resulting in an increase in the area of the actin mesh predicted to be penetrable to vesicles containing IFN-γ. NK cells from MM patients also responded to lenalidomide in this way. This indicates that nanometer-scale rearrangements in cortical actin, a recently discovered step in immune synapse assembly, are a potential new target for therapeutic compounds. PMID:26002964

  17. Lenalidomide augments actin remodeling and lowers NK-cell activation thresholds.

    PubMed

    Lagrue, Kathryn; Carisey, Alex; Morgan, David J; Chopra, Rajesh; Davis, Daniel M

    2015-07-02

    As multiple myeloma (MM) progresses, natural killer (NK)-cell responses decline against malignant plasma cells. The immunomodulatory drug lenalidomide is widely used for treatment of MM but its influence on NK-cell biology is unclear. Here, we report that lenalidomide lowers the threshold for NK-cell activation, causing a 66% decrease in the 50% effective concentration (EC50) for activation through CD16, and a 38% decrease in EC50 for NK group 2 member D (NKG2D)-mediated activation, allowing NK cells to respond to lower doses of ligand. In addition, lenalidomide augments NK-cell responses, causing a twofold increase in the proportion of primary NK cells producing interferon-γ (IFN-γ), and a 20-fold increase in the amount of IFN-γ produced per cell. Importantly, lenalidomide did not trigger IFN-γ production in unstimulated NK cells. Thus, lenalidomide enhances the NK-cell arm of the immune response, without activating NK cells inappropriately. Of particular clinical importance, lenalidomide also allowed NK cells to be activated by lower doses of rituximab, an anti-CD20 monoclonal antibody (mAb) widely used to treat B-cell malignancies. This supports combined use of lenalidomide and rituximab in a clinical setting. Finally, superresolution microscopy revealed that lenalidomide increased the periodicity of cortical actin at immune synapses, resulting in an increase in the area of the actin mesh predicted to be penetrable to vesicles containing IFN-γ. NK cells from MM patients also responded to lenalidomide in this way. This indicates that nanometer-scale rearrangements in cortical actin, a recently discovered step in immune synapse assembly, are a potential new target for therapeutic compounds.

  18. Effects of cardiac Myosin binding protein-C on actin motility are explained with a drag-activation-competition model.

    PubMed

    Walcott, Sam; Docken, Steffen; Harris, Samantha P

    2015-01-06

    Although mutations in cardiac myosin binding protein-C (cMyBP-C) cause heart disease, its role in muscle contraction is not well understood. A mechanism remains elusive partly because the protein can have multiple effects, such as dual biphasic activation and inhibition observed in actin motility assays. Here we develop a mathematical model for the interaction of cMyBP-C with the contractile proteins actin and myosin and the regulatory protein tropomyosin. We use this model to show that a drag-activation-competition mechanism accurately describes actin motility measurements, while models lacking either drag or competition do not. These results suggest that complex effects can arise simply from cMyBP-C binding to actin.

  19. Effects of Cardiac Myosin Binding Protein-C on Actin Motility Are Explained with a Drag-Activation-Competition Model

    PubMed Central

    Walcott, Sam; Docken, Steffen; Harris, Samantha P.

    2015-01-01

    Although mutations in cardiac myosin binding protein-C (cMyBP-C) cause heart disease, its role in muscle contraction is not well understood. A mechanism remains elusive partly because the protein can have multiple effects, such as dual biphasic activation and inhibition observed in actin motility assays. Here we develop a mathematical model for the interaction of cMyBP-C with the contractile proteins actin and myosin and the regulatory protein tropomyosin. We use this model to show that a drag-activation-competition mechanism accurately describes actin motility measurements, while models lacking either drag or competition do not. These results suggest that complex effects can arise simply from cMyBP-C binding to actin. PMID:25564844

  20. Effects of chebulic acid on advanced glycation endproducts-induced collagen cross-links.

    PubMed

    Lee, Ji-Young; Oh, Jun-Gu; Kim, Jin Sook; Lee, Kwang-Won

    2014-01-01

    Advanced glycation end-products (AGEs) have been implicated in the development of diabetic complications. We report the antiglycating activity of chebulic acid (CA), isolated from Terminalia chebula on breaking the cross-links of proteins induced by AGEs and inhibiting the formation of AGEs. Aminoguanidine (AG) reduced 50% of glycated bovine serum albumin (BSA) with glycolaldehyde (glycol-BSA)-induced cross-links of collagen at a concentration of 67.8 ± 2.5 mM, the level of CA required for exerting a similar antiglycating activity was 38.8 ± 0.5 µM. Also, the breaking activity on collagen cross-links induced by glycol-BSA was potent with CA (IC50=1.46 ± 0.05 mM), exhibiting 50-fold stronger breaking activity than with ALT-711, a well-known cross-link breaker (IC50=72.2 ± 2.4 mM). IC50 values of DPPH· scavenging activity for CA and ascorbic acid (AA) were 39.2 ± 4.9 and 19.0 ± 1.2 µg dry matter (DM) mL(-1), respectively, and ferric reducing and antioxidant power (FRAP) activities for CA and AA were 4.70 ± 0.06 and 11.4 ± 0.1 mmol/FeSO4·7H2O/g DM, respectively. The chelating activities of CA, AG and ALT711 on copper-catalyzed oxidation of AA were compared, and in increasing order, ALT-711 (IC50 of 1.92 ± 0.20 mM)cross-linking, the activity of which may be explained in large part by its chelating and antioxidant activities, suggesting that CA may constitute a promising antiglycating candidate in intervening AGE-mediated diabetic complications.

  1. Improved enzyme properties upon glutaraldehyde cross-linking of alginate entrapped xylanase from Bacillus licheniformis.

    PubMed

    Kumar, Sharad; Haq, Izharul; Prakash, Jyoti; Raj, Abhay

    2017-05-01

    Enzyme immobilization is an exciting alternative to improve the stability of enzymatic processes and economic viability in terms of reusability. In the current study, purified xylanase from B. licheniformis Alk-1 was immobilized within glutaraldehyde activated calcium alginate beads and characterized in respect of free enzyme. Immobilization increases the optimum pH and temperature of entrapped and cross-linked enzyme from pH=8.0 to 9.0 and 50-60°C. The kinetics parameter of immobilized (cross-linked) enzyme showed an increase in Km (from 4.36mg/mL to 5.38mg/mL) and decrease in Vmax (from 383 IU/mg/min to 370 IU/mg/min). Immobilization increases the optimum reaction time for xylan degradation of immobilized xylanase from 15 to 30min when compare to free form. The storage stability study suggested that the immobilized enzyme retains 80% of its original activity at 4°C after 30days compared to free enzyme (5%). Further, immobilization improved enzyme stability in presence of different additives. The immobilized (cross-linked) enzyme also exhibited adequate recycling efficiency up to five reaction cycles with 37% retention activity. The finding of this study suggests improvement of overall performance of immobilized xylanase in respect to free form and can be used to make a bioreactor for various applications such as poultry feed preparations.

  2. 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).

  3. A copper-hydrogen peroxide redox system induces dityrosine cross-links and chemokine oligomerisation.

    PubMed

    MacGregor, Helen J; Kato, Yoji; Marshall, Lindsay J; Nevell, Thomas G; Shute, Janis K

    2011-12-01

    The activity of the chemoattractant cytokines, the chemokines, in vivo is enhanced by oligomerisation and aggregation on glycosaminoglycan (GAG), particularly heparan sulphate, side chains of proteoglycans. The chemokine RANTES (CCL5) is a T-lymphocyte and monocyte chemoattractant, which has a minimum tetrameric structure for in vivo activity and a propensity to form higher order oligomers. RANTES is unusual among the chemokines in having five tyrosine residues, an amino acid susceptible to oxidative cross-linking. Using fluorescence emission spectroscopy, Western blot analysis and LCMS-MS, we show that a copper/H2O2 redox system induces the formation of covalent dityrosine cross-links and RANTES oligomerisation with the formation of tetramers, as well as higher order oligomers. Amongst the transition metals tested, namely copper, nickel, mercury, iron and zinc, copper appeared unique in this respect. At high (400 μM) concentrations of H2O2, RANTES monomers, dimers and oligomers are destroyed, but heparan sulphate protects the chemokine from oxidative damage, promoting dityrosine cross-links and multimer formation under oxidative conditions. Low levels of dityrosine cross-links were detected in copper/H2O2-treated IL-8 (CXCL8), which has one tyrosine residue, and none were detected in ENA-78 (CXCL5), which has none. Redox-treated RANTES was fully functional in Boyden chamber assays of T-cell migration and receptor usage on activated T-cells following RANTES oligomerisation was not altered. Our results point to a protective, anti-oxidant, role for heparan sulphate and a previously unrecognised role for copper in chemokine oligomerisation that may offer an explanation for the known anti-inflammatory effect of copper-chelators such as penicillamine and tobramycin.

  4. 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.

  5. 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.

  6. 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

  7. [Use of native and cross-linked collagen membranes for guided tissue and bone regeneration].

    PubMed

    Schwarz, Frank; Sager, Martin; Rothamel, Daniel; Herten, Monika; Sculean, Anton; Becker, Jürgen

    2006-01-01

    A material which is used as a barrier for GBR/GTR procedures has to satisfy several physicochemical characteristics such as biocompatibility, tissue integration, barrier function, and dimensional stability. Recently, many investigations reported on the use of products derived from type I and type III porcine or bovine collagen. Collagen membranes are predominantly resorbed by enzymatic activity (protease and collagenase). To decrease resorption, various physical and chemical cross-linking techniques have been used. Although nowadays cross-linking of collagen seems to be a commonly used procedure, its impact on physicochemical properties of the membrane is still unknown. The aim of the present literature review is to evaluate the potential use of different collagen membranes for GBR/GTR procedures.

  8. Effect of electron beam-cross-linked gels on the rheological properties of raw natural rubber

    NASA Astrophysics Data System (ADS)

    Mitra, Suman; Chattopadhyay, Santanu; Bharadwaj, Y. K.; Sabharwal, S.; Bhowmick, Anil K.

    2008-05-01

    Electron beam (EB)-cross-linked natural rubber (NR) gels were prepared from latex and characterized by various techniques. The addition of a small amount of these gels to raw NR was found to reduce the apparent shear viscosity and die swell remarkably. This effect was further enhanced with the addition of butyl acrylate as a sensitizer. The apparent shear viscosity first decreased up to 8 phr of gel loading and then increased. However, the percent die swell value decreased steadily upon gel loading. These were explained by calculating principal normal stress difference, the activation energy of melt flow and characteristics of EB-cross-linked gels. These effects were also reflected in the changes of mechanical and dynamic mechanical properties of gel-filled raw NR. Tailoring of the above properties could be done with the help of these gels.

  9. 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.

  10. 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).

  11. 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.

  12. 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.

  13. Glutaraldehyde cross-linking of amniotic membranes affects their nanofibrous structures and limbal epithelial cell culture characteristics.

    PubMed

    Lai, Jui-Yang; Ma, David Hui-Kang

    2013-01-01

    Given that the cells can sense nanometer dimensions, the chemical cross-linking-mediated alteration in fibrillar structure of collagenous tissue scaffolds is critical to determining their cell culture performances. This article explores, for the first time, the effect of nanofibrous structure of glutaraldehyde (GTA) cross-linked amniotic membrane (AM) on limbal epithelial cell (LEC) cultivation. Results of ninhydrin assays demonstrated that the amount of new cross-links formed between the collagen chains is significantly increased with increasing the cross-linking time from 1 to 24 hours. By transmission electron microscopy, the AM treated with GTA for a longer duration exhibited a greater extent of molecular aggregation, thereby leading to a considerable increase in nanofiber diameter and resistance against collagenase degradation. In vitro biocompatibility studies showed that the samples cross-linked with GTA for 24 hours are not well-tolerated by the human corneal epithelial cell cultures. When the treatment duration is less than 6 hours, the biological tissues cross-linked with GTA for a longer time may cause slight reductions in 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt, and anti-inflammatory activities. Nevertheless, significant collagen molecular aggregation also enhances the stemness gene expression, indicating a high ability of these AM matrices to preserve the progenitors of LECs in vitro. It is concluded that GTA cross-linking of collagenous tissue materials may affect their nanofibrous structures and corneal epithelial stem cell culture characteristics. The AM treated with GTA for 6 hours holds promise for use as a niche for the expansion and transplantation of limbal epithelial progenitor cells.

  14. 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.

  15. 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.

  16. 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.

  17. 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.

  18. Two-photon induced collagen cross-linking in bioartificial cardiac tissue

    NASA Astrophysics Data System (ADS)

    Kuetemeyer, Kai; Kensah, George; Heidrich, Marko; Meyer, Heiko; Martin, Ulrich; Gruh, Ina; Heisterkamp, Alexander

    2011-08-01

    Cardiac tissue engineering is a promising strategy for regenerative therapies to overcome the shortage of donor organs for transplantation. Besides contractile function, the stiffness of tissue engineered constructs is crucial to generate transplantable tissue surrogates with sufficient mechanical stability to withstand the high pressure present in the heart. Although several collagen cross-linking techniques have proven to be efficient in stabilizing biomaterials, they cannot be applied to cardiac tissue engineering, as cell death occurs in the treated area. Here, we present a novel method using femtosecond (fs) laser pulses to increase the stiffness of collagen-based tissue constructs without impairing cell viability. Raster scanning of the fs laser beam over riboflavin-treated tissue induced collagen cross-linking by two-photon photosensitized singlet oxygen production. One day post-irradiation, stress-strain measurements revealed increased tissue stiffness by around 40% being dependent on the fibroblast content in the tissue. At the same time, cells remained viable and fully functional as demonstrated by fluorescence imaging of cardiomyocyte mitochondrial activity and preservation of active contraction force. Our results indicate that two-photon induced collagen cross-linking has great potential for studying and improving artificially engineered tissue for regenerative therapies.

  19. Spermidine delays eye lens opacification in vitro by suppressing transglutaminase-catalyzed crystallin cross-linking.

    PubMed

    Lentini, Alessandro; Tabolacci, Claudio; Mattioli, Palma; Provenzano, Bruno; Beninati, Simone

    2011-02-01

    A Ca(2+)-dependent TG activity, identified in the eye lens of several mammalian species, has long been implicated in cataract formation. The precise mechanism of the involvement of this enzyme in this process remains unclear. The purpose of this work was to investigate the modulatory effect of polyamines on TG activity during rabbit eye lens in vitro opacification. We observed, in an in vitro Ca(2+)-induced cataract model, a rapid decrease of the endogenous levels of SPD with the progression of opacification, paralleled by an increase of crystallin cross-linking by bis(γ-glutamyl)SPD. This pattern was reversed adding exogenous SPD to the incubation medium. Indeed, endogenous SPD levels were restored and cross-linking by bis(γ-glutamyl)SPD were drastically reduced. Surprisingly, under this experimental condition, the loss of transparency of lens was delayed. We found that exogenous SPD incubation led to a remarkable increase of mono(γ-glutamyl)SPD, likely responsible of the inhibition of cross-linking of lens crystallins and of the transparency persistence.

  20. Oxidation increases mucin polymer cross-links to stiffen airway mucus gels.

    PubMed

    Yuan, Shaopeng; Hollinger, Martin; Lachowicz-Scroggins, Marrah E; Kerr, Sheena C; Dunican, Eleanor M; Daniel, Brian M; Ghosh, Sudakshina; Erzurum, Serpel C; Willard, Belinda; Hazen, Stanley L; Huang, Xiaozhu; Carrington, Stephen D; Oscarson, Stefan; Fahy, John V

    2015-02-25

    Airway mucus in cystic fibrosis (CF) is highly elastic, but the mechanism behind this pathology is unclear. We hypothesized that the biophysical properties of CF mucus are altered because of neutrophilic oxidative stress. Using confocal imaging, rheology, and biochemical measures of inflammation and oxidation, we found that CF airway mucus gels have a molecular architecture characterized by a core of mucin covered by a web of DNA and a rheological profile characterized by high elasticity that can be normalized by chemical reduction. We also found that high levels of reactive oxygen species in CF mucus correlated positively and significantly with high concentrations of the oxidized products of cysteine (disulfide cross-links). To directly determine whether oxidation can cross-link mucins to increase mucus elasticity, we exposed induced sputum from healthy subjects to oxidizing stimuli and found a marked and thiol-dependent increase in sputum elasticity. Targeting mucin disulfide cross-links using current thiol-amino structures such as N-acetylcysteine (NAC) requires high drug concentrations to have mucolytic effects. We therefore synthesized a thiol-carbohydrate structure (methyl 6-thio-6-deoxy-α-D-galactopyranoside) and found that it had stronger reducing activity than NAC and more potent and fast-acting mucolytic activity in CF sputum. Thus, oxidation arising from airway inflammation or environmental exposure contributes to pathologic mucus gel formation in the lung, which suggests that it can be targeted by thiol-modified carbohydrates.

  1. The spatial response of nonlinear strain propagation in response to actively driven microspheres through entangled actin networks

    NASA Astrophysics Data System (ADS)

    Falzone, Tobias; Blair, Savanna; Robertson-Anderson, Rae

    2015-03-01

    The semiflexible biopolymer actin, a ubiquitous component of nearly all biological organisms, plays an important role in many mechanically-driven processes such as muscle contraction, cancer invasion and cell motility. As such, entangled actin networks, which possess unique and complex viscoelastic properties, have been the subject of much theoretical and experimental work. However, due to this viscoelastic complexity, much is still unknown regarding the correlation of the applied stress on actin networks to the induced filament strain at the molecular and micro scale. Here, we use simultaneous optical trapping and fluorescence microscopy to characterize the link between applied microscopic forces and strain propagation as a function of strain rate and concentration. Specifically, we track fiduciary markers on entangled actin filaments before, during and after actively driving embedded microspheres through the network. These measurements provide much needed insight into the molecular-level dynamics connecting stress and strain in semiflexible polymer networks.

  2. Ethanol increases p190RhoGAP activity, leading to actin cytoskeleton rearrangements.

    PubMed

    Selva, Javier; Egea, Gustavo

    2011-12-01

    We previously reported that cells chronically exposed to ethanol show alterations in actin cytoskeleton organization and dynamics in primary cultures of newborn rat astrocytes, a well-established in vitro model for foetal alcohol spectrum disorders. These alterations were attributed to a decrease in the cellular levels of active RhoA (RhoA-GTP), which in turn was produced by an increase in the total RhoGAP activity. We here provide evidence that p190RhoGAPs are the main factors responsible for such increase. Thus, in astrocytes chronically exposed to ethanol we observe: (i) an increase in p190A- and p190B-associated RhoGAP activity; (ii) a higher binding of p190A and p190B to RhoA-GTP; (iii) a higher p120RasGAP-p190A RhoGAP complex formation; and (iv) the recruitment of both p190RhoGAPs to the plasma membrane. The simultaneous silencing of both p190 isoforms prevents the actin rearrangements and the total RhoGAP activity increase triggered both by ethanol. Therefore, our data directly points p190RhoGAPs as ethanol-exposure molecular targets on glial cells of the CNS.

  3. 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

  4. Improved Enzyme Catalytic Characteristics upon Glutaraldehyde Cross-Linking of Alginate Entrapped Xylanase Isolated from Aspergillus flavus MTCC 9390

    PubMed Central

    Bhushan, Bharat; Pal, Ajay; Jain, Veena

    2015-01-01

    Purified fungal xylanase was entrapped in alginate beads. Its further cross-linking using glutaraldehyde resulted in large enzyme aggregates which may function as both a catalyst and a support material for numerous substrate molecules. Enzyme cross-linking presented a negative impact on enzyme leaching during repeated washings and recovery of enzyme activity was substantial after twelve cycles of usage. The entrapment followed by cross-linking doubled the total bound activity and also greatly improved the enzyme stability at extreme chemical environment. The wide pH stability, better thermo- and storage stability, lowered Km value, and protection from some metal ions are salient achievements of present immobilization. The study shows the efficacy, durability, and sustainability of immobilized catalytic system which could be efficiently used for various juice processing operations. PMID:26347814

  5. Covalent DNA-Protein Cross-Linking by Phosphoramide Mustard and Nornitrogen Mustard in Human Cells.

    PubMed

    Groehler, Arnold; Villalta, Peter W; Campbell, Colin; Tretyakova, Natalia

    2016-02-15

    N,N-Bis-(2-chloroethyl)-phosphorodiamidic acid (phosphoramide mustard, PM) and N,N-bis-(2-chloroethyl)-amine (nornitrogen mustard, NOR) are the two biologically active metabolites of cyclophosphamide, a DNA alkylating drug commonly used to treat lymphomas, breast cancer, certain brain cancers, and autoimmune diseases. PM and NOR are reactive bis-electrophiles capable of cross-linking cellular biomolecules to form covalent DNA-DNA and DNA-protein cross-links (DPCs). In the present work, a mass spectrometry-based proteomics approach was employed to characterize PM- and NOR-mediated DNA-protein cross-linking in human cells. Following treatment of human fibrosarcoma cells (HT1080) with cytotoxic concentrations of PM, over 130 proteins were found to be covalently trapped to DNA, including those involved in transcriptional regulation, RNA splicing/processing, chromatin organization, and protein transport. HPLC-ESI(+)-MS/MS analysis of proteolytic digests of DPC-containing DNA from NOR-treated cells revealed a concentration-dependent formation of N-[2-[cysteinyl]ethyl]-N-[2-(guan-7-yl)ethyl]amine (Cys-NOR-N7G) conjugates, confirming that it cross-links cysteine thiols of proteins to the N7 position of guanines in DNA. Cys-NOR-N7G adduct numbers were higher in NER-deficient xeroderma pigmentosum cells (XPA) as compared with repair proficient cells. Furthermore, both XPA and FANCD2 deficient cells were sensitized to PM treatment as compared to that of wild type cells, suggesting that Fanconi anemia and nucleotide excision repair pathways are involved in the removal of cyclophosphamide-induced DNA damage.

  6. Nuclear envelope lamin-A couples actin dynamics with immunological synapse architecture and T cell activation.

    PubMed

    González-Granado, José M; Silvestre-Roig, Carlos; Rocha-Perugini, Vera; Trigueros-Motos, Laia; Cibrián, Danay; Morlino, Giulia; Blanco-Berrocal, Marta; Osorio, Fernando G; Freije, José M P; López-Otín, Carlos; Sánchez-Madrid, Francisco; Andrés, Vicente

    2014-04-22

    In many cell types, nuclear A-type lamins regulate multiple cellular functions, including higher-order genome organization, DNA replication and repair, gene transcription, and signal transduction; however, their role in specialized immune cells remains largely unexplored. We showed that the abundance of A-type lamins was almost negligible in resting naïve T lymphocytes, but was increased upon activation of the T cell receptor (TCR). The increase in lamin-A was an early event that accelerated formation of the immunological synapse between T cells and antigen-presenting cells. Polymerization of F-actin in T cells is a critical step for immunological synapse formation, and lamin-A interacted with the linker of nucleoskeleton and cytoskeleton (LINC) complex to promote F-actin polymerization. We also showed that lamin-A expression accelerated TCR clustering and led to enhanced downstream signaling, including extracellular signal-regulated kinase 1/2 (ERK1/2) signaling, as well as increased target gene expression. Pharmacological inhibition of the ERK pathway reduced lamin-A-dependent T cell activation. Moreover, mice lacking lamin-A in immune cells exhibited impaired T cell responses in vivo. These findings underscore the importance of A-type lamins for TCR activation and identify lamin-A as a previously unappreciated regulator of the immune response.

  7. Evidences for a role of protein cross-links in transglutaminase-related disease.

    PubMed

    Tabolacci, Claudio; Lentini, Alessandro; Provenzano, Bruno; Beninati, Simone

    2012-02-01

    Transglutaminases (TGs) are a large family of related and ubiquitous enzymes that catalyze the cross-linking of a glutaminyl residue of a protein/peptide substrate to a lysyl residue of a protein/peptide co-substrate. Considerable and intense progress has been made in the understanding of the chemistry, molecular biology and cell biology of TGs. The knowledge that very different physiological and pathological processes are dependent on the presence of adequate levels of these cross-linking enzymes and on the amount of both free and protein-conjugated polyamines by TG, has generated an incredible amount of original research and review articles. It is clear that TG-mediated reactions are essential for some biological processes, such as blood coagulation, skin barrier formation and extracellular matrix assembly, but may also be involved in pathogenetic mechanisms responsible for several human diseases, such as cancer, AIDS, neurodegenerative disorders, celiac disease, and eye lens opacification. We present here a comprehensive review of recent insights into the pathophysiology of TGs related to their protein cross-linking activity.

  8. Protein cross-linking by chlorinated polyamines and transglutamylation stabilizes neutrophil extracellular traps

    PubMed Central

    Csomós, Krisztián; Kristóf, Endre; Jakob, Bernadett; Csomós, István; Kovács, György; Rotem, Omri; Hodrea, Judit; Bagoly, Zsuzsa; Muszbek, Laszlo; Balajthy, Zoltán; Csősz, Éva; Fésüs, László

    2016-01-01

    Neutrophil extracellular trap (NET) ejected from activated dying neutrophils is a highly ordered structure of DNA and selected proteins capable to eliminate pathogenic microorganisms. Biochemical determinants of the non-randomly formed stable NETs have not been revealed so far. Studying the formation of human NETs we have observed that polyamines were incorporated into the NET. Inhibition of myeloperoxidase, which is essential for NET formation and can generate reactive chlorinated polyamines through hypochlorous acid, decreased polyamine incorporation. Addition of exogenous primary amines that similarly to polyamines inhibit reactions catalyzed by the protein cross-linker transglutaminases (TGases) has similar effect. Proteomic analysis of the highly reproducible pattern of NET components revealed cross-linking of NET proteins through chlorinated polyamines and ɛ(γ-glutamyl)lysine as well as bis-γ-glutamyl polyamine bonds catalyzed by the TGases detected in neutrophils. Competitive inhibition of protein cross-linking by monoamines disturbed the cross-linking pattern of NET proteins, which resulted in the loss of the ordered structure of the NET and significantly reduced capacity to trap bacteria. Our findings provide explanation of how NETs are formed in a reproducible and ordered manner to efficiently neutralize microorganisms at the first defense line of the innate immune system. PMID:27512953

  9. Leveraging cross-link modification events in CLIP-seq for motif discovery.

    PubMed

    Bahrami-Samani, Emad; Penalva, Luiz O F; Smith, Andrew D; Uren, Philip J

    2015-01-01

    High-throughput protein-RNA interaction data generated by CLIP-seq has provided an unprecedented depth of access to the activities of RNA-binding proteins (RBPs), the key players in co- and post-transcriptional regulation of gene expression. Motif discovery forms part of the necessary follow-up data analysis for CLIP-seq, both to refine the exact locations of RBP binding sites, and to characterize them. The specific properties of RBP binding sites, and the CLIP-seq methods, provide additional information not usually present in the classic motif discovery problem: the binding site structure, and cross-linking induced events in reads. We show that CLIP-seq data contains clear secondary structure signals, as well as technology- and RBP-specific cross-link signals. We introduce Zagros, a motif discovery algorithm specifically designed to leverage this information and explore its impact on the quality of recovered motifs. Our results indicate that using both secondary structure and cross-link modifications can greatly improve motif discovery on CLIP-seq data. Further, the motifs we recover provide insight into the balance between sequence- and structure-specificity struck by RBP binding.

  10. 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.

  11. Sequence selectivity, cross-linking efficiency and cytotoxicity of DNA-targeted 4-anilinoquinoline aniline mustards.

    PubMed

    McClean, S; Costelloe, C; Denny, W A; Searcey, M; Wakelin, L P

    1999-06-01

    We have investigated the sequence selectivity, DNA binding site characteristics, interstrand cross-linking ability and cytotoxicity of four 4-anilinoquinoline aniline mustards related to the AT-selective minor groove-binding bisquaternary ammonium heterocycles. The compounds studied include two full mustards that differ in alkylating power, a half mustard and a quaternary anilinoquinolinium bismustard. We have also compared their cytotoxicity with their precursor diols and their toxicity and cross-linking ability with the classical alkylating agents melphalan and chlorambucil. We find that the anilinoquinoline aniline mustards weakly and non-specifically alkylate guanines in the major groove and that they bind strongly to AT-rich sequences in the minor groove, where they alkylate both adenines and guanines at the N3 position. The most preferred sites are classical minor groove binder AT-tracts to which all four ligands bind equally well. The remaining sites are AT-rich, but include GC base pairs, to which the ligands bind with preferences depending on their structure. The full mustards alkylate at the 3' ends of the binding site in an orientation that depends on the spatial disposition of the purines within the two strands. Generally speaking guanines are found to be much less reactive than adenines. The anilinoquinoline aniline mustards are interstrand cross-linking agents that are 60- to 100-fold more effective than melphalan, with the quaternary compound being the most efficacious. However, the type of binding site at which the cross-links occur is not clear, since distamycin challenge fails to antagonize them fully. The full mustards are 20- to 50-fold more cytotoxic than their diol precursors, are more cytotoxic than the half mustard and are 20- to 30-fold more active than melphalan and chlorambucil. The quaternary ligand is the most potent. Given the evidence to hand, it appears that antitumour activity correlates with capacity to cause interstrand cross-links

  12. [The effect of structure of benzimidazoles on the character of forming intramolecular cross-links in DNA and chromatin].

    PubMed

    Mil', E M; Zhil'tsova, V M; Biniukov, V I; Zhizhina, G P; Stoliarova, L G; Kuznetsov, Iu P

    1994-01-01

    An investigation of a number of benzimidazole class preparations, being distinguished by a position of aminomethyl substitutes, has been carried out. It has been shown, that the non-substituted preparation BIO-10 does not form UV-cross-links in DNA and chromatine; BIO-40, having one substitute in the position 2, causes the formation of inter-molecular cross-links DNA-DNA. The preparation BIO-50, having 2 aminomethyl groups in the imidazole nucleus positions 2 and 6, forms cross-links DNA-DNA and DNA-protein in chromatine. The generation of radicals by the preparations BIO-10 and BIO-50 has been studied by the EPR-method by use of spin trap. It has been demonstrated, that BIO-10, unlike BIO-50, actively generates superoxide. A supposition has been made, that an UV-formation of superoxide-radical in the presence of BIO-10 might be a reason of DNA-macromolecule destruction.

  13. 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'.

  14. 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.

  15. 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.

  16. 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

  17. 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 ...

  18. A Facile and Efficient Approach for the Production of Reversible Disulfide Cross-linked Micelles.

    PubMed

    Li, Yuanpei; Bharadwaj, Gaurav; Lee, Joyce S

    2016-12-23

    -loaded, disulfide cross-linked micelles demonstrated less hemolytic activity when compared to their non-cross-linked counterparts.

  19. Stabilization of dentin matrix after cross-linking treatments, in vitro

    PubMed Central

    Scheffel, Débora L.S.; Hebling, Josimeri; Scheffel, Régis H.; Agee, Kelli A.; Cadenaro, Milena; Turco, Gianluca; Breschi, Lorenzo; Mazzoni, Annalisa; de Souza Costa, Carlos A.; Pashley, David H.

    2014-01-01

    Objectives To evaluate the effect of EDC on elastic modulus (E), MMPs activity, hydroxyproline (HYP) release and thermal denaturation temperature of demineralized dentin collagen. Methods Dentin beams were obtained from human molars and completely demineralized in 10 wt% H3PO4 for 18 h. The initial E and MMP activity were determined with three-point bending and microcolorimetric assay, respectively. Extra demineralized beams were dehydrated and the initial dry mass (DM) was determined. All the beams were distributed into groups (n = 10) and treated for 30 s or 60 s with: water, 0.5 M, 1 M or 2 M EDC or 10% glutaraldehyde (GA). After treatment, the new E and MMP activity were redetermined. The beams submitted to DM measurements were storage for 1 week in artificial saliva, after that the mass loss and HYP release were evaluated. The collagen thermal denaturation temperature (TDT) was determined by DSC analysis. Data for E, MMP activity and HYP release were submitted to Wilcoxon and Kruskal–Wallis or Mann–Whitney tests. Mass loss and TDT data were submitted to ANOVA and Tukey tests at the 5% of significance. Results EDC was able to significantly increase collagen stiffness in 60 s. 10% GA groups obtained the highest E values after both 30 and 60 s. All cross-linking agents decreased MMP activity and HYP release and increased TDT temperature. Significant differences were identified among EDC groups after 30 or 60 s of cross-linking, 1 M or 2 M EDC showed the lowest MMP activity. Significance Cross-linking agents are capable of preventing dentin collagen degradation. EDC treatment may be clinically useful to increase resin-dentin stability. PMID:24332989

  20. Effects of maturase binding and Mg2+ concentration on group II intron RNA folding investigated by UV cross-linking.

    PubMed

    Noah, James W; Lambowitz, Alan M

    2003-11-04

    The Lactococcus lactis Ll.LtrB group II intron encodes a reverse transcriptase/maturase (LtrA protein) that promotes RNA splicing by stabilizing the catalytically active RNA structure. Here, we mapped 17 UV cross-links induced in both wild-type Ll.LtrB RNA and Ll.LtrB-Delta2486 RNA, which has a branch-point deletion that prevents splicing, and we used these cross-links to follow tertiary structure formation under different conditions in the presence or absence of the LtrA protein. Twelve of the cross-links are long-range, with six near known tertiary interaction sites in the active RNA structure. In a reaction medium containing 0.5 M NH(4)Cl, eight of the 17 cross-links were detected in the absence of Mg(2+) or the presence of EDTA, and all were detected at 5 mM Mg(2+), where efficient splicing requires the LtrA protein. The frequencies of all but four cross-links increased with increasing Mg(2+) concentrations, becoming maximal between 4 and 50 mM Mg(2+), where the intron is self-splicing. These findings suggest that a high Mg(2+) concentration induces self-splicing by globally stabilizing tertiary structure, including key tertiary interactions that are required for catalytic activity. Significantly, the binding of the maturase under protein-dependent splicing conditions (0.5 M NH(4)Cl and 5 mM Mg(2+)) increased the frequency of only nine cross-links, seven of which are long-range, suggesting that, in contrast to a high Mg(2+) concentration, LtrA promotes splicing by stabilizing critical tertiary structure interactions, while leaving other regions of the intron relatively flexible. This difference may contribute to the high rate of protein-dependent splicing, relative to the rate of self-splicing. The propensity of the intron RNA to form tertiary structure even at relatively low Mg(2+) concentrations raises the possibility that the maturase functions at least in part by tertiary structure capture. Finally, an abundant central wheel cross-link, present in >50% of

  1. Coiled-Coil–Mediated Dimerization Is Not Required for Myosin VI to Stabilize Actin during Spermatid Individualization in Drosophila melanogaster

    PubMed Central

    Noguchi, Tatsuhiko; Frank, Deborah J.; Isaji, Mamiko

    2009-01-01

    Myosin VI is a pointed-end–directed actin motor that is thought to function as both a transporter of cargoes and an anchor, capable of binding cellular components to actin for long periods. Dimerization via a predicted coiled coil was hypothesized to regulate activity and motor properties. However, the importance of the coiled-coil sequence has not been tested in vivo. We used myosin VI's well-defined role in actin stabilization during Drosophila spermatid individualization to test the importance in vivo of the predicted coiled coil. If myosin VI functions as a dimer, a forced dimer should fully rescue myosin VI loss of function defects, including actin stabilization, actin cone movement, and cytoplasmic exclusion by the cones. Conversely, a molecule lacking the coiled coil should not rescue at all. Surprisingly, neither prediction was correct, because each rescued partially and the molecule lacking the coiled coil functioned better than the forced dimer. In extracts, no cross-linking into higher molecular weight forms indicative of dimerization was observed. In addition, a sequence required for altering nucleotide kinetics to make myosin VI dimers processive is not required for myosin VI's actin stabilization function. We conclude that myosin VI does not need to dimerize via the predicted coiled coil to stabilize actin in vivo. PMID:19005209

  2. FSGS3/CD2AP is a barbed-end capping protein that stabilizes actin and strengthens adherens junctions.

    PubMed

    Tang, Vivian W; Brieher, William M

    2013-12-09

    By combining in vitro reconstitution biochemistry with a cross-linking approach, we have identified focal segmental glomerulosclerosis 3/CD2-associated protein (FSGS3/CD2AP) as a novel actin barbed-end capping protein responsible for actin stability at the adherens junction. FSGS3/CD2AP colocalizes with E-cadherin and α-actinin-4 at the apical junction in polarized Madin-Darby canine kidney (MDCK) cells. Knockdown of FSGS3/CD2AP compromised actin stability and decreased actin accumulation at the adherens junction. Using a novel apparatus to apply mechanical stress to cell-cell junctions, we showed that knockdown of FSGS3/CD2AP compromised adhesive strength, resulting in tearing between cells and disruption of barrier function. Our results reveal a novel function of FSGS3/CD2AP and a previously unrecognized role of barbed-end capping in junctional actin dynamics. Our study underscores the complexity of actin regulation at cell-cell contacts that involves actin activators, inhibitors, and stabilizers to control adhesive strength, epithelial behavior, and permeability barrier integrity.

  3. Treatment of actinic cheilitis by photodynamic therapy with 5-aminolevulinic acid and blue light activation.

    PubMed

    Zaiac, Martin; Clement, Annabelle

    2011-11-01

    Actinic cheilitis (AC), a common disorder of the lower lip, should be treated early to prevent progression to invasive squamous cell carcinoma. This study evaluated the safety and efficacy of photodynamic therapy (PDT) with 5-aminolevulinic acid (ALA) activated by blue light for the treatment of AC. Fifteen patients with clinically evident or biopsy-proven AC received two treatments with ALA PDT with blue light activation. Treatments were spaced three to five weeks apart. Most patients achieved 65% to 75% clearance three to five weeks after the first treatment and all achieved more than 75% clearance one month after the second treatment. Three patients achieved complete clearance. Pain and burning during irradiation were absent or mild. All patients said they would repeat the procedure. ALA PDT with 417 nm blue light is a promising option for the treatment of AC of the lower lip.

  4. Rho/Rho-dependent kinase affects locomotion and actin-myosin II activity of Amoeba proteus.

    PubMed

    Kłopocka, W; Redowicz, M J

    2004-10-01

    The highly motile free-living unicellular organism Amoeba proteus has been widely used as a model to study cell motility. However, the molecular mechanisms underlying its unique locomotion are still scarcely known. Recently, we have shown that blocking the amoebae's endogenous Rac- and Rho-like proteins led to distinct and irreversible changes in the appearance of these large migrating cells as well as to a significant inhibition of their locomotion. In order to elucidate the mechanism of the Rho pathway, we tested the effects of blocking the endogenous Rho-dependent kinase (ROCK) by anti-ROCK antibodies and Y-27632, (+)-(R)-trans-4-(1-aminoethyl)-N-(4-pyridyl)cyclohexanecarboxamide dihydrochloride, a specific inhibitor of ROCK, on migrating amoebae and the effect of the Rho and ROCK inhibition on the actin-activated Mg-ATPase of the cytosolic fraction of the amoebae. Amoebae microinjected with anti-ROCK inhibitors remained contracted and strongly attached to the glass surface and exhibited an atypical locomotion. Despite protruding many pseudopodia that were advancing in various directions, the amoebae could not effectively move. Immunofluorescence studies showed that ROCK-like protein was dispersed throughout the cytoplasm and was also found in the regions of actin-myosin II interaction during both isotonic and isometric contraction. The Mg-ATPase activity was about two- to threefold enhanced, indicating that blocking the Rho/Rho-dependent kinase activated myosin. It is possible then that in contrast to the vertebrate cells, the inactivation of Rho/Rho-dependent kinase in amoebae leads to the activation of myosin II and to the observed hypercontracted cells which cannot exert effective locomotion.

  5. 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.

  6. 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.

  7. 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.

  8. Proton conducting sulfonated poly (imide-benzimidazole) with tunable density of covalent/ionic cross-linking for fuel cell membranes

    NASA Astrophysics Data System (ADS)

    Yue, Zhouying; Cai, Yang-Ben; Xu, Shiai

    2015-07-01

    Ionic cross-linked sulfonated polyimides containing bis-benzimidazole rings have been prepared from 1,4,5,8-naphthalenetetracarboxylic dianhydride (NTDA), 6,6‧-bis[2-(4-aminophenyl)benzimidazole] (BAPBI) and 3,3‧-bis(4-sulfophenoxy)- benzidine (BSPOB). A new cross-linker, 4,4‧-bibromomethenyl diphenyl ether, is used to induce covalent cross-linking between halogen and imidazole groups in SPIBI chains via a facile thermally activated reaction. The resulted covalent and ionic cross-linked membranes show an improved resistance to hydrolytic attack in deionized water at 80 °C (more than two months) and free radical attack in Fenton's solution (more than 690 min) as compared to non-cross-linked SPIBIs (less than two days and 270 min, respectively). Cross-linking also results in a reduction in proton conductivity due to the blockage of a hydrophilic channel. However, all the prepared CBr-ySPIBI-x membranes show a proton conductivity higher than 10-2 S cm-1 under hydrous condition. This could be attributed to the fact that more cross-linking sites are contained in each repeating unit, which ensures enough cross-linking degree at high sulfonation level. All these results suggest that CBr-ySPIBI-x membranes have a great potential for applications in the proton exchange membrane fuel cells.

  9. Biocompatibility of Genipin and Glutaraldehyde Cross-Linked Chitosan Materials in the Anterior Chamber of the Eye

    PubMed Central

    Lai, Jui-Yang

    2012-01-01

    Chitosan is a naturally occurring cationic polysaccharide and has attracted much attention in the past decade as an important ophthalmic biomaterial. We recently demonstrated that the genipin (GP) cross-linked chitosan is compatible with human retinal pigment epithelial cells. The present work aims to further investigate the in vivo biocompatibility of GP-treated chitosan (GP-chi group) by adopting the anterior chamber of a rabbit eye model. The glutaraldehyde (GTA) cross-linked samples (GTA-chi group) were used for comparison. The 7-mm-diameter membrane implants made from either non-cross-linked chitosan or chemically modified materials with a cross-linking degree of around 80% were inserted in the ocular anterior chamber for 24 weeks and characterized by slit-lamp and specular microscopic examinations, intraocular pressure measurements, and corneal thickness measurements. The interleukin-6 expressions at mRNA level were also detected by quantitative real-time reverse transcription polymerase chain reaction. Results of clinical observations showed that the overall ocular scores in the GTA-chi groups were relatively high. In contrast, the rabbits bearing GP-chi implants in the anterior chamber of the eye exhibited no signs of ocular inflammation. As compared to the non-cross-linked counterparts, the GP-chi samples improved the preservation of corneal endothelial cell density and possessed better anti-inflammatory activities, indicating the benefit action of the GP cross-linker. In summary, the intracameral tissue response to the chemically modified chitosan materials strongly depends on the selection of cross-linking agents. PMID:23109832

  10. Covalent and ionic co-cross-linking--an original way to prepare chitosan-gelatin hydrogels for biomedical applications.

    PubMed

    Jătariu Cadinoiu, Anca N; Popa, Marcel; Curteanu, Silvia; Peptu, Cătălina A

    2011-09-01

    The first goal of this work was to develop a method for obtaining interpenetrating gelatin (G)-chitosan (CS) networks prepared by double cross-linking (covalent followed by ionic) that exhibit hydrogel character. The second goal was to modulate their properties as a function of the preparation parameters by using neural network models. This study was therefore carried out by experiment and simulation. The covalent cross-linking resulted from the reaction between the carbonyl groups of glutaraldehyde with amino groups belonging to both polymers; the ionic cross-linking is based on the interaction between tripolyphosphate anions and protonated amine groups (ammonium ions) of the polymers. The total cross-linking density (indirectly assessed by estimating the water swelling capacity) and the ability to include hydrosoluble bioactive principles are influenced by the following process parameters: the CS/G ratio, the amount of ionic cross-linker, and the ionic cross-linking time. The prepared hydrogels were characterized with respect to their structural, morphological, and some physical properties. The hydrogels ability to load high amounts of water-soluble drugs indicates their potential use as carriers for biologically active principles in the human body. A neural network methodology was applied to model the swelling degree and caffeine loading/release capacity depending on reaction conditions; in addition, applying this method, the optimal preparation conditions have been determined, targeting pre-established values for swelling degree or maximum caffeine value. The accuracy of the results obtained through this technique proves that the neural networks are suitable tools for modeling cross-linking processes taking place complex nonlinear polymers.

  11. The F-BAR protein Hof1 tunes formin activity to sculpt actin cables during polarized growth

    PubMed Central

    Graziano, Brian R.; Yu, Hoi-Ying E.; Alioto, Salvatore L.; Eskin, Julian A.; Ydenberg, Casey A.; Waterman, David P.; Garabedian, Mikael; Goode, Bruce L.

    2014-01-01

    Asymmetric cell growth and division rely on polarized actin cytoskeleton remodeling events, the regulation of which is poorly understood. In budding yeast, formins stimulate the assembly of an organized network of actin cables that direct polarized secretion. Here we show that the Fer/Cip4 homology–Bin amphiphysin Rvs protein Hof1, which has known roles in cytokinesis, also functions during polarized growth by directly controlling the activities of the formin Bnr1. A mutant lacking the C-terminal half of Hof1 displays misoriented and architecturally altered cables, along with impaired secretory vesicle traffic. In vitro, Hof1 inhibits the actin nucleation and elongation activities of Bnr1 without displacing the formin from filament ends. These effects depend on the Src homology 3 domain of Hof1, the formin homology 1 (FH1) domain of Bnr1, and Hof1 dimerization, suggesting a mechanism by which Hof1 “restrains” the otherwise flexible FH1-FH2 apparatus. In vivo, loss of inhibition does not alter actin levels in cables but, instead, cable shape and functionality. Thus Hof1 tunes formins to sculpt the actin cable network. PMID:24719456

  12. Total synthesis of (-)-doliculide, structure-activity relationship studies and its binding to F-actin.

    PubMed

    Matcha, Kiran; Madduri, Ashoka V R; Roy, Sayantani; Ziegler, Slava; Waldmann, Herbert; Hirsch, Anna K H; Minnaard, Adriaan J

    2012-11-26

    Actin, an abundant protein in most eukaryotic cells, is one of the targets in cancer research. Recently, a great deal of attention has been paid to the synthesis and function of actin-targeting compounds and their use as effective molecular probes in chemical biology. In this study, we have developed an efficient synthesis of (-)-doliculide, a very potent actin binder with a higher cell-membrane permeability than phalloidin. Actin polymerization assays with (-)-doliculide and two analogues on HeLa and BSC-1 cells, together with a prediction of their binding mode to F-actin by unbiased computational docking, show that doliculide stabilizes F-actin in a similar way to jasplakinolide and chondramide C.

  13. Hydration, Ionic Valence and Cross-Linking Propensities of Cations Determine the Stability of Lipopolysaccharide (LPS) Membranes

    SciTech Connect

    Nascimento, Agrinaldo; Pontes, Frederico J.; Lins, Roberto D.; Soares, Thereza A.

    2013-10-29

    The supra-molecular structure of LPS aggregates governs outer membrane permeability and activation of the host immune response during Gram-negative bacterial infections. Molecular dynamics simulations unveil at atomic resolution 10 the subtle balance between cation hydration and cross-link ability in modulating phase transitions of LPS membranes.

  14. Spatially Defined EGF Receptor Activation Reveals an F-Actin-Dependent Phospho-Erk Signaling Complex

    PubMed Central

    Singhai, Amit; Wakefield, Devin L.; Bryant, Kirsten L.; Hammes, Stephen R.; Holowka, David; Baird, Barbara

    2014-01-01

    We investigated the association of signaling proteins with epidermal growth factor (EGF) receptors (EGFR) using biotinylated EGF bound to streptavidin that is covalently coupled in an ordered array of micron-sized features on silicon surfaces. Using NIH-3T3 cells stably expressing EGFR, we observe concentration of fluorescently labeled receptors and stimulated tyrosine phosphorylation that are spatially confined to the regions of immobilized EGF and quantified by cross-correlation analysis. We observe recruitment of phosphorylated paxillin to activated EGFR at these patterned features, as well as β1-containing integrins that preferentially localize to more peripheral EGF features, as quantified by radial fluorescence analysis. In addition, we detect recruitment of EGFP-Ras, MEK, and phosphorylated Erk to patterned EGF in a process that depends on F-actin and phosphoinositides. These studies reveal and quantify the coformation of multiprotein EGFR signaling complexes at the plasma membrane in response to micropatterned growth factors. PMID:25468343

  15. Proper regulation of Cdc42 activity is required for tight actin concentration at the equator during cytokinesis in adherent mammalian cells.

    PubMed

    Zhu, Xiaodong; Wang, Junxia; Moriguchi, Kazuki; Liow, Lu Ting; Ahmed, Sohail; Kaverina, Irina; Murata-Hori, Maki

    2011-10-01

    Cytokinesis in mammalian cells requires actin assembly at the equatorial region. Although functions of RhoA in this process have been well established, additional mechanisms are likely involved. We have examined if Cdc42 is involved in actin assembly during cytokinesis. Depletion of Cdc42 had no apparent effects on the duration of cytokinesis, while overexpression of constitutively active Cdc42 (CACdc42) caused cytokinesis failure in normal rat kidney epithelial cells. Cells depleted of Cdc42 displayed abnormal cell morphology and caused a failure of tight accumulation of actin and RhoA at the equator. In contrast, in cells overexpressing CACdc42, actin formed abnormal bundles and RhoA was largely eliminated from the equator. Our results suggest that accurate regulation of Cdc42 activity is crucial for proper equatorial actin assembly and RhoA localization during cytokinesis. Notably, our observations also suggest that tight actin concentration is not essential for cytokinesis in adherent mammalian cells.

  16. Mechanosensitive channels are activated by stress in the actin stress fibres, and could be involved in gravity sensing in plants.

    PubMed

    Tatsumi, H; Furuichi, T; Nakano, M; Toyota, M; Hayakawa, K; Sokabe, M; Iida, H

    2014-01-01

    Mechanosensitive (MS) channels are expressed in a variety of cells. The molecular and biophysical mechanism involved in the regulation of MS channel activities is a central interest in basic biology. MS channels are thought to play crucial roles in gravity sensing in plant cells. To date, two mechanisms have been proposed for MS channel activation. One is that tension development in the lipid bilayer directly activates MS channels. The second mechanism proposes that the cytoskeleton is involved in the channel activation, because MS channel activities are modulated by pharmacological treatments that affect the cytoskeleton. We tested whether tension in the cytoskeleton activates MS channels. Mammalian endothelial cells were microinjected with phalloidin-conjugated beads, which bound to stress fibres, and a traction force to the actin cytoskeleton was applied by dragging the beads with optical tweezers. MS channels were activated when the force was applied, demonstrating that a sub-pN force to the actin filaments activates a single MS channel. Plants may use a similar molecular mechanism in gravity sensing, since the cytoplasmic Ca(2+) concentration increase induced by changes in the gravity vector was attenuated by potential MS channel inhibitors, and by actin-disrupting drugs. These results support the idea that the tension increase in actin filaments by gravity-dependent sedimentation of amyloplasts activates MS Ca(2+) -permeable channels, which can be the molecular mechanism of a Ca(2+) concentration increase through gravistimulation. We review recent progress in the study of tension sensing by actin filaments and MS channels using advanced biophysical methods, and discuss their possible roles in gravisensing.

  17. Inhibition of DNA cross-linking by mitomycin C by peroxidase-mediated oxidation of mitomycin C hydroquinone.

    PubMed

    Penketh, P G; Hodnick, W F; Belcourt, M F; Shyam, K; Sherman, D H; Sartorelli, A C

    2001-09-14

    Mitomycin C requires reductive activation to cross-link DNA and express anticancer activity. Reduction of mitomycin C (40 microm) by sodium borohydride (200 microm) in 20 mm Tris-HCl, 1 mm EDTA at 37 degrees C, pH 7.4, gives a 50-60% yield of the reactive intermediate mitomycin C hydroquinone. The hydroquinone decays with first order kinetics or pseudo first order kinetics with a t(12) of approximately 15 s under these conditions. The cross-linking of T7 DNA in this system followed matching kinetics, with the conversion of mitomycin C hydroquinone to leuco-aziridinomitosene appearing to be the rate-determining step. Several peroxidases were found to oxidize mitomycin C hydroquinone to mitomycin C and to block DNA cross-linking to various degrees. Concentrations of the various peroxidases that largely blocked DNA cross-linking, regenerated 10-70% mitomycin C from the reduced material. Thus, significant quantities of products other than mitomycin C were produced by the peroxidase-mediated oxidation of mitomycin C hydroquinone or products derived therefrom. Variations in the sensitivity of cells to mitomycin C have been attributed to differing levels of activating enzymes, export pumps, and DNA repair. Mitomycin C hydroquinone-oxidizing enzymes give rise to a new mechanism by which oxic/hypoxic toxicity differentials and resistance can occur.

  18. Ras GTPase-Activating Protein Regulation of Actin Cytoskeleton and Hyphal Polarity in Aspergillus nidulans▿ †

    PubMed Central

    Harispe, Laura; Portela, Cecilia; Scazzocchio, Claudio; Peñalva, Miguel A.; Gorfinkiel, Lisette

    2008-01-01

    Aspergillus nidulans gapA1, a mutation leading to compact, fluffy colonies and delayed polarity establishment, maps to a gene encoding a Ras GTPase-activating protein. Domain organization and phylogenetic analyses strongly indicate that GapA regulates one or more “true” Ras proteins. A gapAΔ strain is viable. gapA colonies are more compact than gapA1 colonies and show reduced conidiation. gapAΔ strains have abnormal conidiophores, characterized by the absence of one of the two layers of sterigmata seen in the wild type. gapA transcript levels are very low in conidia but increase during germination and reach their maximum at a time coincident with germ tube emergence. Elevated levels persist in hyphae. In germinating conidiospores, gapAΔ disrupts the normal coupling of isotropic growth, polarity establishment, and mitosis, resulting in a highly heterogeneous cell population, including malformed germlings and a class of giant cells with no germ tubes and a multitude of nuclei. Unlike wild-type conidia, gapAΔ conidia germinate without a carbon source. Giant multinucleated spores and carbon source-independent germination have been reported in strains carrying a rasA dominant active allele, indicating that GapA downregulates RasA. gapAΔ cells show a polarity maintenance defect characterized by apical swelling and subapical branching. The strongly polarized wild-type F-actin distribution is lost in gapAΔ cells. As GapA-green fluorescent protein shows cortical localization with strong predominance at the hyphal tips, we propose that GapA-mediated downregulation of Ras signaling at the plasma membrane of these tips is involved in the polarization of the actin cytoskeleton that is required for hyphal growth and, possibly, for asexual morphogenesis. PMID:18039943

  19. 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

  20. Actin polymerization plays a significant role in asbestos-induced inflammasome activation in mesothelial cells in vitro.

    PubMed

    MacPherson, Maximilian; Westbom, Catherine; Kogan, Helen; Shukla, Arti

    2016-12-24

    Asbestos exposure leads to malignant mesothelioma (MM), a deadly neoplasm of mesothelial cells of various locations. Although there is no doubt about the role of asbestos in MM tumorigenesis, mechanisms are still not well explored. Recently, our group demonstrated that asbestos causes inflammasome priming and activation in mesothelial cells, which in part is dependent on oxidative stress. Our current study sheds light on yet another mechanism of inflammasome activation by asbestos. Here we show the role of actin polymerization in asbestos-induced activation of the nod-like receptor pyrin domain-containing protein 3 (NLRP3) inflammasome. Using human mesothelial cells, we first demonstrate that asbestos and carbon nanotubes induced caspase-1 activation and high-mobility group box 1, interleukin 1 beta and interleukin 18 secretion was blocked by Cytochalasin D (Cyto D) an actin polymerization inhibitor. Next, to understand the mechanism, we assessed whether phagocytosis of fibers by mesothelial cells is affected by actin polymerization inhibition. Transmission electron microscopy showed the inhibition of fiber uptake by mesothelial cells in the presence of Cyto D. Furthermore, localization of components of the inflammasome, apoptotic speck-like protein containing a CARD domain (ASC) and NLRP3, to the perinuclear space in mitochondria or endoplasmic reticulum in response to fiber exposure was also interrupted in the presence of Cyto D. Taken together, our studies suggest that actin polymerization plays important roles in inflammasome activation by fibers via regulation of phagocytosis and/or spatial localization of inflammasome components.

  1. Actin enables the antimicrobial action of LL-37 peptide in the presence of microbial proteases.

    PubMed

    Sol, Asaf; Skvirsky, Yaniv; Nashef, Rizan; Zelentsova, Katya; Burstyn-Cohen, Tal; Blotnick, Edna; Muhlrad, Andras; Bachrach, Gilad

    2014-08-15

    Host defense peptides play an important host-protective role by their microcidal action, immunomodulatory functions, and tissue repair activities. Proteolysis is a common strategy of pathogens used to neutralize host defense peptides. Here, we show that actin, the most abundant structural protein in eukaryotes, binds the LL-37 host defense peptide, protects it from degradation by the proteases of Pseudomonas aeruginosa and Porphyromonas gingivalis, and enables its antimicrobial activity despite the presence of the proteases. Co-localization of LL-37 with extracellular actin was observed in necrotized regions of samples from oral lesions. Competition assays, cross-linking experiments, limited proteolysis, and mass spectrometry revealed that LL-37 binds by specific hydrophobic interactions to the His-40-Lys-50 segment of actin, located in the DNase I binding loop. The integrity of the binding site of both LL-37 and actin is a prerequisite to the binding. Our results demonstrate that actin, presumably released by dead cells and abundant in infected sites, might be utilized by the immune system to enhance spatio-temporal immunity in an attempt to arrest infection and control inflammation.

  2. Water dispersible cross-linked magnetic chitosan beads for increasing the antimicrobial efficiency of aminoglycoside antibiotics.

    PubMed

    Grumezescu, Alexandru Mihai; Andronescu, Ecaterina; Holban, Alina Maria; Ficai, Anton; Ficai, Denisa; Voicu, Georgeta; Grumezescu, Valentina; Balaure, Paul Cătălin; Chifiriuc, Carmen Mariana

    2013-09-15

    The aim of this study was to obtain a nano-active system to improve antibiotic activity of certain drugs by controlling their release. Magnetic composite nanomaterials based on magnetite core and cross-linked chitosan shell were synthesized via the co-precipitation method and characterized by Fourier transform infrared spectroscopy (FT-IR), infrared microscopy (IRM), scanning electron microscopy (SEM), dynamic light scattering (DLS), thermogravimetric analysis (TGA) and X-ray diffraction (XRD). The prepared magnetic composite nanomaterials exhibit a significant potentiating effect on the activity of two cationic (kanamycin and neomycin) drugs, reducing the amount of antibiotics necessary for the antimicrobial effect. The increase in the antimicrobial activity was explained by the fact that the obtained nanosystems provide higher surface area to volume ratio, resulting into higher surface charge density thus increasing affinity to microbial cell and also by controlling their release. In addition to the nano-effect, the positive zeta potential of the synthesized magnetite/cross-linked chitosan core/shell magnetic nanoparticles allows for a more favorable interaction with the usually negatively charged cell wall of bacteria. The novelty of the present contribution is just the revealing of this synergistic effect exhibited by the synthesized water dispersible magnetic nanocomposites on the activity of different antibiotics against Gram-positive and Gram-negative bacterial strains. The results obtained in this study recommend these magnetic water dispersible nanocomposite materials for applications in the prevention and treatment of infectious diseases.

  3. Superior Catalytic Performance of Gold Nanoparticles Within Small Cross-Linked Lysozyme Crystals.

    PubMed

    Liu, Mingyue; Wang, Libing; Huang, Renliang; Yu, Yanjun; Su, Rongxin; Qi, Wei; He, Zhimin

    2016-10-08

    Bionanomaterials synthesized by bio-inspired templating methods have emerged as a novel class of composite materials with varied applications in catalysis, detection, drug delivery, and biomedicine. In this study, two kinds of cross-linked lysozyme crystals (CLLCs) of different sizes were applied for the in situ growth of Au nanoparticles (AuNPs). The resulting composite materials were characterized by light microscopy, scanning electron microscopy, transmission electron microscopy, thermogravimetric analysis, and X-ray photoelectron spectroscopy. The catalytic properties of the prepared materials were examined in the catalytic reduction of 4-nitrophenol (4-NP) to 4-aminophenol (4-AP). It was found that the size of the AuNPs increased with an increase in Au loading for both small and large crystals. In addition, small crystals favored homogeneous adsorption and distribution of the metal precursors. And the size of the AuNPs within small crystals could be maintained below 2.5 nm by managing the HAuCl4/lysozyme molar ratio. Furthermore, the lysozyme functional groups blocked the AuNP activity sites, therefore reducing their catalytic activity. This effect was more pronounced for small AuNPs. Moreover, the mass transfer of reactants (4-NP) from solution to AuNPs within the crystals restricted their catalytic reduction, leading to superior catalytic performance of the AuNPs within small cross-linked lysozyme crystals (Au@S-CLLCs) compared to those within large cross-linked lysozyme crystals (Au@L-CLLCs) at similar Au loadings. Finally, an increase in Au loading clogged the crystal channels with increased quantities of larger aggregated AuNPs, thus impeding the catalytic performance of Au@S-CLLCs.

  4. 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.

  5. 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.

  6. 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.

  7. 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.

  8. Nuclear actin depolymerization in transcriptionally active avian and amphibian oocytes leads to collapse of intranuclear structures

    PubMed Central

    Maslova, Antonina; Krasikova, Alla

    2012-01-01

    Actin, which is normally depleted in the nuclei of somatic cells, accumulates in high amounts in giant nuclei of amphibian oocytes. The supramolecular organization and functions of this nuclear pool of actin in growing vertebrate oocyte are controversial. Here, we investigated the role of nuclear actin in the maintenance of the spatial architecture of intranuclear structures in avian and amphibian growing oocytes. A meshwork of filamentous actin was not detected in freshly isolated or fixed oocyte nuclei of Xenopus, chicken or quail. We found that the actin meshwork inside the oocyte nucleus could be induced by phalloidin treatment. Actin polymerization is demonstrated to be required to stabilize the specific spatial organization of nuclear structures in avian and amphibian growing oocytes. In experiments with the actin depolymerizing drugs cytochalasin D and latrunculin A, we showed that disassembly of nuclear actin polymers led to chromosome condensation and their transportation to a limited space within the oocyte nucleus. Experimentally induced “collapsing” of chromosomes and nuclear bodies, together with global inhibition of transcription, strongly resembled the process of karyosphere formation during oocyte growth. PMID:22572951

  9. 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

  10. 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.

  11. 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

  12. Properties of soluble and membrane bound dopamine-beta-monooxygenase from bovine adrenal medulla cross-linked with dimethyl suberimidate.

    PubMed

    Miras-Portugal, M T; Millaruelo, A; Vara, F

    1980-12-10

    Bovine dopamine-beta-monooxygenase from chromaffin granules in its soluble and membrane-bound forms was cross-linked with the bifunctional reagent dimethyl suberimidate, and its structural and kinetic properties were studied. 1. The cross-linking reaction does not affect the activity of soluble dopamine-beta-monooxygenase; it produces a ten percent inactivation in the membrane-bound enzyme, possibly because the linkage to other membrane proteins hinders its activity. 2. The soluble dopamine-beta-monooxygenase reaction mixture was analyzed by sodium dodecyl sulfate gel electrophoresis, showing appreciable amounts of dimer and tetramer, but only small amounts of trimer. In membrane-bound dopamine-beta-monooxygenase, subjected to the same treatment, appreciable amounts of dimer and higher aggregates were found. 3. The kinetic properties of soluble dopamine-beta-monooxygenase after the crosslinking reaction are the same as those of the native enzyme, with a ping-pong kinetic mechanism and the same real Michaelis constants for tyramine and ascorbate: KmT = 0.36 mM and KmA = 0.32 mM. Membrane-bound dopamine-beta-monooxygenase does not present a ping-pong mechanism before or after cross-linking; its real Michaelis constants are slightly modified by the cross-linking reaction: KmT = 0.4 mM and KMA = 0.4 mM.

  13. Nuclear Envelope Lamin-A Couples Actin Dynamics with Immunological Synapse Architecture and T Cell Activation

    PubMed Central

    González-Granado, José María; Trigueros-Motos, Laia; Cibrián, Danay; Morlino, Giulia; Blanco-Berrocal, Marta; Osorio, Fernando Garcia; Freije, José María Pérez; López-Otín, Carlos; Sánchez-Madrid, Francisco; Andrés, Vicente

    2014-01-01

    In many cell types, nuclear A-type lamins have been implicated in structural and functional activities, including higher-order genome organization, DNA replication and repair, gene transcription, and signal transduction. However, their role in specialized immune cells remains largely unexplored. Here, we showed that the abundance of A-type lamins is almost negligible in resting naïve T lymphocytes, but that it is substantially increased upon activation of the T cell receptor (TCR), and is an early event that accelerates formation of the immunological synapse between T cells and antigen-presenting cells. We found that lamin-A enhanced the polymerization of F-actin in T cells, a critical step for immunological synapse formation, by physically connecting the nucleus to the plasma membrane through the linker of nucleoskeleton and cytoskeleton (LINC) complex. We also showed that lamin-A played a key role in other membrane, cytoplasmic, and nuclear events related to TCR activation, including receptor-clustering, downstream signaling, and target gene expression. Notably, the presence of lamin-A was associated with enhanced extracellular signal–regulated kinase 1/2 signaling, and pharmacological inhibition of this pathway reduced the extent of lamin-A–dependent T cell activation. Moreover, mice deficient in lamin-A exhibited impaired T cell responses in vivo. These findings underscore the importance of A-type lamins for TCR activation, and identify lamin-A as a previously unappreciated regulator of the immune response. PMID:24757177

  14. Cross-linked bromelain inhibits lipopolysaccharide-induced cytokine production involving cellular signaling suppression in rats.

    PubMed

    Hou, Rolis Chien-Wei; Chen, Yuh-Shuen; Huang, Jing-Rong; Jeng, Kee-Ching G

    2006-03-22

    Bromelain has been reported to have anti-inflammatory and immunomodulatory effects. It has been cross-linked with organic acids and polysaccharides by gamma irradiation. The cross-linked (CL)-bromelain preparation resisted an acidic environment of pH 3 for 2 h and preserved 80% of its enzyme activity. Pretreatment of rats with CL-bromelain intragastrically for 7 days significantly reduced serum cytokine production induced by injected i.p. with 2.5 mg/kg of lipopolysaccharide (LPS). Bromelain significantly reduced serum glutamate-oxalacetate transaminase induced by LPS. The anti-inflammatory effect of CL-bromelain was correlated with reduced LPS-induced NF-kappaB activity and cyclooxygenase 2 (COX-2) mRNA expression in rat livers. In addition, CL-bromelain dose-dependently inhibited LPS-induced COX-2 mRNA and prostaglandin E2 (PGE2) in BV-2 microglial cells. CL-Bromelain also suppressed the LPS-activated extracellular signal-regulated kinase (ERK), c-Jun N-terminal kinase (JNK), and p38 mitogen-activated protein kinase (MAPK). In conclusion, the anti-inflammatory effects of the CL-bromelain preparation in vivo and in vitro suggest its therapeutic potentials.

  15. 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

  16. Utilization of DNA-protein cross-links as a biomarker of chromium exposure.

    PubMed Central

    Zhitkovich, A; Voitkun, V; Kluz, T; Costa, M

    1998-01-01

    Human exposure to carcinogenic Cr(VI) compounds is found among workers in a large number of professional groups, and it can also occur through environmental pollution. A significant number of toxic waste sites contain Cr as a major contaminant. In this paper we summarize our efforts to apply measurements of DNA-protein cross-links (DPC) as test for biologically active doses of Cr(VI). DPC were found at elevated levels in lymphocytes in several human populations with low to medium Cr exposures. At high exposure to Cr(VI), exemplified by a group of Bulgarian chromeplaters, DPC plateaued and adducts' levels were similar to those found in environmentally exposed individuals. Lymphocytic DPC correlated strongly with Cr levels in erythrocytes that are indicative of Cr(VI) exposure. DPC in lymphocytes were not confounded by such variables as smoking, age, body weight, gender, or ethnicity. A new version of the cross-link assay offers improved sensitivity and requires a small amount of biologic material. Preliminary results indicate that the ability of DPC to reach detectable levels at low levels of Cr exposure could be related to a lack of repair of these lesions in lymphoid cells. Cr(III)-mediated cross-links of DNA with peptide glutathione or single amino acids were mutagenic in human cells, with a relationship of higher molecular weight of the peptide/amino acid correlating with a more potent mutagenic response. We speculate that bulky DPC could also have a significant promutagenic effect. The current methodology does not allow specific determination of Cr-induced DPC; however, demonstrated sensitivity of DPC measurements and the assay's large sample capacity may allow this assay to be used as the initial screening test for the occurrence of DNA damage in Cr(VI)-exposed populations. PMID:9703480

  17. Specific disulfide cross-linking to constrict the mobile carrier domain of nonribosomal peptide synthetases

    PubMed Central

    Tarry, Michael J.; Schmeing, T. Martin

    2015-01-01

    Nonribosomal peptide synthetases are large, multi-domain enzymes that produce peptide molecules with important biological activity such as antibiotic, antiviral, anti-tumor, siderophore and immunosuppressant action. The adenylation (A) domain catalyzes two reactions in the biosynthetic pathway. In the first reaction, it activates the substrate amino acid by adenylation and in the second reaction it transfers the amino acid onto the phosphopantetheine arm of the adjacent peptide carrier protein (PCP) domain. The conformation of the A domain differs significantly depending on which of these two reactions it is catalyzing. Recently, several structures of A–PCP di-domains have been solved using mechanism-based inhibitors to trap the PCP domain in the A domain active site. Here, we present an alternative strategy to stall the A–PCP di-domain, by engineering a disulfide bond between the native amino acid substrate and the A domain. Size exclusion studies showed a significant shift in apparent size when the mutant A–PCP was provided with cross-linking reagents, and this shift was reversible in the presence of high concentrations of reducing agent. The cross-linked protein crystallized readily in several of the conditions screened and the best crystals diffracted to ≈8 Å. PMID:25713404

  18. Mus308 Mutants of Drosophila Exhibit Hypersensitivity to DNA Cross-Linking Agents and Are Defective in a Deoxyribonuclease

    PubMed Central

    Boyd, J. B.; Sakaguchi, K.; Harris, P. V.

    1990-01-01

    Mutagen-sensitive strains that identify 16 different Drosophila genes have been screened for alterations in DNA metabolic enzymes. A characteristic defect in an acid-active deoxyribonuclease was observed in strains carrying the six available mutant alleles of the mus308 gene. Since that enzyme is detected at normal levels in a mutant strain that is deficient in the previously identified enzymes DNase 1 and DNase 2, it represents a new Drosophila nuclease that is designated Nuclease 3. The mus308 mutants were originally distinguished from all other mutagen-sensitive mutants of Drosophila because they exhibit hypersensitivity to the DNA cross-linking agent nitrogen mustard without expressing a concurrent sensitivity to the monofunctional agent methyl methanesulfonate. Further observations of hypersensitivity to the mutagens trimethylpsoralen, diepoxybutane and cis-platinum now establish a more general sensitivity of these mutants to agents capable of generating DNA cross-links. In spite of the hypersensitivity of the mus308 mutants to DNA cross-linking agents, the initial incision step of DNA cross-link repair is normal in mus308 cells as assayed by the alkaline elution procedure. The Drosophila mus308 mutants show promise of providing a useful model for analogous defects in other organisms including man. PMID:2397884

  19. Mechanisms of DNA strand breakage and interstrand cross-linking by diaziridinylbenzoquinone (diaziquone) in isolated nuclei from human cells.

    PubMed

    Szmigiero, L; Kohn, K W

    1984-10-01

    AZQ had been found to produce DNA strand breaks and interstrand cross-links in intact cells; evidence had indicated that these two DNA lesions arise by different chemical mechanisms and vary independently in degree in different cell types. In the present work, the mechanisms of the production of DNA strand breaks and interstrand cross-links by AZQ were studied in isolated cell nuclei. This system avoided the problem of poor penetration of test substances into cells. The DNA lesions were measured by means of the alkaline elution technique. It was found that the production of DNA strand breaks by AZQ in isolated nuclei required the addition of a reducing agent such as NADPH and was almost completely prevented by superoxide dismutase. This indicates that the mechanism of DNA strand breakage involves transfer of an electron from a reduced form of AZQ to molecular oxygen. Unexpectedly, interstrand cross-linking also was enhanced greatly by previous reduction of AZQ by NADPH or NaBH4. However, this reaction was not inhibited by superoxide dismutase. General alkylating activity of AZQ also was stimulated by reduction; the pH-dependence of this reaction was determined. The mechanism of DNA interstrand cross-linking by AZQ was surmised to stem from alkylation reactions of the two aziridine groups. The findings suggest the possibility that AZQ or related compounds may function as bioreductive alkylating agents which might be selectively toxic to hypoxic tissues.

  20. Cortactin Adopts a Globular Conformation and Bundles Actin into Sheets*S⃞

    PubMed Central

    Cowieson, Nathan P.; King, Gordon; Cookson, David; Ross, Ian; Huber, Thomas; Hume, David A.; Kobe, Bostjan; Martin, Jennifer L.

    2008-01-01

    Cortactin is a filamentous actin-binding protein that plays a pivotal role in translating environmental signals into coordinated rearrangement of the cytoskeleton. The dynamic reorganization of actin in the cytoskeleton drives processes including changes in cell morphology, cell migration, and phagocytosis. In general, structural proteins of the cytoskeleton bind in the N-terminal region of cortactin and regulatory proteins in the C-terminal region. Previous structural studies have reported an extended conformation for cortactin. It is therefore unclear how cortactin facilitates cross-talk between structural proteins and their regulators. In the study presented here, circular dichroism, chemical cross-linking, and small angle x-ray scattering are used to demonstrate that cortactin adopts a globular conformation, thereby bringing distant parts of the molecule into close proximity. In addition, the actin bundling activity of cortactin is characterized, showing that fully polymerized actin filaments are bundled into sheet-like structures. We present a low resolution structure that suggests how the various domains of cortactin interact to coordinate its array of binding partners at sites of actin branching. PMID:18375393

  1. 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

  2. The effects of phosphorylation and dephosphorylation of brain myosin on its actin-activated Mg2+-ATPase and contractile activities.

    PubMed

    Matsumura, S; Takashima, T; Ohmori, H; Kumon, A

    1988-02-01

    Purified bovine brain myosin contained approximately 1 and 3 mol of protein-bound phosphate/mol myosin in the light chains and heavy chains, respectively. Large portions of this light chain- and heavy chain-bound phosphate (about 0.8 and 2.4 mol, respectively) were removed by incubation with a brain phosphoprotein phosphatase and potato acid phosphatase, respectively. Upon phosphorylation of the dephosphorylated brain myosin with myosin light chain kinase and casein kinase II, about 1.6 and 3.0 mol of phosphate was incorporated into the light chains and heavy chains, respectively, while much lower levels of phosphate were incorporated into the non-dephosphorylated brain myosin under the same conditions. The actin-activated Mg2+-ATPase activity of brain myosin rephosphorylated with myosin light chain kinase was about twice as high as that of dephosphorylated brain myosin (about 30 and 15 nmol phosphate/mg/min, respectively). On the other hand, whereas the rephosphorylated brain myosin superprecipitated rapidly with F-actin, the rate of superprecipitation of the dephosphorylated brain myosin was extremely low. Under appropriate conditions, a loose network of tiny superprecipitates, which formed initially throughout the solution, contracted to form eventually a large and dense particle. These results indicate that phosphorylation of the light chains of brain myosin is a prerequisite for the contraction of brain actomyosin. The role of phosphorylation of the heavy chains by casein kinase II remains to be elucidated.

  3. Wear Measurement of Highly Cross-linked UHMWPE using a 7Be Tracer Implantation Technique

    SciTech Connect

    Wimmer, Markus A.; Laurent, Michael P.; Dwivedi, Yasha; Gallardo, Luis A.; Chipps, K.; Blackmon, Jeffery C; Kozub, R. L.; Bardayan, Daniel W; Gross, Carl J; Stracener, Daniel W; Smith, Michael Scott; Nesaraja, Caroline D; Erikson, Luke; Patel, Nidhi; Rehm, Karl E.; Ahmad, Irshad; Greene, John P.; Greife, Uwe

    2013-01-01

    The very low wear rates achieved with the current highly cross-linked ultrahigh molecular weight polyethylenes (UHMWPE) used in joint prostheses have proven to be difficult to measure accurately by gravimetry. Tracer methods are there- fore being explored. The purpose of this study was to perform a proof-of-concept experiment on the use of the radioactive tracer beryllium-7 (7Be) for the determination of in vitro wear in a highly cross-linked orthopedic UHMWPE. Three cross-linked and four conventional UHMWPE pins made from compression- molded GUR 1050, were activated with 109 to 1010 7Be nuclei using a new implantation setup that produced a homogenous distribution of implanted nuclei up to 8.5 lm below the surface. The pins were tested for wear in a six-station pin-on-flat appara- tus for up to 7.1 million cycles (178 km). A Germanium gamma detector was employed to determine activity loss of the UHMWPE pins at preset intervals during the wear test. The wear of the cross-linked UHMWPE pins was readily detected and esti- mated to be 17 6 3 lg per million cycles. The conventional-to- cross-linked ratio of the wear rates was 13.1 6 0.8, in the expected range for these materials. Oxidative degradation dam- age from implantation was negligible; however, a weak depend- ence of wear on implantation dose was observed limiting the number of radioactive tracer atoms that can be introduced. Future applications of this tracer technology may include the analysis of location-specific wear, such as loss of material in the post or backside of a tibial insert.

  4. Angiotensin I-converting enzyme inhibitor derived from cross-linked oyster protein.

    PubMed

    Xie, Cheng-Liang; Kim, Jin-Soo; Ha, Jong-Myung; Choung, Se-Young; Choi, Yeung-Joon

    2014-01-01

    Following cross-linking by microbial transglutaminase, modified oyster proteins were hydrolyzed to improve inhibitory activity against angiotensin-converting enzyme (ACE) inhibitory activity with the use of a single protease, or a combination of six proteases. The oyster hydrolysate with the lowest 50% ACE inhibitory concentration (IC50) of 0.40 mg/mL was obtained by two-step hydrolysis of the cross-linked oyster protein using Protamex and Neutrase. Five ACE inhibitory peptides were purified from the oyster hydrolysate using a multistep chromatographic procedure comprised of ion-exchange, size exclusion, and reversed-phase liquid chromatography. Their sequences were identified as TAY, VK, KY, FYN, and YA, using automated Edman degradation and mass spectrometry. These peptides were synthesized, and their IC50 values were measured to be 16.7, 29.0, 51.5, 68.2, and 93.9 μM, respectively. Toxicity of the peptides on the HepG2 cell line was not detected. The oyster hydrolysate also significantly decreased the systolic blood pressure of spontaneously hypertensive rats (SHR). The antihypertensive effect of the oyster hydrolysate on SHR was rapid and long-lasting, compared to commercially obtained sardine hydrolysate. These results suggest that the oyster hydrolysate could be a source of effective nutraceuticals against hypertension.

  5. Covalent Immobilization of Catalase onto Regenerated Silk Fibroins via Tyrosinase-Catalyzed Cross-Linking.

    PubMed

    Wang, Ping; Qi, Chenglong; Yu, Yuanyuan; Yuan, Jiugang; Cui, Li; Tang, Gengtie; Wang, Qiang; Fan, Xuerong

    2015-09-01

    Regenerated silk fibroins could be used as medical scaffolds and carrier materials for enzyme immobilization. In the present work, tyrosinase enzyme was used for enzymatic oxidation of silk fibroins, followed by immobilization of catalase onto the fibroin surfaces through physical adsorption and covalent cross-linking as well. Spectrophotometry, SDS-PAGE, and Fourier transform infrared spectroscopy (FTIR) were used to examine the efficiency of enzymatic oxidation and catalase immobilization, respectively. The results indicate that tyrosine residues in silk fibroins could be oxidized and converted to the active o-quinones. Incubating silk fibroins with catalase and tyrosinase led to a noticeable change of molecular weight distribution, indicating the occurrence of the cross-links between silk fibroins and catalase molecules. Two different pathways were proposed for the catalase immobilizations, and the method based on grafting of catalase onto the freeze-dried fibroin membrane is more acceptable. The residual enzyme activity for the immobilized catalase exhibited higher than that of the control after repeated washing cycles. Meanwhile, the thermal stability and alkali resistance were also slightly improved as compared to free catalase. The mechanisms of enzymatic immobilization are also concerned.

  6. 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.

  7. Entrapment of cross-linked cellulase colloids in alginate beads for hydrolysis of cellulose.

    PubMed

    Nguyen, Le Truc; Lau, Yun Song; Yang, Kun-Lin

    2016-09-01

    Entrapment of enzymes in calcium alginate beads is a popular enzyme immobilization method. However, leaching of immobilized enzymes from the alginate beads is a common problem because enzyme molecules are much smaller than the pore size of alginate beads (∼200nm). To address this issue, we employ a millifluidic reactor to prepare cross-linked cellulase aggregate (XCA) colloids with a uniform size (∼300nm). Subsequently, these colloids are immobilized in calcium alginate beads as biocatalysts to hydrolyze cellulose substrates. By using fluorescent microscopy, we conclude that the immobilized XCA colloids distribute uniformly inside the beads and do not leach out from the beads after long-term incubation. Meanwhile, the pore size of the alginate beads is big enough for the cellulose substrates and fibers to diffuse into the beads for hydrolysis. For example, palm oil fiber and microcrystalline cellulose can be hydrolyzed within 48h and release reducing sugar concentrations up to 2.48±0.08g/l and 4.99±0.09g/l, respectively. Moreover, after 10 cycles of hydrolysis, 96.4% of the XCA colloids remain inside the alginate beads and retain 67% of the original activity. In contrast, free cellulase immobilized in the alginate beads loses its activity completely after 10 cycles. The strategy can also be used to prepare other types of cross-linked enzyme aggregates with high uniformity.

  8. Fixation of allosteric states of the nicotinic acetylcholine receptor by chemical cross-linking

    PubMed Central

    Watty, Anke; Methfessel, Christoph; Hucho, Ferdinand

    1997-01-01

    Receptor activity can be described in terms of ligand-induced transitions between functional states. The nicotinic acetylcholine receptor (nAChR), a prototypic ligand-gated ion channel, is an “unconventional allosteric protein” which exists in at least three interconvertible conformations, referred to as resting (low agonist affinity, closed channel), activated (open channel), and desensitized (high agonist affinity, closed channel). Here we show that 3,3′-dimethyl suberimidate (DMS) is an agonistic bifunctional cross-linking reagent, which irreversibly “freezes” the nAChR in a high agonist affinity/closed-channel state. The monofunctional homologue methyl acetoimidate, which is also a weak cholinergic agonist, has no such irreversible effect. Glutardialdehyde, a cross-linker that is not a cholinergic effector, fixes the receptor in a low-affinity state in the absence of carbamoylcholine, but, like DMS, in a high-affinity state in its presence. Covalent cross-linking thus allows us to arrest the nAChR in defined conformational states. PMID:9223339

  9. Angiotensin I-Converting Enzyme Inhibitor Derived from Cross-Linked Oyster Protein

    PubMed Central

    Xie, Cheng-Liang; Kim, Jin-Soo; Ha, Jong-Myung; Choung, Se-Young

    2014-01-01

    Following cross-linking by microbial transglutaminase, modified oyster proteins were hydrolyzed to improve inhibitory activity against angiotensin-converting enzyme (ACE) inhibitory activity with the use of a single protease, or a combination of six proteases. The oyster hydrolysate with the lowest 50% ACE inhibitory concentration (IC50) of 0.40 mg/mL was obtained by two-step hydrolysis of the cross-linked oyster protein using Protamex and Neutrase. Five ACE inhibitory peptides were purified from the oyster hydrolysate using a multistep chromatographic procedure comprised of ion-exchange, size exclusion, and reversed-phase liquid chromatography. Their sequences were identified as TAY, VK, KY, FYN, and YA, using automated Edman degradation and mass spectrometry. These peptides were synthesized, and their IC50 values were measured to be 16.7, 29.0, 51.5, 68.2, and 93.9 μM, respectively. Toxicity of the peptides on the HepG2 cell line was not detected. The oyster hydrolysate also significantly decreased the systolic blood pressure of spontaneously hypertensive rats (SHR). The antihypertensive effect of the oyster hydrolysate on SHR was rapid and long-lasting, compared to commercially obtained sardine hydrolysate. These results suggest that the oyster hydrolysate could be a source of effective nutraceuticals against hypertension. PMID:25140307

  10. Thermal activation energy for bidirectional movement of actin along bipolar tracks of myosin filaments.

    PubMed

    Okubo, Hiroyuki; Iwai, Masanori; Iwai, Sosuke; Chaen, Shigeru

    2010-05-28

    Previous in vitro motility assays using bipolar myosin thick filaments demonstrated that actin filaments were capable of moving in both directions along the myosin filament tracks. The movements; however, were slower in the direction leading away from the central bare zone than towards it. To understand the mechanism underlying these different direction-dependent motilities, we have examined the effects of temperature on the velocities of the bidirectional movements along reconstituted myosin filaments. Activation energies of the movements were determined by Arrhenius plots at high and low concentrations of ATP. As a result, the thermal activation energy of the movement away from the central bare zone was significantly higher than that of the movement toward the zone. Given that the backward movement away from the central bare zone would cause the myosin heads to be constrained and the stiffness of the cross-bridges to increase, these results suggest that elastic energy required for the cross-bridge transition is supplied by thermal fluctuations.

  11. Impaired tropomyosin-troponin interactions reduce activation of the actin thin filament.

    PubMed

    Robaszkiewicz, Katarzyna; Ostrowska, Zofia; Cyranka-Czaja, Anna; Moraczewska, Joanna

    2015-05-01

    Tropomyosin and troponin are bound to the actin filament to control the contraction of striated muscle in the Ca-dependent manner. The interactions between both regulatory proteins important for the regulation process are not fully understood. To gain more insight into the mechanisms of the thin filament regulation by skeletal α-tropomyosin and troponin, we analyzed effects of seven myopathy-related substitutions: Leu99Met, Ala155Thr, Arg167Gly, Arg167Cys, Arg167His, Lys168Glu, and Arg244Gly. All substitutions reduced Ca-dependent activation of the actomyosin ATPase. The effects of mutations in Arg167 and Lys168 were the most severe. The amino acid substitutions did not significantly affect troponin binding to the whole filament, but reduced 1.2-2.8 fold the affinity of troponin to tropomyosin alone. The excimer fluorescence of N-(1-pyrene)iodoacetamide, a probe attached to the central Cys190, demonstrated that substitutions located near the troponin core domain-binding region strongly affected conformational changes accompanying the tropomyosin-troponin interactions. The thermal stability of all tropomyosin mutants was lower than the stability of the wild type tropomyosin, with TM reduced by 5.3-8.5°C. Together the analyses demonstrated that the myopathy-causing mutations affected tropomyosin structure and led to changes in interactions between tropomyosin and troponin, which impaired the transition of the thin filament from the inactive off to the active on state.

  12. Analysis of Protein-protein Interaction Interface between Yeast Mitochondrial Proteins Rim1 and Pif1 Using Chemical Cross-linking Mass Spectrometry.

    PubMed

    Zybailov, Boris; Gokulan, Kuppan; Wiese, Jadon; Ramanagoudr-Bhojappa, Ramanagouda; Byrd, Alicia K; Glazko, Galina; Jaiswal, Mihir; Mackintosh, Samuel; Varughese, Kottayil I; Raney, Kevin D

    2015-11-01

    Defining protein-protein contacts is a challenging problem and cross-linking is a promising solution. Here, we present a case of mitochondrial single strand binding protein Rim1 and helicase Pif1, an interaction first observed in immuno-affinity pull-down from yeast cells using Pif1 bait. We found that only the short succinimidyl-diazirine cross-linker or formaldehyde captured the interaction between recombinant Rim1 and Pif1. In addition, Pif1 needed to be stripped of its N-terminal and C-terminal domains, and Rim1's C-terminus needed to be modified for the cross-linked product to become visible. Our report is an example of a non-trivial analysis, where a previously identified stable interaction escapes initial capture with cross-linking agents and requires substantial modification to recombinant proteins and fine-tuning of the mass spectrometry-based methods for the cross-links to become detectable. We used high resolution mass spectrometry to detect the cross-linked peptides. A 1:1 mixture of (15)N and (14)N-labeled Rim1 was used to validate the cross-links by their mass shift in the LC-MS profiles. Two sites on Rim1 were confirmed: 1) the N-terminus, and 2) the K29 residue. Performing cross-linking with a K29A variant visibly reduced the cross-linked product. Further, K29A-Rim1 showed a five-fold lower affinity to single stranded DNA compared to wild-type Rim1. Both the K29A variant and wild type Rim1 showed similar degrees of stimulation of Pif1 helicase activity. We propose structural models of the Pif1-Rim1 interaction and discuss its functional significance. Our work represents a non-trivial protein-protein interface analysis and demonstrates utility of short and non-specific cross-linkers.

  13. Susceptibility of the individual caseins in reconstituted skim milk to cross-linking by transglutaminase: influence of temperature, pH and mineral equilibria.

    PubMed

    Hinz, Katharina; Huppertz, Thom; Kelly, Alan L

    2012-11-01

    The susceptibility of total casein and the individual caseins in reconstituted skim milk to transglutaminase (TGase)-induced cross-linking was studied as a function of incubation temperature (5-40 °C), pH (5·0-7·0) and mineral addition. Within the ranges studied, the level of total casein cross-linked increased with increasing temperature, pH and concentration of added trisodium citrate, whereas adding calcium chloride had the opposite effect. These effects can be largely related to the effects of these parameters on TGase activity. In addition, the parameters were also found to influence the susceptibility of κ-casein, and to a lesser extent β-casein, to cross-linking, whereas the susceptibility of αs1-casein was not affected. The susceptibility of κ-casein to cross-linking increased with increasing temperature and calcium chloride addition, but decreased with increasing pH and citrate content, whereas the susceptibility of β-casein to TGase-induced cross-linking decreased with increasing temperature, but was not affected by other parameters. These findings highlight the fact that selection of environmental conditions during cross-linking can be applied to tailor the surface, and hence possibly colloidal stability, of casein micelles in TGase-treated milk.

  14. Yeast mitochondria contain ATP-sensitive, reversible actin-binding activity.

    PubMed Central

    Lazzarino, D A; Boldogh, I; Smith, M G; Rosand, J; Pon, L A

    1994-01-01

    Sedimentation assays were used to demonstrate and characterize binding of isolated yeast mitochondria to phalloidin-stabilized yeast F-actin. These actin-mitochondrial interactions are ATP sensitive, saturable, reversible, and do not depend upon mitochondrial membrane potential. Protease digestion of mitochondrial outer membrane proteins or saturation of myosin-binding sites on F-actin with the S1 subfragment of skeletal myosin block binding. These observations indicate that a protein (or proteins) on the mitochondrial surface mediates ATP-sensitive, reversible binding of mitochondria to the lateral surface of microfilaments. Actin copurifies with mitochondria during subcellular fractionation and is released from the organelle upon treatment with ATP. Thus, actin-mitochondrial interactions resembling those observed in vitro may also exist in intact yeast cells. Finally, a yeast mutant bearing a temperature-sensitive mutation in the actin-encoding ACT1 gene (act1-3) displays temperature-dependent defects in transfer of mitochondria from mother cells to newly developed buds during yeast cell mitosis. Images PMID:7812049

  15. 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.

  16. 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.

  17. 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.

  18. 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.

  19. 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.

  20. Jak3 enables chemokine-dependent actin cytoskeleton reorganization by regulating cofilin and Rac/Rhoa GTPases activation.

    PubMed

    Ambriz-Peña, Xochitl; García-Zepeda, Eduardo Alberto; Meza, Isaura; Soldevila, Gloria

    2014-01-01

    We have previously shown that Jak3 is involved in the signaling pathways of CCR7, CCR9 and CXCR4 in murine T lymphocytes and that Jak3⁻/⁻ lymphocytes display an intrinsic defect in homing to peripheral lymph nodes. However, the molecular mechanism underlying the defective migration observed in Jak3⁻/⁻ lymphocytes remains elusive. Here, it is demonstrated for the first time, that Jak3 is required for the actin cytoskeleton reorganization in T lymphocytes responding to chemokines. It was found that Jak3 regulates actin polymerization by controlling cofilin inactivation in response to CCL21 and CXCL12. Interestingly, cofilin inactivation was not precluded in PTX- treated cells despite their impaired actin polymerization. Additionally, Jak3 was required for small GTPases Rac1 and RhoA activation, which are indispensable for acquisition of the migratory cell phenotype and the generation of a functional leading edge and uropod, respectively. This defect correlates with data obtained by time-lapse video-microscopy showing an incompetent uropod formation and impaired motility in Jak3-pharmacologically inhibited T lymphocytes. Our data support a new model in which Jak3 and heterotrimeric G proteins can use independent, but complementary, signaling pathways to regulate actin cytoskeleton dynamics during cell migration in response to chemokines.

  1. Tyrosine kinases activate store-mediated Ca2+ entry in human platelets through the reorganization of the actin cytoskeleton.

    PubMed Central

    Rosado, J A; Graves, D; Sage, S O

    2000-01-01

    We have recently reported that store-mediated Ca(2+) entry in platelets is likely to be mediated by a reversible trafficking and coupling of the endoplasmic reticulum with the plasma membrane, a model termed 'secretion-like coupling'. In this model the actin cytoskeleton plays a key regulatory role. Since tyrosine kinases have been shown to be important for Ca(2+) entry in platelets and other cells, we have now investigated the possible involvement of tyrosine kinases in the secretion-like-coupling model. Treatment of platelets with thrombin or thapsigargin induced actin polymerization by a calcium-independent pathway. Methyl 2,5-dihydroxycinnamate, a tyrosine kinase inhibitor, prevented thrombin- or thapsigargin-induced actin polymerization. The effects of tyrosine kinases in store-mediated Ca(2+) entry were found to be entirely dependent on the actin cytoskeleton. PP1, an inhibitor of the Src family of proteins, partially inhibited store-mediated Ca(2+) entry. In addition, depletion of intracellular Ca(2+) stores stimulated cytoskeletal association of the cytoplasmic tyrosine kinase pp60(src), a process that was sensitive to treatment with cytochalasin D and PP1, but not to inhibition of Ras proteins using prenylcysteine analogues. Finally, combined inhibition of both Ras proteins and tyrosine kinases resulted in complete inhibition of Ca(2+) entry, suggesting that these two families of proteins have independent effects in the activation of store-mediated Ca(2+) entry in human platelets. PMID:11023829

  2. 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.

  3. 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

  4. 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.

  5. 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.

  6. 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.

  7. Abacavir forms novel cross-linking abacavir protein adducts in patients.

    PubMed

    Meng, Xiaoli; Lawrenson, Alexandre S; Berry, Neil G; Maggs, James L; French, Neil S; Back, David J; Khoo, Saye H; Naisbitt, Dean J; Park, B Kevin

    2014-04-21

    Abacavir (ABC), a nucleoside-analogue reverse transcriptase inhibitor, is associated with severe hypersensitivity reactions that are thought to involve the activation of CD8+ T cells in a HLA-B*57:01-restricted manner. Recent studies have claimed that noncovalent interactions of ABC with HLA-B*57:01 are responsible for the immunological reactions associated with ABC. However, the formation of hemoglobin-ABC aldehyde (ABCA) adducts in patients exposed to ABC suggests that protein conjugation might represent a pathway for antigen formation. To further characterize protein conjugation reactions, we used mass spectrometric methods to define ABCA modifications in patients receiving ABC therapy. ABCA formed a novel intramolecular cross-linking adduct on human serum albumin (HSA) in patients and in vitro via Michael addition, followed by nucleophilic adduction of the aldehyde with a neighboring protein nucleophile. Adducts were detected on Lys159, Lys190, His146, and Cys34 residues in the subdomain IB of HSA. Only a cysteine adduct and a putative cross-linking adduct were detected on glutathione S-transferase Pi (GSTP). These findings reveal that ABC forms novel types of antigens in all patients taking the drug. It is therefore vital that the immunological consequences of such pathways of haptenation are explored in the in vitro models that have been used by various groups to define new mechanisms of drug hypersensitivity exemplified by ABC.

  8. Isodityrosine cross-linking mediates insolubilization of cell walls in Chlamydomonas.

    PubMed

    Waffenschmidt, S; Woessner, J P; Beer, K; Goodenough, U W

    1993-07-01

    Enzymatic removal of the cell wall induces vegetative Chlamydomonas reinhardtii cells to transcribe wall genes and synthesize new hydroxyproline-rich glycoproteins (HRGPs) related to the extensins found in higher plant cell walls. A cDNA expression library made from such induced cells was screened with antibodies to an oligopeptide containing the (SP)x repetitive domains found in Chlamydomonas wall proteins. One of the selected cDNAs encodes an (SP)x-rich polypeptide that also displays a repeated YGG motif. Ascorbate, a peroxidase inhibitor, and tyrosine derivatives were shown to inhibit insolubilization of both the vegetative and zygotic cell walls of Chlamydomonas, suggesting that oxidative cross-linking of tyrosines is occurring. Moreover, insolubilization of both walls was concomitant with a burst in H2O2 production and in extracellular peroxidase activity. Finally, both isodityrosine and dityrosine were found in hydrolysates of the insolubilized vegetative wall layer. We propose that the formation of tyrosine cross-links is essential to Chlamydomonas HRGP insolubilization.

  9. Continuous Sensing Photonic Lab-on-a-Chip Platform Based on Cross-Linked Enzyme Crystals.

    PubMed

    Conejero-Muriel, Mayte; Rodríguez-Ruiz, Isaac; Verdugo-Escamilla, Cristóbal; Llobera, Andreu; Gavira, José A

    2016-12-06

    Microfluidics or lab-on-a-chip technology offer clear advantages over conventional systems such as a dramatic reduction of reagent consumption or a shorter analysis time, which are translated into cost-effective systems. In this work, we present a photonic enzymatic lab-on-a-chip reactor based on cross-linked enzyme crystals (CLECs), able to work in continuous flow, as a highly sensitive, robust, reusable, and stable platform for continuous sensing with superior performance as compared to the state of the art. The microreactor is designed to facilitate the in situ crystallization and crystal cross-linking generating enzymatically active material that can be stored for months/years. Thus, and by means of monolithically integrated micro-optics elements, continuous enzymatic reactions can be spectrophotometrically monitored. Lipase, an enzyme with industrial significance for catalyzed transesterification, hydrolysis, and esterification reactions, is used to demonstrate the potential of the microplatforms as both a continuous biosensor and a microreactor for the synthesis of high value compounds.

  10. The transport of octamethylcyclotetrasiloxane (D4) and polydimethylsiloxane (PDMS) in lightly cross-linked silicone rubber.

    PubMed

    Wolf, C J; Jerina, K L; Brandon, H J; Young, V L

    2001-01-01

    The transport of octamethylcyclotetrasiloxane (D4), one of the major constituents of silicone fluids and rubbers, and low viscosity polydimethylsiloxane oil into a silica filled cross-linked silicone elastomeric rubber was measured as a function of temperature, cross-link density of the rubber, and concentration of the D4 in methanol solution. A small amount of material, approximately 3 wt%, is extracted from the rubber with hexane. The extraction process has a large effect upon D4 solubility in the rubber, increasing from approximately 160 to 180 wt% after extraction. The heats of solution for both penetrants into the rubber are essentially zero and the activation energies for diffusion are small, approximately 8 and 15 kJ molt(-1) for D4 and PDMS, respectively. The diffusion process is Fickian and the diffusion coefficient of D4 into silicone/silica rubbers is essentially independent of concentration over the concentration investigated, i.e. from 1 to 100 vol% D4 in methanol. The permeability, i.e. the product of the diffusion coefficient and the solubility, decreases rapidly for D4 concentrations less than 50 vol% (0.1 mol fraction). This suggests that the permeation of D4 out of any encapsulation device, such as a silicone breast implant, is linearly dependent upon the concentration of D4 in the prosthesis. Swelling is isotropic and was measured by dimensional changes in rectangular samples and correlates well with the volume of D4 sorbed.

  11. Isodityrosine cross-linking mediates insolubilization of cell walls in Chlamydomonas.

    PubMed Central

    Waffenschmidt, S; Woessner, J P; Beer, K; Goodenough, U W

    1993-01-01

    Enzymatic removal of the cell wall induces vegetative Chlamydomonas reinhardtii cells to transcribe wall genes and synthesize new hydroxyproline-rich glycoproteins (HRGPs) related to the extensins found in higher plant cell walls. A cDNA expression library made from such induced cells was screened with antibodies to an oligopeptide containing the (SP)x repetitive domains found in Chlamydomonas wall proteins. One of the selected cDNAs encodes an (SP)x-rich polypeptide that also displays a repeated YGG motif. Ascorbate, a peroxidase inhibitor, and tyrosine derivatives were shown to inhibit insolubilization of both the vegetative and zygotic cell walls of Chlamydomonas, suggesting that oxidative cross-linking of tyrosines is occurring. Moreover, insolubilization of both walls was concomitant with a burst in H2O2 production and in extracellular peroxidase activity. Finally, both isodityrosine and dityrosine were found in hydrolysates of the insolubilized vegetative wall layer. We propose that the formation of tyrosine cross-links is essential to Chlamydomonas HRGP insolubilization. PMID:7689882

  12. Can a mixed damage interfere with DNA-protein cross-links repair?

    PubMed

    Marzano, C; Severin, E; Bordin, F

    2001-01-01

    Some photochemical and photobiological properties of 4,5',8-trimethylpsoralen (TMP) have been studied in comparison with 1,4,6,8-tetramethyl-2H-furo[2,3-h]quinolin-2 one (FQ) and 8-methoxypsoralen (8-MOP). TMP and FQ can photobind to mammalian cell DNA in vivo, by UVA irradiation, forming DNA-protein cross-links (DPC), but only TMP shows a strong capacity of inducing interstrand cross-links (ISC). The mechanism of DPC formation was studied using the double irradiation method in Chinese hamster ovary (CHO) cells, and DPC were detected by alkaline elution. Both TMP and FQ induce covalent diadducts linking together DNA and proteins. Studying the formation of double strand breaks (DSB) in CHO cells we observed that TMP induced a low amount of DSB, similar to 8-MOP. TMP and 8-MOP induced chromosomal aberrations in CHO cells to the same extent, while FQ appeared to be more active. Our data suggest that the ISC induced by TMP could trap enzymes involved in DPC repair.

  13. XPF-ERCC1 participates in the Fanconi anemia pathway of cross-link repair.

    PubMed

    Bhagwat, Nikhil; Olsen, Anna L; Wang, Anderson T; Hanada, Katsuhiro; Stuckert, Patricia; Kanaar, Roland; D'Andrea, Alan; Niedernhofer, Laura J; McHugh, Peter J

    2009-12-01

    Interstrand cross-links (ICLs) prevent DNA strand separation and, therefore, transcription and replication, making them extremely cytotoxic. The precise mechanism by which ICLs are removed from mammalian genomes largely remains elusive. Genetic evidence implicates ATR, the Fanconi anemia proteins, proteins required for homologous recombination, translesion synthesis, and at least two endonucleases, MUS81-EME1 and XPF-ERCC1. ICLs cause replication-dependent DNA double-strand breaks (DSBs), and MUS81-EME1 facilitates DSB formation. The subsequent repair of these DSBs occurs via homologous recombination after the ICL is unhooked by XPF-ERCC1. Here, we examined the effect of the loss of either nuclease on FANCD2 monoubiquitination to determine if the nucleolytic processing of ICLs is required for the activation of the Fanconi anemia pathway. FANCD2 was monoubiquitinated in Mus81(-/-), Ercc1(-/-), and XPF-deficient human, mouse, and hamster cells exposed to cross-linking agents. However, the monoubiquitinated form of FANCD2 persisted longer in XPF-ERCC1-deficient cells than in wild-type cells. Moreover, the levels of chromatin-bound FANCD2 were dramatically reduced and the number of ICL-induced FANCD2 foci significantly lower in XPF-ERCC1-deficient cells. These data demonstrate that the unhooking of an ICL by XPF-ERCC1 is necessary for the stable localization of FANCD2 to the chromatin and subsequent homologous recombination-mediated DSB repair.

  14. Reconstitution of peptidoglycan cross-linking leads to improved fluorescent probes of cell wall synthesis.

    PubMed

    Lebar, Matthew D; May, Janine M; Meeske, Alexander J; Leiman, Sara A; Lupoli, Tania J; Tsukamoto, Hirokazu; Losick, Richard; Rudner, David Z; Walker, Suzanne; Kahne, Daniel

    2014-08-06

    The peptidoglycan precursor, Lipid II, produced in the model Gram-positive bacterium Bacillus subtilis differs from Lipid II found in Gram-negative bacteria such as Escherichia coli by a single amidation on the peptide side chain. How this difference affects the cross-linking activity of penicillin-binding proteins (PBPs) that assemble peptidoglycan in cells has not been investigated because B. subtilis Lipid II was not previously available. Here we report the synthesis of B. subtilis Lipid II and its use by purified B. subtilis PBP1 and E. coli PBP1A. While enzymes from both organisms assembled B. subtilis Lipid II into glycan strands, only the B. subtilis enzyme cross-linked the strands. Furthermore, B. subtilis PBP1 catalyzed the exchange of both D-amino acids and D-amino carboxamides into nascent peptidoglycan, but the E. coli enzyme only exchanged D-amino acids. We exploited these observations to design a fluorescent D-amino carboxamide probe to label B. subtilis PG in vivo and found that this probe labels the cell wall dramatically better than existing reagents.

  15. CLC CI-/H+ Transporters Constrained by Covalent Cross-Linking

    SciTech Connect

    Nguitragool,W.; Miller, C.

    2007-01-01

    CLC Cl-/H+ exchangers are homodimers with Cl--binding and H+-coupling residues contained within each subunit. It is not known whether the transport mechanism requires conformational rearrangement between subunits or whether each subunit operates as a separate exchanger. We designed various cysteine substitution mutants on a cysteine-less background of CLC-ec1, a bacterial CLC exchanger of known structure, with the aim of covalently linking the subunits. The constructs were cross-linked in air or with exogenous oxidant, and the cross-linked proteins were reconstituted to assess their function. In addition to conventional disulfides, a cysteine-lysine cross-bridge was formed with I2 as an oxidant. The constructs, all of which contained one, two, or four cross-bridges, were functionally active and kinetically competent with respect to Cl- turnover rate, Cl-/H+ exchange stoichiometry, and H+ pumping driven by a Cl- gradient. These results imply that large quaternary rearrangements, such as those known to occur for 'common gating' in CLC channels, are not necessary for the ion transport cycle and that it is therefore likely that the transport mechanism is carried out by the subunits working individually, as with 'fast gating' of the CLC channels.

  16. Novel actin depolymerizing macrolide aplyronine A.

    PubMed

    Saito, S; Watabe, S; Ozaki, H; Kigoshi, H; Yamada, K; Fusetani, N; Karaki, H

    1996-09-01

    Aplyronine A is a macrolide isolated from Aplysia kurodai. By monitoring fluorescent intensity of pyrenyl-actin, it was found that aplyronine A inhibited both the velocity and the degree of actin polymerization. Aplyronine A also quickly depolymerized F-actin. The kinetics of depolymerization suggest that aplyronine A severs F-actin. The relationship between the concentration of total actin and F-actin at different concentrations of aplyronine A suggests that aplyronine A forms a 1:1 complex with G-actin. From these results, it is concluded that aplyronine A inhibits actin polymerization and depolymerizes F-actin by nibbling. Comparison of the chemical structure of aplyronine A and another actin-depolymerizing macrolide, mycalolide B, suggests that the side-chain but not the macrolide ring of aplyronine A may account for its actin binding and severing activity.

  17. Actin Mechanics and Fragmentation*

    PubMed Central

    De La Cruz, Enrique M.; Gardel, Margaret L.

    2015-01-01

    Cell physiological processes require the regulation and coordination of both mechanical and dynamical properties of the actin cytoskeleton. Here we review recent advances in understanding the mechanical properties and stability of actin filaments and how these properties are manifested at larger (network) length scales. We discuss how forces can influence local biochemical interactions, resulting in the formation of mechanically sensitive dynamic steady states. Understanding the regulation of such force-activated chemistries and dynamic steady states reflects an important challenge for future work that will provide valuable insights as to how the actin cytoskeleton engenders mechanoresponsiveness of living cells. PMID:25957404

  18. Evaluation of cross-linked aggregates from purified Bacillus subtilis levansucrase mutants for transfructosylation reactions

    PubMed Central

    Ortiz-Soto, Maria Elena; Rudiño-Piñera, Enrique; Rodriguez-Alegria, Maria Elena; Munguia, Agustin Lopez

    2009-01-01

    Background Increasing attention has been focused on inulin and levan-type oligosaccharides, including fructosyl-xylosides and other fructosides due to their nutraceutical properties. Bacillus subtilis levansucrase (LS) catalyzes the synthesis of levan from sucrose, but it may also transfer the fructosyl moiety from sucrose to acceptor molecules included in the reaction medium. To study transfructosylation reactions with highly active and robust derivatives, cross-linked enzyme aggregates (CLEAs) were prepared from wild LS and two mutants. CLEAs combine the catalytic features of pure protein preparations in terms of specific activity with the mechanical behavior of industrial biocatalysts. Results Two types of procedures were used for the preparation of biocatalysts from purified wild type LS (WT LS) B. subtilis and the R360K and Y429N LS mutants: purified enzymes aggregated with glutaraldehyde (cross-linked enzyme aggregates: CLEAs), and covalently immobilized enzymes in Eupergit C®. The biocatalysts were characterized and used for fructoside synthesis using xylose as an acceptor model. CLEAs were able to catalyze the synthesis of fructosides as efficiently as soluble enzymes. The specific activity of CLEAs prepared from wild type LS (44.9 U/mg of CLEA), R360K (56.5 U/mg of CLEA) and Y429N (1.2 U/mg of CLEA) mutants were approximately 70, 40 and 200-fold higher, respectively, than equivalent Eupergit C® immobilized enzyme preparations (U/mg of Eupergit), where units refer to global LS activity. In contrast, the specific activity of the free enzymes was 160, 171.2 and 1.5 U/mg of protein, respectively. Moreover, all CLEAs had higher thermal stability than corresponding soluble enzymes. In the long term, the operational stability was affected by levan synthesis. Conclusion This is the first report of cross-linked transglycosidases aggregates. CLEAs prepared from purified LS and mutants have the highest specific activity for immobilized fructosyltransferases (FTFs

  19. 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.

  20. 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.

  1. 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.

  2. 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.

  3. 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.

  4. 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

  5. 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.

  6. The RhoA effector mDia is induced during T cell activation and regulates actin polymerization and cell migration in T lymphocytes.

    PubMed

    Vicente-Manzanares, Miguel; Rey, Mercedes; Pérez-Martínez, Manuel; Yáñez-Mó, María; Sancho, David; Cabrero, José Román; Barreiro, Olga; de la Fuente, Hortensia; Itoh, Kazuyuki; Sánchez-Madrid, Francisco

    2003-07-15

    Regulation of actin polymerization is critical for many different functions of T lymphocytes, including cell migration. Here we show that the RhoA effector mDia is induced in vitro in activated PBL and is highly expressed in vivo in diseased tissue-infiltrating activated lymphocytes. mDia localizes at the leading edge of polarized T lymphoblasts in an area immediately posterior to the leading lamella, in which its effector protein profilin is also concentrated. Overexpression of an activated mutant of mDia results in an inhibition of both spontaneous and chemokine-directed T cell motility. mDia does not regulate the shape of the cell, which involves another RhoA effector, p160 Rho-coiled coil kinase, and is not involved in integrin-mediated cell adhesion. However, mDia activation blocked CD3- and PMA-mediated cell spreading. mDia activation increased polymerized actin levels, which resulted in the blockade of chemokine-induced actin polymerization by depletion of monomeric actin. Moreover, mDia was shown to regulate the function of the small GTPase Rac1 through the control of actin availability. Together, our data demonstrate that RhoA is involved in the control of the filamentous actin/monomeric actin balance through mDia, and that this balance is critical for T cell responses.

  7. Activation of the MKL1/actin signaling pathway induces hormonal escape in estrogen-responsive breast cancer cell lines.

    PubMed

    Kerdivel, Gwenneg; Boudot, Antoine; Habauzit, Denis; Percevault, Frederic; Demay, Florence; Pakdel, Farzad; Flouriot, Gilles

    2014-06-05

    Estrogen receptor alpha (ERα) is generally considered to be a good prognostic marker because almost 70% of ERα-positive tumors respond to anti-hormone therapies. Unfortunately, during cancer progression, mammary tumors can escape from estrogen control, resulting in resistance to treatment. In this study, we demonstrate that activation of the actin/megakaryoblastic leukemia 1 (MKL1) signaling pathway promotes the hormonal escape of estrogen-sensitive breast cancer cell lines. The actin/MKL1 signaling pathway is silenced in differentiated ERα-positive breast cancer MCF-7 and T47D cell lines and active in ERα-negative HMT-3522 T4-2 and MDA-MB-231 breast cancer cells, which have undergone epithelial-mesenchymal transition. We showed that MKL1 activation in MCF-7 cells, either by modulating actin dynamics or using MKL1 mutants, down-regulates ERα expression and abolishes E2-dependent cell growth. Interestingly, the constitutively active form of MKL1 represses PR and HER2 expression in these cells and increases the expression of HB-EGF, TGFβ, and amphiregulin growth factors in an E2-independent manner. The resulting expression profile (ER-, PR-, HER2-) typically corresponds to the triple-negative breast cancer expression profile.

  8. 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

  9. 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.

  10. 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

  11. 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.

  12. 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.

  13. Reduction of exportin 6 activity leads to actin accumulation via failure of RanGTP restoration and NTF2 sequestration in the nuclei of senescent cells.

    PubMed

    Park, Su Hyun; Park, Tae Jun; Lim, In Kyoung

    2011-04-15

    We have previously reported that G-actin accumulation in nuclei is a universal phenomenon of cellular senescence. By employing primary culture of human diploid fibroblast (HDF) and stress-induced premature senescence (SIPS), we explored whether the failure of actin export to cytoplasm is responsible for actin accumulation in nuclei of senescent cells. Expression of exportin 6 (Exp6) and small G-protein, Ran, was significantly reduced in the replicative senescence, but not yet in SIPS, whereas nuclear import of actin by cofilin was already increased in SIPS. After treatment of young HDF cells with H(2)O(2), rapid reduction of nuclear RanGTP was observed along with cytoplasmic increase of RanGDP. Furthermore, significantly reduced interaction of Exp6 with RanGTP was found by GST-Exp6 pull-down analysis. Failure of RanGTP restoration was accompanied with inhibition of ATP synthesis and NTF2 sequestration in the nuclei along with accordant change of senescence morphology. Indeed, knockdown of Exp6 expression significantly increased actin molecule in the nuclei of young HDF cells. Therefore, actin accumulation in nuclei of senescent cells is most likely due to the failure of RanGTP restoration with ATP deficiency and NTF2 accumulation in nuclei, which result in the decrease of actin export via Exp6 inactivation, in addition to actin import by cofilin activation.

  14. Reduction of exportin 6 activity leads to actin accumulation via failure of RanGTP restoration and NTF2 sequestration in the nuclei of senescent cells

    SciTech Connect

    Park, Su Hyun; Park, Tae Jun; Lim, In Kyoung

    2011-04-15

    We have previously reported that G-actin accumulation in nuclei is a universal phenomenon of cellular senescence. By employing primary culture of human diploid fibroblast (HDF) and stress-induced premature senescence (SIPS), we explored whether the failure of actin export to cytoplasm is responsible for actin accumulation in nuclei of senescent cells. Expression of exportin 6 (Exp6) and small G-protein, Ran, was significantly reduced in the replicative senescence, but not yet in SIPS, whereas nuclear import of actin by cofilin was already increased in SIPS. After treatment of young HDF cells with H{sub 2}O{sub 2}, rapid reduction of nuclear RanGTP was observed along with cytoplasmic increase of RanGDP. Furthermore, significantly reduced interaction of Exp6 with RanGTP was found by GST-Exp6 pull-down analysis. Failure of RanGTP restoration was accompanied with inhibition of ATP synthesis and NTF2 sequestration in the nuclei along with accordant change of senescence morphology. Indeed, knockdown of Exp6 expression significantly increased actin molecule in the nuclei of young HDF cells. Therefore, actin accumulation in nuclei of senescent cells is most likely due to the failure of RanGTP restoration with ATP deficiency and NTF2 accumulation in nuclei, which result in the decrease of actin export via Exp6 inactivation, in addition to actin import by cofilin activation.

  15. A core cross-linked polymeric micellar platium(IV) prodrug with enhanced anticancer efficiency.

    PubMed

    Hou, Jie; Shang, Jincai; Jiao, Chengbin; Jiang, Peiyue; Xiao, Huijie; Luo, Lan; Liu, Tongjun

    2013-07-01

    A core cross-linked polymeric micellar cisplatin(IV) conjugate prodrug is prepared by attaching the cisplatin(IV) to mPEG-b-PLL biodegradable copolymers to form micellar nanoparticles that can disintegrate to release the active anticancer agent cisplatin(II) in a mild reducing environment. Moreover, in vitro studies show that this cisplatin(IV) conjugate prodrug displays enhanced cytotoxicity against HepG2 cancer cells compared with cisplatin(II). Further studies demonstrate that the high cellular uptake and platinum-DNA adduct of this cisplatin(IV) conjugate prodrug can induce more cancer-cell apoptosis than cisplatin(II), which is responsible for its enhanced anticancer activity.

  16. Wiskott-Aldrich syndrome protein is required for NK cell cytotoxicity and colocalizes with actin to NK cell-activating immunologic synapses

    NASA Astrophysics Data System (ADS)

    Orange, Jordan S.; Ramesh, Narayanaswamy; Remold-O'Donnell, Eileen; Sasahara, Yoji; Koopman, Louise; Byrne, Michael; Bonilla, Francisco A.; Rosen, Fred S.; Geha, Raif S.; Strominger, Jack L.

    2002-08-01

    The Wiskott-Aldrich syndrome (WAS) is a primary immunodeficiency disorder caused by a mutation in WAS protein (WASp) that results in defective actin polymerization. Although the function of many hematopoietic cells requires WASp, the specific expression and function of this molecule in natural killer (NK) cells is unknown. Here, we report that WAS patients have increased percentages of peripheral blood NK cells and that fresh enriched NK cells from two patients with a WASp mutation have defective cytolytic function. In normal NK cells, WASp was expressed and localized to the activating immunologic synapse (IS) with filamentous actin (F-actin). Perforin also localized to the NK cell-activating IS but at a lesser frequency than F-actin and WASp. The accumulation of F-actin and WASp at the activating IS was decreased significantly in NK cells that had been treated with the inhibitor of actin polymerization, cytochalasin D. NK cells from WAS patients lacked expression of WASp and accumulated F-actin at the activating IS infrequently. Thus, WASp has an important function in NK cells. In patients with WASp mutations, the resulting NK cell defects are likely to contribute to their disease.

  17. Apical domain polarization localizes actin-myosin activity to drive ratchet-like apical constriction.

    PubMed

    Mason, Frank M; Tworoger, Michael; Martin, Adam C

    2013-08-01

    Apical constriction promotes epithelia folding, which changes tissue architecture. During Drosophila gastrulation, mesoderm cells exhibit repeated contractile pulses that are stabilized such that cells apically constrict like a ratchet. The transcription factor Twist is required to stabilize cell shape. However, it is unknown how Twist spatially coordinates downstream signals to prevent cell relaxation. We find that during constriction, Rho-associated kinase (Rok) is polarized to the middle of the apical domain (medioapical cortex), separate from adherens junctions. Rok recruits or stabilizes medioapical myosin II (Myo-II), which contracts dynamic medioapical actin cables. The formin Diaphanous mediates apical actin assembly to suppress medioapical E-cadherin localization and form stable connections between the medioapical contractile network and adherens junctions. Twist is not required for apical Rok recruitment, but instead polarizes Rok medioapically. Therefore, Twist establishes radial cell polarity of Rok/Myo-II and E-cadherin and promotes medioapical actin assembly in mesoderm cells to stabilize cell shape fluctuations.

  18. Interaction between activated chemokine receptor 1 and FcεRI at membrane rafts promotes communication and F-actin-rich cytoneme extensions between mast cells

    PubMed Central

    Beer, Freddy; Ono, Shoichiro; Ono, Santa J.

    2010-01-01

    Chemokines play important regulatory roles in immunity, but their contributions to mast cell function remain poorly understood. We examined the effects of FcεRI–chemokine receptor (CCR) 1 co-stimulation on receptor localization and cellular morphology of bone marrow-derived mast cells. Whereas FcεRI and CCR1 co-localized at the plasma membrane in unsensitized cells, sensitization with IgE promoted internalization of CCR1 molecules. Co-stimulation of FcεRI and CCR1 with antigen and macrophage inflammatory protein-1α was more effective than FcεRI stimulation alone in causing leading edge formation, flattened morphology, membrane ruffles and ganglioside (GM1+) lipid mediator release. Co-stimulation resulted in phalloidin-positive cytoneme-like cellular extensions, also known as tunneling nanotubes, which originated at points of calcium accumulation. This is the first report of cytoneme formation by mast cells. To determine the importance of lipid rafts for mast cell function, the cells were cholesterol depleted. Cholesterol depletion enhanced degranulation in resting, sensitized and co-stimulated cells, but not in FcεRI-cross-linked cells, and inhibited formation of filamentous actin+ cytonemes but not GM1+ cytonemes. Treatment with latrunculin A to sequester globular-actin abolished cytoneme formation. The cytonemes may participate in intercellular communication during allergic and inflammatory responses, and their presence in the co-stimulated mast cells suggests new roles for CCRs in immunopathology. PMID:20173038

  19. Regulation of the actin-activated MgATPase activity of Acanthamoeba myosin II by phosphorylation of serine 639 in motor domain loop 2.

    PubMed

    Liu, Xiong; Lee, Duck-Yeon; Cai, Shutao; Yu, Shuhua; Shu, Shi; Levine, Rodney L; Korn, Edward D

    2013-01-02

    It had been proposed previously that only filamentous forms of Acanthamoeba myosin II have actin-activated MgATPase activity and that this activity is inhibited by phosphorylation of up to four serine residues in a repeating sequence in the C-terminal nonhelical tailpiece of the two heavy chains. We have reinvestigated these issues using recombinant WT and mutant myosins. Contrary to the earlier proposal, we show that two nonfilamentous forms of Acanthamoeba myosin II, heavy meromyosin and myosin subfragment 1, have actin-activated MgATPase that is down-regulated by phosphorylation. By mass spectroscopy, we identified five serines in the heavy chains that can be phosphorylated by a partially purified kinase preparation in vitro and also are phosphorylated in endogenous myosin isolated from the amoebae: four serines in the nonhelical tailpiece and Ser639 in loop 2 of the motor domain. S639A mutants of both subfragment 1 and full-length myosin had actin-activated MgATPase that was not inhibited by phosphorylation of the serines in the nonhelical tailpiece or their mutation to glutamic acid or aspartic acid. Conversely, S639D mutants of both subfragment 1 and full-length myosin were inactive, irrespective of the phosphorylation state of the serines in the nonhelical tailpiece. To our knowledge, this is the first example of regulation of the actin-activated MgATPase activity of any myosin by modification of surface loop 2.

  20. Structural features of covalently cross-linked hydroxylase and reductase proteins of soluble methane monooxygenase as revealed by mass spectrometric analysis.

    PubMed

    Kopp, Daniel A; Berg, Eric A; Costello, Catherine E; Lippard, Stephen J

    2003-06-06

    Soluble methane monooxygenase requires complexes between its three component proteins for efficient catalysis. The hydroxylase (MMOH) must bind both to the reductase (MMOR) and to the regulatory protein (MMOB) to facilitate oxidation of methane to methanol. Although structures of MMOH, MMOB, and one domain of MMOR have been determined, less geometric information is available for the complexes. To address this deficiency, MMOH and MMOR were cross-linked by a carbodiimide reagent and analyzed by specific proteolysis, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry, and capillary high performance liquid chromatography mass spectrometry. Tandem mass spectra conclusively identified two amine-to-carboxylate cross-linked sites involving the alpha subunit of MMOH and the [2Fe-2S] domain of MMOR (MMOR-Fd). In particular, the N terminus of the MMOH alpha subunit forms cross-links to the side chains of MMOR-Fd residues Glu-56 and Glu-91. These Glu residues are close to one another on the surface of MMOR-Fd and >25 A from the [2Fe-2S] cluster. Because the N terminus of the alpha subunit of MMOH was not located in the crystal structure of MMOH, a detailed structural model of the complex based on the cross-link was precluded; however, a previously proposed binding site for MMOR on MMOH could be ruled out. Based on the cross-linking results, a MMOR E56Q/E91Q double mutant was generated. The mutant retains >80% of MMOR NADH oxidase activity but reduces sMMO activity to approximately 65% of the level supported by the wild type reductase. Cross-linking to MMOH was diminished but not abolished in the double mutant, indicating that other residues of MMOR also form cross-links to MMOH.

  1. 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.

  2. 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...

  3. 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...

  4. 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...

  5. 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...

  6. 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...

  7. 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...

  8. 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.

  9. 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.

  10. 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

  11. A novel method for immobilization of proteins via entrapment of magnetic nanoparticles through epoxy cross-linking.

    PubMed

    Iype, Tessy; Thomas, Jaiby; Mohan, Sangeetha; Johnson, Kochurani K; George, Ligi E; Ambattu, Lizebona A; Bhati, Aniruddha; Ailsworth, Kristen; Menon, Bindu; Rayabandla, Sunayana M; Jesudasan, Rachel A; Santhosh, Sam; Ramchand, Chaniyilparampu N

    2017-02-15

    A method for immobilization of functional proteins by chemical cross-linking of the protein of interest and uncoated iron oxide nanoparticles in the presence of Epichlorohydrin is described. As a result of the cross-linking, the proteins form a matrix in which the particles get entrapped. The optimum concentration of Epichlorohydrin that facilitates immobilization of protein without affecting the functional properties of the protein was determined. This method was used to immobilize several functional proteins and the development and functional activity of Protein A-magnetic nanoparticles (MNPs) is described here in detail. The Protein A-MNPs possess high binding capacity due to the increased surface area of uncoated nanoparticles and robust magnetic separation due to the absence of polymeric coating materials. Protein A-MNPs were successfully used for purification of antibodies and also for immunoprecipitation. We also immobilized enzymes such as horse radish peroxidase and esterase and found that by providing the optimum incubation time, temperature and protein to nanoparticle ratio, we can retain the activity and improve the stability of the enzyme. This study is the first demonstration that Epichlorohydrin can be used to entrap nanoparticles in a cross-linked matrix of protein without impairing the activity of immobilized protein.

  12. 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.

  13. Spinoculation Triggers Dynamic Actin and Cofilin Activity That Facilitates HIV-1 Infection of Transformed and Resting CD4 T Cells▿

    PubMed Central

    Guo, Jia; Wang, Weifeng; Yu, Dongyang; Wu, Yuntao

    2011-01-01

    Centrifugal inoculation, or spinoculation, is widely used in virology research to enhance viral infection. However, the mechanism remained obscure. Using HIV-1 infection of human T cells as a model, we demonstrate that spinoculation triggers dynamic actin and cofilin activity, probably resulting from cellular responses to centrifugal stress. This actin activity also leads to the upregulation of the HIV-1 receptor and coreceptor, CD4 and CXCR4, enhancing viral binding and entry. We also demonstrate that an actin inhibitor, jasplakinolide, diminishes spin-mediated enhancement. In addition, small interfering RNA (siRNA) knockdown of LIMK1, a cofilin kinase, decreases the enhancement. These results suggest that spin-mediated enhancement cannot be explained simply by a virus-concentrating effect; rather, it is coupled with spin-induced cytoskeletal dynamics that promote receptor mobilization, viral entry, and postentry processes. Our results highlight the importance of cofilin and a dynamic cytoskeleton for the initiation of viral infection. Our results also indicate that caution needs to be taken in data interpretation when cells are spinoculated; some of the spin-induced cellular permissiveness may be beyond the natural capacity of an infecting virus. PMID:21795326

  14. A novel actin barbed-end-capping activity in EPS-8 regulates apical morphogenesis in intestinal cells of Caenorhabditis elegans.

    PubMed

    Croce, Assunta; Cassata, Giuseppe; Disanza, Andrea; Gagliani, Maria Cristina; Tacchetti, Carlo; Malabarba, Maria Grazia; Carlier, Marie-France; Scita, Giorgio; Baumeister, Ralf; Di Fiore, Pier Paolo

    2004-12-01

    Redundant gene function frequently hampers investigations of the physiological roles of mammalian proteins. This is the case for Eps8, a receptor tyrosine kinase (RTK) substrate that participates in the activation of the Rac-specific guanine nucleotide-exchange function of Sos1 (refs 2-5), thereby regulating actin remodelling by RTKs. EPS8-knockout mice, however, exhibit no evident phenotype, owing to the redundant function of three other EPS8-related genes. Here we show that in the nematode Caenorhabditis elegans, only one orthologue of the EPS8 gene exists, which gives rise to two alternatively spliced isoforms, EPS-8A and EPS-8B, differing at their carboxyl termini. In the nematode, eps-8 is essential for embryonic development. Furthermore, EPS-8A, but not EPS-8B, is specifically required for proper apical morphogenesis in the intestinal cells. This latter phenotype could be precisely correlated with a previously unknown actin barbed-end-capping activity, which is present in the C terminus of the EPS-8A isoform. Therefore, nematode genetics allowed not only the unmasking of distinct EPS-8-linked phenotypes, but also the definition of a novel function for this molecule in actin dynamics.

  15. 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

  16. 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

  17. 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.

  18. 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.

  19. 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.

  20. 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.

  1. 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.

  2. Dual pools of actin at presynaptic terminals.

    PubMed

    Bleckert, Adam; Photowala, Huzefa; Alford, Simon

    2012-06-01

    We investigated actin's function in vesicle recycling and exocytosis at lamprey synapses and show that FM1-43 puncta and phalloidin-labeled filamentous actin (F-actin) structures are colocalized, yet recycling vesicles are not contained within F-actin clusters. Additionally, phalloidin also labels a plasma membrane-associated cortical actin. Injection of fluorescent G-actin revealed activity-independent dynamic actin incorporation into presynaptic synaptic vesicle clusters but not into cortical actin. Latrunculin-A, which sequesters G-actin, dispersed vesicle-associated actin structures and prevented subsequent labeled G-actin and phalloidin accumulation at presynaptic puncta, yet cortical phalloidin labeling persisted. Dispersal of presynaptic F-actin structures by latrunculin-A did not disrupt vesicle clustering or recycling or alter the amplitude or kinetics of excitatory postsynaptic currents (EPSCs). However, it slightly enhanced release during repetitive stimulation. While dispersal of presynaptic actin puncta with latrunculin-A failed to disperse synaptic vesicles or inhibit synaptic transmission, presynaptic phalloidin injection blocked exocytosis and reduced endocytosis measured by action potential-evoked FM1-43 staining. Furthermore, phalloidin stabilization of only cortical actin following pretreatment with latrunculin-A was sufficient to inhibit synaptic transmission. Conversely, treatment of axons with jasplakinolide, which induces F-actin accumulation but disrupts F-actin structures in vivo, resulted in increased synaptic transmission accompanied by a loss of phalloidin labeling of cortical actin but no loss of actin labeling within vesicle clusters. Marked synaptic deficits seen with phalloidin stabilization of cortical F-actin, in contrast to the minimal effects of disruption of a synaptic vesicle-associated F-actin, led us to conclude that two structurally and functionally distinct pools of actin exist at presynaptic sites.

  3. The Effects of Cross-Linking in a Supramolecular Binder on Cycle Life in Silicon Microparticle Anodes.

    PubMed

    Lopez, Jeffrey; Chen, Zheng; Wang, Chao; Andrews, Sean C; Cui, Yi; Bao, Zhenan

    2016-01-27

    Self-healing supramolecular binder was previously found to enhance the cycling stability of micron-sized silicon particles used as the active material in lithium-ion battery anodes. In this study, we systematically control the density of cross-linking junctions in a modified supramolecular polymer binder in order to better understand how viscoelastic materials properties affect cycling stability. We found that binders with relaxation times on the order of 0.1 s gave the best cycling stability with 80% capacity maintained for over 175 cycles using large silicon particles (∼0.9 um). We attributed this to an improved balance between the viscoelastic stress relaxation in the binder and the stiffness needed to maintain mechanical integrity of the electrode. The more cross-linked binder showed markedly worse performance confirming the need for liquid-like flow in order for our self-healing polymer electrode concept to be effective.

  4. Resin bond strength to water versus ethanol-saturated human dentin pretreated with three different cross-linking agents

    PubMed Central

    Venigalla, Bhuvan Shome; Jyothi, Pinnamreddy; Kamishetty, Shekhar; Reddy, Smitha; Cherukupalli, Ravi Chandra; Reddy, Depa Arun

    2016-01-01

    Context: Resin-dentin bonds are unstable owing to hydrolytic and enzymatic degradation. Several approaches such as collagen cross-linking and ethanol-wet bonding (EWB) have been developed to overcome this problem. Collagen cross-linking improves the intrinsic properties of the collagen matrix. However, it leaves a water-rich collagen matrix with incomplete resin infiltration making it susceptible to fatigue degradation. Since EWB is expected to overcome the drawbacks of water-wet bonding (WWB), a combination of collagen cross-linking with EWB was tested. Aim: The aim of this study was to compare the effect of pretreatment with different cross-linking agents such as ultraviolet A (UVA)-activated 0.1% riboflavin, 1 M carbodiimide, and 6.5 wt% proanthocyanidin on the immediate and long-term bond strengths of an etch and rinse adhesive system to water- versus ethanol-saturated dentin within clinically relevant application time periods. Settings and Design: Long-term in vitro study evaluating the microtensile bond strength of adhesive-dentin interface after different surface pretreatments. Subjects and Methods: Eighty freshly extracted human molars were prepared to expose dentin, etched with 37% phosphoric acid for 15 s rinsed, and grouped randomly. They were blot-dried and pretreated with different cross-linkers: 0.1% riboflavin for 2 min followed by UVA activation for 2 min; 1 M carbodiimide for 2 min; 6.5 wt% proanthocyanidin for 2 min and rinsed. They were then bonded with Adper Single Bond Adhesive (3M ESPE), by either WWB or EWB, followed by resin composite build-ups (Filtek Z350, 3M ESPE). Bonded specimens in each group were then sectioned and divided into two halves. Microtensile bond strength was tested in one half after 24 h and the other after 6 months storage in artificial saliva. Statistical Analysis Used: Analysis was done using SPSS version 18 software (SPSS Inc., Chicago, IL, USA). Intergroup comparison of bond strength was done using ANOVA with post hoc

  5. The Fanconi anemia pathway promotes replication-dependent DNA interstrand cross-link repair.

    PubMed

    Knipscheer, Puck; Räschle, Markus; Smogorzewska, Agata; Enoiu, Milica; Ho, The Vinh; Schärer, Orlando D; Elledge, Stephen J; Walter, Johannes C

    2009-12-18

    Fanconi anemia is a human cancer predisposition syndrome caused by mutations in 13 Fanc genes. The disorder is characterized by genomic instability and cellular hypersensitivity to chemicals that generate DNA interstrand cross-links (ICLs). A central event in the activation of the Fanconi anemia pathway is the mono-ubiquitylation of the FANCI-FANCD2 complex, but how this complex confers ICL resistance remains enigmatic. Using a cell-free system, we showed that FANCI-FANCD2 is required for replication-coupled ICL repair in S phase. Removal of FANCD2 from extracts inhibits both nucleolytic incisions near the ICL and translesion DNA synthesis past the lesion. Reversal of these defects requires ubiquitylated FANCI-FANCD2. Our results show that multiple steps of the essential S-phase ICL repair mechanism fail when the Fanconi anemia pathway is compromised.

  6. Hierarchical cross-linking in physical alginate gels: a rheological and dynamic light scattering investigation

    NASA Astrophysics Data System (ADS)

    Larobina, Domenico; Cipelletti, Luca

    We investigate the dynamics of alginate gels, an important class of biopolymer-based viscoelastic materials, by combining mechanical tests and non-conventional, time-resolved light scattering methods. Two relaxation modes are observed upon applying a compressive or shear stress. Dynamic light scattering and diffusive wave spectroscopy measurements reveal that these modes are associated with discontinuous rearrangement events that restructure the gel network via anomalous, non-diffusive microscopic dynamics. We show that these dynamics are due to both thermal activation and internal stress stored during gelation and propose a scenario where a hierarchy of cross-links with different life times is responsible for the observed complex behavior. Measurements at various temperatures and sample ages are presented to support this scenario.

  7. 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.

  8. Effect of D-penicillamine on rat lung elastin cross-linking during the perinatal period.

    PubMed

    Koçtürk, Semra; Oktay, Gülgün; Güner, Gül; Pekçetin, Cetin; Güre, Ataman

    2006-01-01

    This study was designed to clarify the effects of D-penicillamine (DPA), a drug used for treatment of various pathological events, on lung elastin formation and maturation of the newborn in the perinatal period. The investigation was conducted on 20 newborn rats bred from 40 female and six male rats. DPA doses 400 mg kg(-1) day(-1) and physiological saline were given intraperitoneally (i.p) to experimental and control groups. To assess newborn maturation, their body and lung weights were determined. Serum Cu levels were measured by atomic absorption spectroscopy and ceruloplasmin (Cp) activities were measured spectrophotometrically. Newborn lung tissue elastin, desmosine (DES) and isodesmosine (IDES) levels were measured by HPLC. The results showed that DPA treatment caused loss of skin elasticity and reduction in body and lung weight in newborns of the experimental group. The serum Cu levels and Cp activity were found to be significantly lower in both maternal and newborn of the experimental groups compared with the control group. The lung DES, IDES and elastin values of newborns in the experimental group were decreased compared with the control group. In conclusion, our results indicate that 400 mg kg(-1) day(-1) DPA, a dose that is used in the treatment of Wilson's disease, rheumatoid arthritis and cystinuria, caused the retardation of newborn maturation, a decrease in DES-IDES cross-links and levels of lung elastin of offspring in the perinatal period. Another conclusion to be drawn from this study is that even low levels of Cu depletion due to DPA administration induces a change in cross-linking in lung elastin during the perinatal period.

  9. Molt cycle-associated changes in calcium-dependent proteinase activity that degrades actin and myosin in crustacean muscle

    SciTech Connect

    Mykles, D.L.; Skinner, D.M.

    1982-01-01

    The role of calcium-dependent proteinase (CDP) in the proecdysial atrophy of crustacean claw muscle has been investigated. During atrophy the molar ratio of actin to myosin heavy chain decreased 31%, confirming earlier ultrastructural observations that the ratio of thin:thick myofilaments declined from 9:1 to 6:1 (D.L. Mykles and D.M. Skinner, 1981, J. Ultrastruct. Res. 75, 314 to 325). The release of TCA-soluble material in muscle homogenates at neutral pH was stimulated by Ca/sup 2 +/ and completely inhibited by EGTA. The specific degradation of the major myofibrillar proteins (actin, myosin heavy and light chains, paramyosin, tropomyosin, troponin-T, and troponin-I) was demonstrated by SDS-polyacrylamide gel electrophoresis. Proteolytic activity was more than twofold greater in proecdysial muscle homogenates. Degradation of myofibrillar proteins was inhibited by EGTA, and the two inhibitors of crysteine proteinases, leupeptin, and antipain, but not pepstatin, an inhibitor of aspartic proteinases. Unlike CDPs from vertebrate muscle, the CDP(s) in crab claw muscle degrades actin and myosin in addition to other myofibrillar proteins.

  10. 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

  11. Bifunctional alkylating agent-mediated MGMT-DNA cross-linking and its proteolytic cleavage in 16HBE cells.

    PubMed

    Cheng, Jin; Ye, Feng; Dan, Guorong; Zhao, Yuanpeng; Wang, Bin; Zhao, Jiqing; Sai, Yan; Zou, Zhongmin

    2016-08-15

    Nitrogen mustard (NM), a bifunctional alkylating agent (BAA), contains two alkyl arms and can act as a cross-linking bridge between DNA and protein to form a DNA-protein cross-link (DPC). O(6)-methylguanine-DNA methyltransferase (MGMT), a DNA repair enzyme for alkyl adducts removal, is found to enhance cell sensitivity to BAAs and to promote damage, possibly due to its stable covalent cross-linking with DNA mediated by BAAs. To investigate MGMT-DNA cross-link (mDPC) formation and its possible dual roles in NM exposure, human bronchial epithelial cell line 16HBE was subjected to different concentrations of HN2, a kind of NM, and we found mDPC was induced by HN2 in a concentration-dependent manner, but the mRNA and total protein of MGMT were suppressed. As early as 1h after HN2 treatment, high mDPC was achieved and the level maintained for up to 24h. Quick total DPC (tDPC) and γ-H2AX accumulation were observed. To evaluate the effect of newly predicted protease DVC1 on DPC cleavage, we applied siRNA of MGMT and DVC1, MG132 (proteasome inhibitor), and NMS-873 (p97 inhibitor) and found that proteolysis plays a role. DVC1 was proven to be more important in the cleavage of mDPC than tDPC in a p97-dependent manner. HN2 exposure induced DVC1 upregulation, which was at least partially contributed to MGMT cleavage by proteolysis because HN2-induced mDPC level and DNA damage was closely related with DVC1 expression. Homologous recombination (HR) was also activated. Our findings demonstrated that MGMT might turn into a DNA damage promoter by forming DPC when exposed to HN2. Proteolysis, especially DVC1, plays a crucial role in mDPC repair.

  12. Photosensitized UVA-Induced Cross-Linking between Human DNA Repair and Replication Proteins and DNA Revealed by Proteomic Analysis

    PubMed Central

    2016-01-01

    Long wavelength ultraviolet radiation (UVA, 320–400 nm) interacts with chromophores present in human cells to induce reactive oxygen species (ROS) that damage both DNA and proteins. ROS levels are amplified, and the damaging effects of UVA are exacerbated if the cells are irradiated in the presence of UVA photosensitizers such as 6-thioguanine (6-TG), a strong UVA chromophore that is extensively incorporated into the DNA of dividing cells, or the fluoroquinolone antibiotic ciprofloxacin. Both DNA-embedded 6-TG and ciprofloxacin combine synergistically with UVA to generate high levels of ROS. Importantly, the extensive protein damage induced by these photosensitizer+UVA combinations inhibits DNA repair. DNA is maintained in intimate contact with the proteins that effect its replication, transcription, and repair, and DNA–protein cross-links (DPCs) are a recognized reaction product of ROS. Cross-linking of DNA metabolizing proteins would compromise these processes by introducing physical blocks and by depleting active proteins. We describe a sensitive and statistically rigorous method to analyze DPCs in cultured human cells. Application of this proteomics-based analysis to cells treated with 6-TG+UVA and ciprofloxacin+UVA identified proteins involved in DNA repair, replication, and gene expression among those most vulnerable to cross-linking under oxidative conditions. PMID:27654267

  13. Development of a novel photoreactive calmodulin derivative: Cross-linking of purified adenylate cyclase from bovine brain

    SciTech Connect

    Harrison, J.K.; Lawton, R.G.; Gnegy, M.E. )

    1989-07-11

    A novel photoreactive calmodulin (CaM) derivative was developed and used to label the purified CaM-sensitive adenylate cyclase from bovine cortex. {sup 125}I-CaM was conjugated with the heterobifunctional cross-linking agent p-nitrophenyl 3-diazopyruvate (DAPpNP). Spectral data indicated that diazopyruvoyl (DAP) groups were incorporated into the CaM molecule. Iodo-CaM-DAPs behaved like native CaM with respect to (1) Ca{sup 2+}-dependent enhanced mobility on sodium dodecyl sulfate-polyacrylamide gels and (2) Ca{sup 2+}-dependent stimulation of adenylate cyclase activity. {sup 125}I-CaM-DAP photochemically cross-linked to CaM-binding proteins in a manner that was both Ca{sup 2+} dependent and CaM specific. Photolysis of forskolin-agarose-purified adenylate cyclase from bovine cortex with {sup 125}I-CaM-DAP produced a single cross-linked product which migrates on sodium dodecyl sulfate-polyacrylamide gels with an apparent molecular weight of approximately 140,000.

  14. Mimicking the hierarchical functions of dentin collagen cross-links with plant derived phenols and phenolic acids

    PubMed Central

    Vidal, Cristina M. P.; Leme, Ariene A.; Aguiar, Thaiane R.; Phansalkar, Rasika; Nam, Joo-Won; Bisson, Jonathan; McAlpine, James B.; Chen, Shao-Nong; Pauli, Guido F.; Bedran-Russo, Ana

    2015-01-01

    Proanthocyanidins (PACs) are secondary plant metabolites that mediate non-enzymatic collagen cross-linking and enhance the properties of collagen based tissue, such as dentin. The extent and nature of cross-linking is influenced by the composition and specific chemical structure of the bioactive compounds present in certain PAC-rich extracts. This study investigated the effect of the molecular weight and stereochemistry of polyphenol compounds on two important properties of dentin, biomechanics and biostability. For that, purified phenols, a phenolic acid and some of its derivatives were selected: PACs dimers (A1, A2, B1 and B2) and a trimer (C1), gallic acid (Ga), its esters methyl gallate (MGa) and propyl gallate (PGa), and a pentagalloyl ester of glucose (PGG). Synergism was assessed by combination of the most active PAC and gallic acid derivative. Mechanical properties of dentin organic matrix were determined by the modulus of elasticity obtained in a flexural test. Biostability was evaluated by resistance to collagenase degradation. PACs significantly enhanced dentin mechanical properties and decreased collagen digestion. Among the gallic acid derivatives, only PGG had a significant enhancing effect. The lack of observed C1:PGG synergy indicates that both compounds have similar mechanisms of interaction with the dentin matrix. These findings reveal that the molecular weight of polyphenols have a determinant effect on their interaction with type I collagen and modulate the mechanism of cross-linking at the molecular, inter-molecular, and inter-micro-fibrillar levels. PMID:25379878

  15. Calcineurin-dependent cofilin activation and increased retrograde actin flow drive 5-HT-dependent neurite outgrowth in Aplysia bag cell neurons.

    PubMed

    Zhang, Xiao-Feng; Hyland, Callen; Van Goor, David; Forscher, Paul

    2012-12-01

    Neurite outgrowth in response to soluble growth factors often involves changes in intracellular Ca(2+); however, mechanistic roles for Ca(2+) in controlling the underlying dynamic cytoskeletal processes have remained enigmatic. Bag cell neurons exposed to serotonin (5-hydroxytryptamine [5-HT]) respond with a threefold increase in neurite outgrowth rates. Outgrowth depends on phospholipase C (PLC) → inositol trisphosphate → Ca(2+) → calcineurin signaling and is accompanied by increased rates of retrograde actin network flow in the growth cone P domain. Calcineurin inhibitors had no effect on Ca(2+) release or basal levels of retrograde actin flow; however, they completely suppressed 5-HT-dependent outgrowth and F-actin flow acceleration. 5-HT treatments were accompanied by calcineurin-dependent increases in cofilin activity in the growth cone P domain. 5-HT effects were mimicked by direct activation of PLC, suggesting that increased actin network treadmilling may be a widespread mechanism for promoting neurite outgrowth in response to neurotrophic factors.

  16. 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.

  17. 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.

  18. 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

  19. 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.

  20. 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

  1. 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.

  2. 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.

  3. 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.

  4. 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.

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

    PubMed Central

    Ri