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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 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

  2. 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. PMID:19883801

  3. Arabidopsis CROLIN1, a Novel Plant Actin-binding Protein, Functions in Cross-linking and Stabilizing Actin Filaments*

    PubMed Central

    Jia, Honglei; Li, Jisheng; Zhu, Jingen; Fan, Tingting; Qian, Dong; Zhou, Yuelong; Wang, Jiaojiao; Ren, Haiyun; Xiang, Yun; An, Lizhe

    2013-01-01

    Higher order actin filament structures are necessary for cytoplasmic streaming, organelle movement, and other physiological processes. However, the mechanism by which the higher order cytoskeleton is formed in plants remains unknown. In this study, we identified a novel actin-cross-linking protein family (named CROLIN) that is well conserved only in the plant kingdom. There are six isovariants of CROLIN in the Arabidopsis genome, with CROLIN1 specifically expressed in pollen. In vitro biochemical analyses showed that CROLIN1 is a novel actin-cross-linking protein with binding and stabilizing activities. Remarkably, CROLIN1 can cross-link actin bundles into actin networks. CROLIN1 loss of function induces pollen germination and pollen tube growth hypersensitive to latrunculin B. All of these results demonstrate that CROLIN1 may play an important role in stabilizing and remodeling actin filaments by binding to and cross-linking actin filaments. PMID:24072702

  4. 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. PMID:26607837

  5. Polymorphism of Cross-Linked Actin Networks in Giant Vesicles

    NASA Astrophysics Data System (ADS)

    Limozin, Laurent; Sackmann, Erich

    2002-09-01

    Actin networks cross-linked by natural linkers α-actinin and filamin are generated in giant vesicles by polymerization through ionophore-mediated influx of Mg2+. α-actinin induces the formation of randomly linked networks at 25 °C which transform at <15 °C into spiderweblike gels or ringlike bundles depending on the vesicle size. Muscle filamin forms ringlike structures under all experimental conditions which can supercoil by subsequent Mg2+ addition. The polymorphism is rationalized in terms of recent models of bivalent ion coupled semiflexible polyelectrolytes and by considering the topology of the linkers.

  6. H2O2-treated actin: assembly and polymer interactions with cross-linking proteins.

    PubMed Central

    DalleDonne, I; Milzani, A; Colombo, R

    1995-01-01

    During inflammation, hydrogen peroxide, produced by polymorphonuclear leukocytes, provokes cell death mainly by disarranging filamentous (polymerized) actin (F-actin). To show the molecular mechanism(s) by which hydrogen peroxide could alter actin dynamics, we analyzed the ability of H2O2-treated actin samples to polymerize as well as the suitability of actin polymers (from oxidized monomers) to interact with cross-linking proteins. H2O2-treated monomeric (globular) actin (G-actin) shows an altered time course of polymerization. The increase in the lag phase and the lowering in both the polymerization rate and the polymerization extent have been evidenced. Furthermore, steady-state actin polymers, from oxidized monomers, are more fragmented than control polymers. This seems to be ascribable to the enhanced fragility of oxidized filaments rather than to the increase in the nucleation activity, which markedly falls. These facts; along with the unsuitability of actin polymers from oxidized monomers to interact with both filamin and alpha-actinin, suggest that hydrogen peroxide influences actin dynamics mainly by changing the F-actin structure. H2O2, via the oxidation of actin thiols (in particular, the sulfhydryl group of Cys-374), likely alters the actin C-terminus, influencing both subunit/subunit interactions and the spatial structure of the binding sites for cross-linking proteins in F-actin. We suggest that most of the effects of hydrogen peroxide on actin could be explained in the light of the "structural connectivity," demonstrated previously in actin. Images FIGURE 3 FIGURE 9 PMID:8599677

  7. Actin cross-link assembly and disassembly mechanics for alpha-Actinin and fascin.

    PubMed

    Courson, David S; Rock, Ronald S

    2010-08-20

    Self-assembly of complex structures is commonplace in biology but often poorly understood. In the case of the actin cytoskeleton, a great deal is known about the components that include higher order structures, such as lamellar meshes, filopodial bundles, and stress fibers. Each of these cytoskeletal structures contains actin filaments and cross-linking proteins, but the role of cross-linking proteins in the initial steps of structure formation has not been clearly elucidated. We employ an optical trapping assay to investigate the behaviors of two actin cross-linking proteins, fascin and alpha-actinin, during the first steps of structure assembly. Here, we show that these proteins have distinct binding characteristics that cause them to recognize and cross-link filaments that are arranged with specific geometries. alpha-Actinin is a promiscuous cross-linker, linking filaments over all angles. It retains this flexibility after cross-links are formed, maintaining a connection even when the link is rotated. Conversely, fascin is extremely selective, only cross-linking filaments in a parallel orientation. Surprisingly, bundles formed by either protein are extremely stable, persisting for over 0.5 h in a continuous wash. However, using fluorescence recovery after photobleaching and fluorescence decay experiments, we find that the stable fascin population can be rapidly competed away by free fascin. We present a simple avidity model for this cross-link dissociation behavior. Together, these results place constraints on how cytoskeletal structures assemble, organize, and disassemble in vivo. PMID:20551315

  8. 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.

  9. A Combination of Actin Treadmilling and Cross-Linking Drives Contraction of Random Actomyosin Arrays.

    PubMed

    Oelz, Dietmar B; Rubinstein, Boris Y; Mogilner, Alex

    2015-11-01

    We investigate computationally the self-organization and contraction of an initially random actomyosin ring. In the framework of a detailed physical model for a ring of cross-linked actin filaments and myosin-II clusters, we derive the force balance equations and solve them numerically. We find that to contract, actin filaments have to treadmill and to be sufficiently cross linked, and myosin has to be processive. The simulations reveal how contraction scales with mechanochemical parameters. For example, they show that the ring made of longer filaments generates greater force but contracts slower. The model predicts that the ring contracts with a constant rate proportional to the initial ring radius if either myosin is released from the ring during contraction and actin filaments shorten, or if myosin is retained in the ring, while the actin filament number decreases. We demonstrate that a balance of actin nucleation and compression-dependent disassembly can also sustain contraction. Finally, the model demonstrates that with time pattern formation takes place in the ring, worsening the contractile process. The more random the actin dynamics are, the higher the contractility will be. PMID:26536259

  10. Intrastrand cross-linked actin between Gln-41 and Cys-374. I. Mapping of sites cross-linked in F-actin by N-(4-azido-2-nitrophenyl) putrescine.

    PubMed

    Hegyi, G; Mák, M; Kim, E; Elzinga, M; Muhlrad, A; Reisler, E

    1998-12-22

    A new heterobifunctional photo-cross-linking reagent, N-(4-azido-2-nitrophenyl)-putrescine (ANP), was synthesized and covalently bound to Gln-41 of rabbit skeletal muscle actin by a bacterial transglutaminase-mediated reaction. Up to 1.0 mol of the reagent was incorporated per mole of G-actin; at least 90% of it was bound to Gln-41 while a minor fraction (about 8%) was attached to Gln-59. The labeled G-actin was polymerized, and the resulting F-actin was intermolecularly cross-linked by irradiation with UV light. The labeled and cross-linked peptides were isolated from either a complete or limited tryptic digest of cross-linked actin. In the limited digest the tryptic cleavage was restricted to arginine by succinylation of the lysyl residues. N-terminal sequencing and mass spectrometry indicated that the cross-linked peptides contained residues 40-50 (or 40-62 in the arginine limited digest) and residues 373-375, and that the actual cross-linking took place between Gln-41 and Cys-374. This latter finding was also supported by the inhibition of Cys-374 labeling with a fluorescent probe in the cross-linked actin. The dynamic length of ANP, between 11.1 and 12.5 A, constrains to that range the distance between the gamma-carboxyl group of Gln-41 in one monomer and the sulfur atom of Cys-374 in an adjacent monomer. This is consistent with the distances between these two residues on adjacent monomers of the same strand in the long-pitch helix in the structural models of F-actin [Holmes, K. C., Popp, D., Gebhard, W., and Kabsch, W. (1990) Nature 347, 44-49 and Lorenz, M., Popp, D., and Holmes, K. C. (1993) J. Mol. Biol. 234, 826-836]. The effect of cross-linking on the function of actin is described in the companion papers. PMID:9922144

  11. Motion in partially and fully cross-linked F-actin networks

    NASA Astrophysics Data System (ADS)

    Morris, Eliza; Ehrlicher, Allen; Weitz, David

    2012-02-01

    Single molecule experiments have measured stall forces and procession rates of molecular motors on isolated cytoskeletal fibers in Newtonian fluids. But in the cell, these motors are transporting cargo through a highly complex cytoskeletal network. To compare these single molecule results to the forces exerted by motors within the cell, an evaluation of the response of the cytoskeletal network is needed. Using magnetic tweezers and fluorescence confocal microscopy we observe and quantify the relationship between bead motion and filament response in F-actin networks both partially and fully cross-linked with filamin We find that when the transition from full to partial cross-linking is brought about by a decrease in cross-linker concentration there is a simultaneous decline in the elasticity of the network, but the response of the bead remains qualitatively similar. However, when the cross-linking is reduced through a shortening of the F-actin filaments the bead response is completely altered. The characteristics of the altered bead response will be discussed here.

  12. 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

  13. Stress Enhanced Gelation in α-Actinin-4 Cross-linked Actin Networks

    NASA Astrophysics Data System (ADS)

    Yao, Norman; Broedersz, Chase; Depken, Martin; Becker, Daniel; Pollak, Martin; Mackintosh, Frederick; Weitz, David

    2012-02-01

    A hallmark of biopolymer networks is their exquisite sensitivity to stress, demonstrated for example, by pronounced nonlinear elastic stiffening. Typically, they also yield under increased static load, providing a mechanism to achieve fluid-like behavior. In this talk, I will demonstrate an unexpected dynamical behavior in biopolymer networks consisting of F-actin cross-linked by a physiological actin binding protein, α-Actinin-4. Applied stress actually enhances gelation of these networks by delaying the onset of structural relaxation and network flow, thereby extending the regime of solid-like behavior to much lower frequencies. By using human kidney disease-associated mutant cross-linkers with varying binding affinities, we propose a molecular origin for this stress-enhanced gelation: It arises from the increased binding affinity of the cross-linker under load, characteristic of catch-bond-like behavior. This property may have important biological implications for intracellular mechanics, representing as it does a qualitatively new class of material behavior.

  14. Microtubule-Actin Cross-Linking Factor 1: Domains, Interaction Partners, and Tissue-Specific Functions.

    PubMed

    Goryunov, Dmitry; Liem, Ronald K H

    2016-01-01

    The cytoskeleton of most eukaryotic cells is composed of three principal filamentous components: actin filaments, microtubules (MTs), and intermediate filaments. It is a highly dynamic system that plays crucial roles in a wide range of cellular processes, including migration, adhesion, cytokinesis, morphogenesis, intracellular traffic and signaling, and structural flexibility. Among the large number of cytoskeleton-associated proteins characterized to date, microtubule-actin cross-linking factor 1 (MACF1) is arguably the most versatile integrator and modulator of cytoskeleton-related processes. MACF1 belongs to the plakin family of proteins, and within it, to the spectraplakin subfamily. These proteins are characterized by the ability to bridge MT and actin cytoskeletal networks in a dynamic fashion, which underlies their involvement in the regulation of cell migration, axonal extension, and vesicular traffic. Studying MACF1 functions has provided insights not only into the regulation of the cytoskeleton but also into molecular mechanisms of both normal cellular physiology and cellular pathology. Multiple MACF1 isoforms exist, composed of a large variety of alternatively spliced domains. Each of these domains mediates a specific set of interactions and functions. These functions are manifested in tissue and cell-specific phenotypes observed in conditional MACF1 knockout mice. The conditional models described to date reveal critical roles of MACF1 in mammalian skin, nervous system, heart muscle, and intestinal epithelia. Complete elimination of MACF1 is early embryonic lethal, indicating an essential role for MACF1 in early development. Further studies of MACF1 domains and their interactions will likely reveal multiple new roles of this protein in various tissues. PMID:26778566

  15. The actinin family of actin cross-linking proteins - a genetic perspective.

    PubMed

    Murphy, Anita C H; Young, Paul W

    2015-01-01

    Actinins are one of the major actin cross-linking proteins found in virtually all cell types and are the ancestral proteins of a larger family that includes spectrin, dystrophin and utrophin. Invertebrates have a single actinin-encoding ACTN gene, while mammals have four. Mutations in all four human genes have now been linked to heritable diseases or traits. ACTN1 mutations cause macrothrombocytopenia, a platelet disorder characterized by excessive bleeding. ACTN2 mutations have been linked to a range of cardiomyopathies, and ACTN4 mutations cause a kidney condition called focal segmental glomerulosclerosis. Intriguingly, approximately 16 % of people worldwide are homozygous for a nonsense mutation in ACTN3 that abolishes actinin-3 protein expression. This ACTN3 null allele has undergone recent positive selection in specific human populations, which may be linked to improved endurance and adaptation to colder climates. In this review we discuss the human genetics of the ACTN gene family, as well as ACTN gene knockout studies in several model organisms. Observations from both of these areas provide insights into the evolution and cellular functions of actinins. PMID:26312134

  16. 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.

  17. An affine continuum mechanical model for cross-linked F-actin networks with compliant linker proteins.

    PubMed

    Holzapfel, Gerhard A; Unterberger, Michael J; Ogden, Ray W

    2014-10-01

    Cross-linked actin networks are important building blocks of the cytoskeleton. In order to gain deeper insight into the interpretation of experimental data on actin networks, adequate models are required. In this paper we introduce an affine constitutive network model for cross-linked F-actin networks based on nonlinear continuum mechanics, and specialize it in order to reproduce the experimental behavior of in vitro reconstituted model networks. The model is based on the elastic properties of single filaments embedded in an isotropic matrix such that the overall properties of the composite are described by a free-energy function. In particular, we are able to obtain the experimentally determined shear and normal stress responses of cross-linked actin networks typically observed in rheometer tests. In the present study an extensive analysis is performed by applying the proposed model network to a simple shear deformation. The single filament model is then extended by incorporating the compliance of cross-linker proteins and further extended by including viscoelasticity. All that is needed for the finite element implementation is the constitutive model for the filaments, the linkers and the matrix, and the associated elasticity tensor in either the Lagrangian or Eulerian formulation. The model facilitates parameter studies of experimental setups such as micropipette aspiration experiments and we present such studies to illustrate the efficacy of this modeling approach. PMID:25043658

  18. A chemo-mechanical constitutive model for transiently cross-linked actin networks and a theoretical assessment of their viscoelastic behaviour.

    PubMed

    Fallqvist, B; Kroon, M

    2013-04-01

    Biological materials can undergo large deformations and also show viscoelastic behaviour. One such material is the network of actin filaments found in biological cells, giving the cell much of its mechanical stiffness. A theory for predicting the relaxation behaviour of actin networks cross-linked with the cross-linker α-actinin is proposed. The constitutive model is based on a continuum approach involving a neo-Hookean material model, modified in terms of concentration of chemically activated cross-links. The chemical model builds on work done by Spiros (Doctoral thesis, University of British Columbia, Vancouver, Canada, 1998) and has been modified to respond to mechanical stress experienced by the network. The deformation is split into a viscous and elastic part, and a thermodynamically motivated rate equation is assigned for the evolution of viscous deformation. The model predictions were evaluated for stress relaxation tests at different levels of strain and found to be in good agreement with experimental results for actin networks cross-linked with α-actinin. PMID:22623110

  19. Actin bundles cross-linked with [Formula: see text]-actinin studied by nanobeam X-ray diffraction.

    PubMed

    Töpperwien, M; Priebe, M; Salditt, T

    2016-07-01

    We have performed scanning nano-beam small-angle X-ray scattering (nano-SAXS) experiments on in vitro-formed actin filaments cross-linked with [Formula: see text]-actinin. The experimental method combines a high resolution in reciprocal space with a real space resolution as given by the spot-size of the nano-focused X-ray beam, and opens up new opportunities to study local super-molecular structures of actin filaments. In this first proof-of-concept, we show that the local orientation of actin bundles formed by the cross-linking can be visualized by the X-ray darkfield maps. The filament bundles give rise to highly anisotropic diffraction patterns showing distinct streaks perpendicular to the bundle axes. Interestingly, some diffraction patterns exhibit a fine structure in the form of intensity modulations allowing for a more detailed analysis of the order within the bundles. A first empirical quantification of these modulations is included in the present work. PMID:26715112

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

    PubMed

    Bershitsky, S Y; Tsaturyan, A K

    1995-09-01

    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

  1. Hierarchical Cross-linked F-actin Networks: Understanding Structure and Assembly

    NASA Astrophysics Data System (ADS)

    Hirst, Linda; Nguyen, Lam

    2009-11-01

    The protein, F-actin provides us with an interesting system in which to investigate the assembly properties of semi-flexible filaments in the presence of cross-linkers. Recently it was observed that F-actin, in the presence of the cross-linker alpha-actinin at high molar ratios will generate a novel hierarchical network of filament bundles. We investigate this system using coarse-grained molecular dynamics (MD) simulation, confocal microscopy and x-ray scattering. We have studied the F-actin/alpha-actinin system in detail with different actin conc. (C) and alpha-actinin/actin molar ratios (gamma). Confocal microscopy and analysis shows that the assembled systems fall into one of 3 phases depending on C and gamma: (1) loosely connected network of F-actin and bundles, (2) loosely connected network of dense domains and (3) uniform network of bundles. This can be explained and replicated using MD simulation. We have also examined different types of cross-linkers to represent the proteins, fascin and filamin. Results show that phase formation is related to the flexibility in binding between F-actin and cross-linkers. This degree of freedom, possible with longer cross-linkers allows the formation of branch points and thus bundle networks.

  2. β1 and β3 Integrins Cooperate to Induce Syndecan-4-Containing Cross-linked Actin Networks in Human Trabecular Meshwork Cells

    PubMed Central

    Filla, Mark S.; Woods, Anne; Kaufman, Paul L.; Peters, Donna M.

    2006-01-01

    Purpose To characterize the molecular composition of cross-linked actin networks (CLANs) and the regulation of their formation by integrins in normal human trabecular meshwork (TM) cells. CLANs have been observed in steroid-treated and glaucomatous TM cells and have been suggested to contribute to decreased outflow facility by altering the contractility of the TM. Methods Immunofluorescence microscopy was used to identify molecular components of CLANs and quantitate CLAN formation in HTM cells plated on coverslips coated with various extracellular matrix (ECM) proteins (fibronectin, types I and IV collagen, and vitronectin), vascular cell adhesion molecule (VCAM)-1, or activating antibodies against β1, β3, or α2β1 integrins. These integrin antibodies were also used as soluble ligands. Results CLAN vertices contained the actin-binding proteins α-actinin and filamin and the signaling molecules syndecan-4 and PIP2. CLANs lacked Arp3 and cortactin. CLAN formation was dependent on the ECM substrate and was significantly higher on fibronectin and VCAM-1 compared with vitronectin, types I or IV collagen. Adsorbed β1 integrin antibodies also induced CLANs, whereas adsorbed β3 or α2β1 integrin antibodies did not. Soluble β3 integrin antibodies, however, induced CLANs and actually enhanced CLAN formation in cells spread on fibronectin, VCAM-1, type I or type IV collagen, or β1 integrin antibodies. Conclusions CLANs are unique actin-branched networks whose formation can be regulated by β1 and β3 integrin signaling pathways. Thus, integrin-mediated signaling events can modulate the organization of the actin cytoskeleton in TM cells and hence could participate in regulating cytoskeletal events previously demonstrated to be involved in controlling outflow facility. PMID:16639003

  3. Viability, Apoptosis, Proliferation, Activation, and Cytokine Secretion of Human Keratoconus Keratocytes after Cross-Linking

    PubMed Central

    Stachon, Tanja; Wang, Jiong; Seitz, Berthold; Szentmáry, Nóra

    2015-01-01

    Purpose. The purpose of this study was to determine the impact of cross-linking (CXL) on viability, apoptosis, proliferation, activation, and cytokine secretion of human keratoconus (KC) keratocytes, in vitro. Methods. Primary KC keratocytes were cultured in DMEM/Ham's F12 medium supplemented with 10% FCS and underwent UVA illumination (370 nm, 2 J/cm2) during exposure to 0.1% riboflavin and 20% Dextran in PBS. Twenty-four hours after CXL, viability was assessed using Alamar blue assay; apoptosis using APO-DIRECT Kit; proliferation using ELISA-BrdU kit; and CD34 and alpha-smooth muscle actin (α-SMA) expression using flow cytometry. Five and 24 hours after CXL, FGFb, HGF, TGFβ1, VEGF, KGF, IL-1β, IL-6, and IL-8 secretion was measured using enzyme-linked-immunoabsorbent assay (ELISA). Results. Following CXL, cell viability and proliferation decreased (P < 0.05; P = 0.009), the percentage of apoptotic keratocytes increased (P < 0.05) significantly, and CD34 and α-SMA expression remained unchanged (P > 0.06). Five hours after CXL, FGFb secretion increased significantly (P = 0.037); however no other cytokine secretion differed significantly from controls after 5 or 24 hours (P > 0.12). Conclusions. Cross-linking decreases viability, triggers apoptosis, and inhibits proliferation, without an impact on multipotent hematopoietic stem cell transformation and myofibroblastic transformation of KC keratocytes. CXL triggers FGFb secretion of KC keratocytes transiently (5 hours), normalizing after 24 hours. PMID:25699261

  4. 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

  5. Bcr is a substrate for Transglutaminase 2 cross-linking activity

    PubMed Central

    2011-01-01

    Background Breakpoint cluster region (Bcr) is a multi-domain protein that contains a C-terminal GTPase activating protein (GAP) domain for Rac. Transglutaminase 2 (TG2) regulates Bcr by direct binding to its GAP domain. Since TG2 has transglutaminase activity that has been implicated in the response to extreme stress, we investigated if Bcr can also act as a substrate for TG2. Results We here report that activation of TG2 by calcium caused the formation of covalently cross-linked Bcr. Abr, a protein related to Bcr but lacking its N-terminal oligomerization domain, was not cross-linked by TG2 even though it forms a complex with it. A Bcr mutant missing the first 62 amino acid residues remained monomeric in the presence of activated TG2, showing that this specific domain is necessary for the cross-linking reaction. Calcium influx induced by a calcium ionophore in primary human endothelial cells caused cross-linking of endogenous Bcr, which was inhibited by the TG2 inhibitor cystamine. Treatment of cells with cobalt chloride, a hypoxia-mimetic that causes cellular stress, also generated high molecular weight Bcr complexes. Cross-linked Bcr protein appeared in the TritonX-100-insoluble cell fraction and further accumulated in cells treated with a proteasome inhibitor. Conclusions Bcr thus represents both an interacting partner under non-stressed conditions and a target of transglutaminase activity for TG2 during extreme stress. PMID:21310073

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

    PubMed Central

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

    2007-01-01

    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. PMID:17873062

  7. 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

  8. Cross-linking and modification of cytochrome c with redox-active metal complexes

    SciTech Connect

    Lukes, A.

    1991-05-02

    This thesis consists of two parts. The first part shows that a redox-active trinuclear metal cluster may be used as a cross-linking reagent for proteins. Electron transfer is observed in the protein oligomers. The second part involves labelling the cysteine residue of baker's yeast cytochrome c with chloromercuriferrocene. Chloromercuriferrocene reacts with cytochrome c in two interesting ways. Symmetrization produces two products; two proteins cross-linked with mercury and diferrocenylmercury. Simple substitution of FeHgCl onto the protein followed by the addition of a proton by electrophilic substitution affords ferrocene and the mercuric chloride modified protein. 16 refs., 3 figs.

  9. Activation of polymeric materials towards enzymatic postgrafting and cross-linking.

    PubMed

    Fatarella, E; Ciabatti, I; Cortez, J

    2012-10-10

    A methodology to activate inert polymeric materials to enzymatic functionalisation is described herein. Plasma irradiation can be used to graft compounds containing a moiety that is reactive towards an enzyme of interest. Subsequently, such enzyme can be used to either postgraft functional compounds or cross-link the polymeric materials. Argon plasma was utilised to graft 2-aminoethyl methacrylate onto cotton and wool fibres, introducing surface alkylamine groups to impart reactivity towards transglutaminase and tyrosinase. The efficiency of plasma grafting was verified by ATR-FTIR. Enzyme postgrafting of fluorescent peptides coupled with confocal microscopy was used to demonstrate transglutaminase activity towards cotton, a material typically inert to this enzyme. The grafting of alkylamines onto wool resulted in additional cross-linking by both enzymes, leading to significantly increased yarn breaking load and elongation at break. This technology permits the activation of inert materials towards enzymatic postgrafting, with applications in fields as diverse as textiles and biomaterials. PMID:22975121

  10. Modulation of UvrD helicase activity by covalent DNA-protein cross-links.

    PubMed

    Kumari, Anuradha; Minko, Irina G; Smith, Rebecca L; Lloyd, R Stephen; McCullough, Amanda K

    2010-07-01

    UvrD (DNA helicase II) has been implicated in DNA replication, DNA recombination, nucleotide excision repair, and methyl-directed mismatch repair. The enzymatic function of UvrD is to translocate along a DNA strand in a 3' to 5' direction and unwind duplex DNA utilizing a DNA-dependent ATPase activity. In addition, UvrD interacts with many other proteins involved in the above processes and is hypothesized to facilitate protein turnover, thus promoting further DNA processing. Although UvrD interactions with proteins bound to DNA have significant biological implications, the effects of covalent DNA-protein cross-links on UvrD helicase activity have not been characterized. Herein, we demonstrate that UvrD-catalyzed strand separation was inhibited on a DNA strand to which a 16-kDa protein was covalently bound. Our sequestration studies suggest that the inhibition of UvrD activity is most likely due to a translocation block and not helicase sequestration on the cross-link-containing DNA substrate. In contrast, no inhibition of UvrD-catalyzed strand separation was apparent when the protein was linked to the complementary strand. The latter result is surprising given the earlier observations that the DNA in this covalent complex is severely bent ( approximately 70 degrees ), with both DNA strands making multiple contacts with the cross-linked protein. In addition, UvrD was shown to be required for replication of plasmid DNAs containing covalent DNA-protein complexes. Combined, these data suggest a critical role for UvrD in the processing of DNA-protein cross-links. PMID:20444702

  11. Development of Long-Circulating Zwitterionic Cross-Linked Micelles for Active-Targeted Drug Delivery.

    PubMed

    Lin, Weifeng; Ma, Guanglong; Kampf, Nir; Yuan, Zhefan; Chen, Shengfu

    2016-06-13

    Blood stability, active targeting, and controlled drug release are the most important features to design desirable drug carriers. Here, we demonstrate a zwitterionic biodegradable cross-linked micelle based on a penta-block copolymer, which utilizes poly(carboxybetaine methacrylate) as hydrophilic segment, poly(ε-caprolactone) as biodegradable hydrophobic segment, poly(S-2-hydroxyethyl-O-ethyl dithiocarbonate methacrylate) (PSODMA) block as thiol protecting segment for cross-linking, and cyclic Arg-Gly-Asp-d-Tyr-Lys [c(RGDyK)] as targeting ligand. As a result, this micelle possessed excellent colloidal stability at high dilution and in 50% fetal bovine serum. In vitro drug release experiment showed no burst release under physiological conditions but accelerated drug release in mimicking tumor tissue environment. In vivo tests showed that the drug-loaded micelles had prolonged half-life in bloodstream, enhanced therapeutic efficiency, and reduced cardiac toxicity and biotoxicity compared with free drug formulation. Taken together, the reported c(RGDyK)-modified zwitterionic interfacially cross-linked micelle has emerged as an appealing platform for cancer therapy. PMID:27050797

  12. Interdomain conformational changes in Akt activation revealed by chemical cross-linking and tandem mass spectrometry.

    PubMed

    Huang, Bill X; Kim, Hee-Yong

    2006-06-01

    Akt, a serine/threonine kinase, plays a critical role in cell survival. Upon growth factor receptor stimulation, cytosolic Akt is recruited to the plasma membrane by phospholipid binding and activated through phosphorylation at Thr(308) and Ser(473). Although crystal structures for the parts of Akt have been reported, neither the three-dimensional structure of the whole molecule nor sequential conformational changes during activation have been demonstrated. In this study, we demonstrated that Akt undergoes dramatic interdomain conformational changes during activation processes by probing the three-dimensional structure of full-length Akt in solution using chemical cross-linking and tandem mass spectrometry. The cross-linking results not only provided new structural information but also revealed distinctive spatial arrangements of individual domains in the Akt molecule in resting, membrane-interacted, phosphorylated, and substrate-bound states. Our data allowed a new model for stepwise interdomain conformational changes in Akt activation sequence, setting a stage for the further investigation on Akt-membrane, Akt-protein, and/or Akt-drug interactions in solution to understand molecular mechanisms involved in physiological and pathophysiological processes of cell survival. PMID:16531397

  13. 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

  14. A bifunctional monocyclic beta-lactam cross-links across the active site of beta-lactamase.

    PubMed

    Ahluwalia, R; Day, R A; Nauss, J

    1995-01-17

    A 4-alkoxy-2-azetidinone behaves as a bifunctional active site-directed inhibitor of the class A beta-lactamase from Bacillus cereus 569/H. It cross-links SER 70 and LYS 234 as it binds in a approximately 1:1 ratio. The cross-linked enzyme is irreversibly inhibited while the secondary structure is partially stabilized under conditions when the native enzyme is otherwise converted to a form with no detectable secondary structure by circular dichroism. PMID:7826374

  15. Caught in the act: covalent cross-linking captures activator-coactivator interactions in vivo.

    PubMed

    Krishnamurthy, Malathy; Dugan, Amanda; Nwokoye, Adaora; Fung, Yik-Hong; Lancia, Jody K; Majmudar, Chinmay Y; Mapp, Anna K

    2011-12-16

    Currently there are few methods suitable for the discovery and characterization of transient, moderate affinity protein-protein interactions in their native environment, despite their prominent role in a host of cellular functions including protein folding, signal transduction, and transcriptional activation. Here we demonstrate that a genetically encoded photoactivatable amino acid, p-benzoyl-l-phenylalanine, can be used to capture transient and/or low affinity binding partners in an in vivo setting. In this study, we focused on ensnaring the coactivator binding partners of the transcriptional activator VP16 in S. cerevisiae. The interactions between transcriptional activators and coactivators in eukaryotes are moderate in affinity and short-lived, and due in part to these characteristics, identification of the direct binding partners of activators in vivo has met with only limited success. We find through in vivo photo-cross-linking that VP16 contacts the Swi/Snf chromatin-remodeling complex through the ATPase Snf2(BRG1/BRM) and the subunit Snf5 with two distinct regions of the activation domain. An analogous experiment with Gal4 reveals that Snf2 is also a target of this activator. These results suggest that Snf2 may be a valuable target for small molecule probe discovery given the prominent role the Swi/Snf complex family plays in development and in disease. More significantly, the successful implementation of the in vivo cross-linking methodology in this setting demonstrates that it can be applied to the discovery and characterization of a broad range of transient and/or modest affinity protein-protein interactions. PMID:21977905

  16. Cross-linked enzyme aggregates (CLEAs) of Pencilluim notatum lipase enzyme with improved activity, stability and reusability characteristics.

    PubMed

    Rehman, Saima; Bhatti, Haq Nawaz; Bilal, Muhammad; Asgher, Muhammad

    2016-10-01

    Cross-linked enzyme aggregates (CLEAs) are considered as an effective tool for the immobilization of enzyme. In this study, Pencillium notatum lipase (PNL) was immobilized as carrier free cross-linked enzyme aggregates using glutaraldehyde (GLA) and Ethylene glycol-bis [succinic acid N-hydroxysuccinimide] (EG-NHS) as cross-linking agents. The optimal conditions for the synthesis of an efficient lipase CLEAs such as precipitant type, the nature and amount of cross-linking reagent, and cross-linking time were optimized. The recovered activities of CLEAs were considerably dependent on the concentration of GLA; however, the activity recovery was not severely affected by EG-NHS as a mild cross-linker. The EG-NHS aggregates displayed superior hydrolytic (52.08±2.52%) and esterification (64.42%) activities as compared to GLA aggregates which showed 23.8±1.86 and 34.54% of hydrolytic and esterification activity, respectively. Morphological analysis by fluorescence and scanning electron microscope revealed that EG-NHS aggregates were smaller in size with larger surface area compared to GLA aggregates. The pH optima of both types of CLEAs were displaced to slightly alkaline region and higher temperature as compared to native enzyme. Highest enzyme activity of CLEAs was achieved at the pH of 9.0 and 42°C temperature. Moreover, a significant improvement in the thermal resistance was also recorded after immobilization. After ten reusability cycles in aqueous medium, GLA and EG-NHS cross-linked lipase CLEAs preserved 63.62% and 70.9% of their original activities, respectively. The results suggest that this novel CLEA-lipase is potentially usable in many industrial applications. PMID:27365121

  17. Codelivery of Doxorubicin and Paclitaxel by Cross-Linked Multilamellar Liposome Enables Synergistic Antitumor Activity

    PubMed Central

    2015-01-01

    Combining chemotherapeutics is a promising method of improving cancer treatment; however, the clinical success of combination therapy is limited by the distinct pharmacokinetics of combined drugs, which leads to nonuniform distribution. In this study, we report a new robust approach to load two drugs with different hydrophilicities into a single cross-linked multilamellar liposomal vesicle (cMLV) to precisely control the drug ratio that reaches the tumor in vivo. The stability of cMLVs improves the loading efficiency and sustained release of doxorubicin (Dox) and paclitaxel (PTX), maximizing the combined therapeutic effect and minimizing the systemic toxicity. Furthermore, we show that the cMLV formulation maintains specific drug ratios in vivo for over 24 h, enabling the ratio-dependent combination synergy seen in vitro to translate to in vivo antitumor activity and giving us control over another parameter important to combination therapy. This combinatorial delivery system may provide a new strategy for synergistic delivery of multiple chemotherapeutics with a ratiometric control over encapsulated drugs to treat cancer and other diseases. PMID:24673622

  18. Curcumin Cross-links Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) Polypeptides and Potentiates CFTR Channel Activity by Distinct Mechanisms*

    PubMed Central

    Bernard, Karen; Wang, Wei; Narlawar, Rajeshwar; Schmidt, Boris; Kirk, Kevin L.

    2009-01-01

    Cystic fibrosis (CF) is caused by loss-of-function mutations in the CFTR chloride channel. Wild type and mutant CFTR channels can be activated by curcumin, a well tolerated dietary compound with some appeal as a prospective CF therapeutic. However, we show here that curcumin has the unexpected effect of cross-linking CFTR polypeptides into SDS-resistant oligomers. This effect occurred for CFTR channels in microsomes as well as in intact cells and at the same concentrations that are effective for promoting CFTR channel activity (5–50 μm). Both mature CFTR polypeptides at the cell surface and immature CFTR protein in the endoplasmic reticulum were cross-linked by curcumin, although the latter pool was more susceptible to this modification. Curcumin cross-linked two CF mutant channels (ΔF508 and G551D) as well as a variety of deletion constructs that lack the major cytoplasmic domains. In vitro cross-linking could be prevented by high concentrations of oxidant scavengers (i.e. reduced glutathione and sodium azide) indicating a possible oxidation reaction with the CFTR polypeptide. Importantly, cyclic derivatives of curcumin that lack the reactive β diketone moiety had no cross-linking activity. One of these cyclic derivatives stimulated the activities of wild type CFTR channels, Δ1198-CFTR channels, and G551D-CFTR channels in excised membrane patches. Like the parent compound, the cyclic derivative irreversibly activated CFTR channels in excised patches during prolonged exposure (>5 min). Our results raise a note of caution about secondary biochemical effects of reactive compounds like curcumin in the treatment of CF. Cyclic curcumin derivatives may have better therapeutic potential in this regard. PMID:19740743

  19. Cisplatin-loaded core cross-linked micelles: comparative pharmacokinetics, antitumor activity, and toxicity in mice

    PubMed Central

    Oberoi, Hardeep S; Nukolova, Natalia V; Laquer, Frederic C; Poluektova, Larisa Y; Huang, Jiangeng; Alnouti, Yazen; Yokohira, Masanao; Arnold, Lora L; Kabanov, Alexander V; Cohen, Samuel M; Bronich, Tatiana K

    2012-01-01

    Polymer micelles with cross-linked ionic cores are shown here to improve the therapeutic performance of the platinum-containing anticancer compound cisplatin. Biodistribution, antitumor efficacy, and toxicity of cisplatin-loaded core cross-linked micelles of poly(ethylene glycol)-b-poly(methacrylic acid) were evaluated in a mouse ovarian cancer xenograft model. Cisplatin-loaded micelles demonstrated prolonged blood circulation, increased tumor accumulation, and reduced renal exposure. Improved antitumor response relative to free drug was seen in a mouse model. Toxicity studies with cisplatin-loaded micelles indicate a significantly improved safety profile and lack of renal abnormalities typical of free cisplatin treatment. Overall, the study supports the fundamental possibility of improving the potential of platinum therapy using polymer micelle-based drug delivery. PMID:22745537

  20. ELMO recruits actin cross-linking family 7 (ACF7) at the cell membrane for microtubule capture and stabilization of cellular protrusions.

    PubMed

    Margaron, Yoran; Fradet, Nadine; Côté, Jean-François

    2013-01-11

    ELMO and DOCK180 proteins form an evolutionarily conserved module controlling Rac GTPase signaling during cell migration, phagocytosis, and myoblast fusion. Here, we identified the microtubule and actin-binding spectraplakin ACF7 as a novel ELMO-interacting partner. A C-terminal polyproline segment in ELMO and the last spectrin repeat of ACF7 mediate a direct interaction between these proteins. Co-expression of ELMO1 with ACF7 promoted the formation of long membrane protrusions during integrin-mediated cell spreading. Quantification of membrane dynamics established that coupling of ELMO and ACF7 increases the persistence of the protruding activity. Mechanistically, we uncovered a role for ELMO in the recruitment of ACF7 to the membrane to promote microtubule capture and stability. Functionally, these effects of ELMO and ACF7 on cytoskeletal dynamics required the Rac GEF DOCK180. In conclusion, our findings support a role for ELMO in protrusion stability by acting at the interface between the actin cytoskeleton and the microtubule network. PMID:23184944

  1. Enhanced esterification activity through interfacial activation and cross-linked immobilization mechanism of Rhizopus oryzae lipase in a nonaqueous medium.

    PubMed

    Kartal, Funda

    2016-07-01

    Interfacial activation via surfactant (Tween 80, Triton X-100) treatment was conducted to improve the esterification activity of Rhizopus oryzae lipase that had undergone immobilization through cross-linked enzyme aggregates (CLEA®) technique. Surfactant pretreated immobilized enzymes exhibited better esterification activity compared to free and non-pretreated immobilized enzyme (Control CLEAs) since higher conversion rates were obtained within shorter times. The superiority of surfactant pretreated CLEAs, especially Tween 80 pretreated CLEAs (T 80 PT CLEAs), were clearly pronounced when longer alcohols were used as substrates. Conversion values exceeded 90% for octyl octanoate, oleyl octanoate and oleyl oleate synthesis with T 80 PT CLEAs whereas Control CLEAs and free enzyme showed no activity. Maximum conversions were achieved in the case equal molars of the substrates or in the case excess of the alcohol to acid in cyclohexane. In solvent free medium containing equal molars of substrates the conversion rates were 85% and 87% with T 80 PT CLEAs respectively for octyl octanoate and oleyl oleate within 2 hours. T 80 PT CLEAs showed 59% of its original activity after 7 consecutive usage for oleyl oleate synthesis. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 32:899-904, 2016. PMID:27111483

  2. Dynamics of active actin networks

    NASA Astrophysics Data System (ADS)

    Koehler, Simone

    2014-03-01

    Local mechanical and structural properties of a eukaryotic cell are determined by its cytoskeleton. To adapt to their environment, cells rely on constant self-organized rearrangement processes of their actin cytoskeleton. To shed light on the principles underlying these dynamic self-organization processes we investigate a minimal reconstituted active system consisting of actin filaments, crosslinking molecules and molecular motor filaments. Using quantitative fluorescence microscopy and image analysis, we show, that these minimal model systems exhibit a generic structure formation mechanism. The competition between force generation by molecular motors and the stabilization of the network by crosslinking proteins results in a highly dynamic reorganization process which is characterized by anomalous transport dynamics with a superdiffusive behavior also found in intracellular dynamics. In vitro, these dynamics are governed by chemical and physical parameters that alter the balance of motor and crosslinking proteins, such as pH. These findings can be expected to have broad implications in our understanding of cytoskeletal regulation in vivo.

  3. A syndecan-4 binding peptide derived from laminin 5 uses a novel PKCε pathway to induce cross-linked actin network (CLAN) formation in human trabecular meshwork (HTM) cells.

    PubMed

    Filla, Mark S; Clark, Ross; Peters, Donna M

    2014-10-01

    In this study, we examined the role(s) of syndecan-4 in regulating the formation of an actin geodesic dome structure called a cross-linked actin network (CLAN) in which syndecan-4 has previously been localized. CLANs have been described in several different cell types, but they have been most widely studied in human trabecular meshwork (HTM) cells where they may play a key role in controlling intraocular pressure by regulating aqueous humor outflow from the eye. In this study we show that a loss of cell surface synedcan-4 significantly reduces CLAN formation in HTM cells. Analysis of HTM cultures treated with or without dexamethasone shows that laminin 5 deposition within the extracellular matrix is increased by glucocorticoid treatment and that a laminin 5-derived, syndecan-4-binding peptide (PEP75), induces CLAN formation in TM cells. This PEP75-induced CLAN formation was inhibited by heparin and the broad spectrum PKC inhibitor Ro-31-7549. In contrast, the more specific PKCα inhibitor Gö 6976 had no effect, thus excluding PKCα as a downstream effector of syndecan-4 signaling. Analysis of PKC isozyme expression showed that HTM cells also expressed both PKCγ and PKCε. Cells treated with a PKCε agonist formed CLANs while a PKCα/γ agonist had no effect. These data suggest that syndecan-4 is essential for CLAN formation in HTM cells and that a novel PKCε-mediated signaling pathway can regulate formation of this unique actin structure. PMID:25128150

  4. A syndecan-4 binding peptide derived from laminin 5 uses a novel PKCε pathway to induce cross-linked actin network (CLAN) formation in human trabecular meshwork (HTM) cells

    PubMed Central

    Filla, Mark S.; Clark, Ross; Peters, Donna M.

    2014-01-01

    In this study, we examined the role(s) of syndecan-4 in regulating the formation of an actin geodesic dome structure called a cross-linked actin network (CLAN) in which syndecan-4 has previously been localized. CLANs have been described in several different cell types, but they have been most widely studied in human trabecular meshwork (HTM) cells where they may play a key role in controlling intraocular pressure by regulating aqueous humor outflow from the eye. In this study we show that a loss of cell surface synedcan-4 significantly reduces CLAN formation in HTM cells. Analysis of HTM cultures treated with or without dexamethasone shows that laminin 5 deposition within the extracellular matrix is increased by glucocorticoid treatment and that a laminin 5-derived, syndecan-4-binding peptide (PEP75), induces CLAN formation in TM cells. This PEP75-induced CLAN formation was inhibited by heparin and the broad spectrum PKC inhibitor Ro-31-7549. In contrast, the more specific PKCα inhibitor Go 6976 had no effect, thus excluding PKCα as a downstream effector of syndecan-4 signaling. Analysis of PKC isozyme expression showed that HTM cells also expressed both PKCγ and PKCε. Cells treated with a PKCε agonist formed CLANs while a PKCα/γ agonist had no effect. These data suggest that syndecan-4 is essential for CLAN formation in HTM cells and that a novel PKCε-mediated signaling pathway can regulate formation of this unique actin structure. PMID:25128150

  5. 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

  6. Constraining the Lateral Helix of Respiratory Complex I by Cross-linking Does Not Impair Enzyme Activity or Proton Translocation.

    PubMed

    Zhu, Shaotong; Vik, Steven B

    2015-08-21

    Complex I (NADH:ubiquinone oxidoreductase) is a multisubunit, membrane-bound enzyme of the respiratory chain. The energy from NADH oxidation in the peripheral region of the enzyme is used to drive proton translocation across the membrane. One of the integral membrane subunits, nuoL in Escherichia coli, has an unusual lateral helix of ∼75 residues that lies parallel to the membrane surface and has been proposed to play a mechanical role as a piston during proton translocation (Efremov, R. G., Baradaran, R., and Sazanov, L. A. (2010) Nature 465, 441-445). To test this hypothesis we have introduced 11 pairs of cysteine residues into Complex I; in each pair one is in the lateral helix, and the other is in a nearby region of subunit N, M, or L. The double mutants were treated with Cu(2+) ions or with bi-functional methanethiosulfonate reagents to catalyze cross-link formation in membrane vesicles. The yields of cross-linked products were typically 50-90%, as judged by immunoblotting, but in no case did the activity of Complex I decrease by >10-20%, as indicated by deamino-NADH oxidase activity or rates of proton translocation. In contrast, several pairs of cysteine residues introduced at other interfaces of N:M and M:L subunits led to significant loss of activity, in particular, in the region of residue Glu-144 of subunit M. The results do not support the hypothesis that the lateral helix of subunit L functions like a piston, but rather, they suggest that conformational changes might be transmitted more directly through the functional residues of the proton translocation apparatus. PMID:26134569

  7. Plastins regulate ectoplasmic specialization via its actin bundling activity on microfilaments in the rat testis.

    PubMed

    Li, Nan; Wong, Chris Kc; Cheng, C Yan

    2016-01-01

    Plastins are a family of actin binding proteins (ABPs) known to cross-link actin microfilaments in mammalian cells, creating actin microfilament bundles necessary to confer cell polarity and cell shape. Plastins also support cell movement in response to changes in environment, involved in cell/tissue growth and development. They also confer plasticity to cells and tissues in response to infection or other pathological conditions (e.g., inflammation). In the testis, the cell-cell anchoring junction unique to the testis that is found at the Sertoli cell-cell interface at the blood-testis barrier (BTB) and at the Sertoli-spermatid (e.g., 8-19 spermatids in the rat testis) is the basal and the apical ectoplasmic specialization (ES), respectively. The ES is an F-actin-rich anchoring junction constituted most notably by actin microfilament bundles. A recent report using RNAi that specifically knocks down plastin 3 has yielded some insightful information regarding the mechanism by which plastin 3 regulates the status of actin microfilament bundles at the ES via its intrinsic actin filament bundling activity. Herein, we provide a brief review on the role of plastins in the testis in light of this report, which together with recent findings in the field, we propose a likely model by which plastins regulate ES function during the epithelial cycle of spermatogenesis via their intrinsic activity on actin microfilament organization in the rat testis. PMID:26608945

  8. Plastins regulate ectoplasmic specialization via its actin bundling activity on microfilaments in the rat testis

    PubMed Central

    Li, Nan; Wong, Chris KC; Cheng, C Yan

    2016-01-01

    Plastins are a family of actin binding proteins (ABPs) known to cross-link actin microfilaments in mammalian cells, creating actin microfilament bundles necessary to confer cell polarity and cell shape. Plastins also support cell movement in response to changes in environment, involved in cell/tissue growth and development. They also confer plasticity to cells and tissues in response to infection or other pathological conditions (e.g., inflammation). In the testis, the cell-cell anchoring junction unique to the testis that is found at the Sertoli cell-cell interface at the blood-testis barrier (BTB) and at the Sertoli-spermatid (e.g., 8–19 spermatids in the rat testis) is the basal and the apical ectoplasmic specialization (ES), respectively. The ES is an F-actin-rich anchoring junction constituted most notably by actin microfilament bundles. A recent report using RNAi that specifically knocks down plastin 3 has yielded some insightful information regarding the mechanism by which plastin 3 regulates the status of actin microfilament bundles at the ES via its intrinsic actin filament bundling activity. Herein, we provide a brief review on the role of plastins in the testis in light of this report, which together with recent findings in the field, we propose a likely model by which plastins regulate ES function during the epithelial cycle of spermatogenesis via their intrinsic activity on actin microfilament organization in the rat testis. PMID:26608945

  9. Colchicine activates actin polymerization by microtubule depolymerization.

    PubMed

    Jung, H I; Shin, I; Park, Y M; Kang, K W; Ha, K S

    1997-06-30

    Swiss 3T3 fibroblasts were treated with the microtubule-disrupting agent colchicine to study any interaction between microtubule dynamics and actin polymerization. Colchicine increased the amount of filamentous actin (F-actin), in a dose- and time-dependent manner with a significant increase at 1 h by about 130% over control level. Confocal microscopic observation showed that colchicine increased F-actin contents by stress fiber formation without inducing membrane ruffling. Colchicine did not activate phospholipase C and phospholipase D, whereas lysophosphatidic acid did, indicating that colchicine may have a different mechanism of actin polymerization regulation from LPA. A variety of microtubule-disrupting agents stimulated actin polymerization in Swiss 3T3 and Rat-2 fibroblasts as did colchicine, but the microtubule-stabilizing agent taxol inhibited actin polymerization induced by the above microtubule-disrupting agents. In addition, colchicine-induced actin polymerization was blocked by two protein phosphatase inhibitors, okadaic acid and calyculin A. These results suggest that microtubule depolymerization activates stress fiber formation by serine/threonine dephosphorylation in fibroblasts. PMID:9264034

  10. Corneal cross-linking.

    PubMed

    Randleman, J Bradley; Khandelwal, Sumitra S; Hafezi, Farhad

    2015-01-01

    Since its inception in the late 1990s, corneal cross-linking has grown from an interesting concept to a primary treatment for corneal ectatic disease worldwide. Using a combination of ultraviolet-A light and a chromophore (vitamin B2, riboflavin), the cornea can be stiffened, usually with a single application, and progressive thinning diseases such as keratoconus arrested. Despite being in clinical use for many years, some of the underlying processes, such as the role of oxygen and the optimal treatment times, are still being worked out. More than a treatment technique, corneal cross-links represent a physiological principle of connective tissue, which may explain the enormous versatility of the method. We highlight the history of corneal cross-linking, the scientific underpinnings of current techniques, evolving clinical treatment parameters, and the use of cross-linking in combination with refractive surgery and for the treatment of infectious keratitis. PMID:25980780

  11. Wear, debris, and biologic activity of cross-linked polyethylene in the knee: benefits and potential concerns.

    PubMed

    Fisher, John; McEwen, Hannah M J; Tipper, Joanne L; Galvin, Alison L; Ingram, Jo; Kamali, Amir; Stone, Martin H; Ingham, Eileen

    2004-11-01

    Cross-linked polyethylene currently is being introduced in knee prostheses. The wear rates, wear debris, and biologic reactivity of non cross-linked, moderately cross-linked, and highly cross-linked polyethylene have been compared in multidirectional wear tests and knee simulators. Multidirectional pin-on-plate wear studies of noncross-linked, moderately cross-linked (5 Mrad), and highly cross-linked (10 Mrad) polyethylene showed a 75% reduction in wear with the highly cross-linked material under kinematics found in the hip, but only a 33% reduction under wear in kinematics representative of the knee. In knee simulator studies, with the fixed-bearing press-fit, condylar Sigma cruciate-retaining knee under high kinematic input conditions, the wear of 5 Mrad moderately cross-linked polyethylene was 13 +/- 4 mm per 1 million cycles, which was lower (p < 0.05) than the wear of clinically used, gamma vacuum foil GUR 1020 polyethylene (23 +/- 6 mm/1 million cycles). For the low-contact stress mobile-bearing knee, the wear of moderately cross-linked polyethylene was 2 +/- 1 mm per 1 million cycles, which was lower (p < 0.05) than GVF GUR 1020 polyethylene (5 +/- 2 mm/1 million cycles). The wear debris isolated from the fixed-bearing knees showed the moderately cross-linked material had a larger percentage volume of particles smaller than 1 mum in size, compared with GVF GUR 1020 polyethylene. Direct cell culture studies of wear debris generated in sterile wear simulators using multidirectional motion showed a increase (p < 0.05) in tumor necrosis factor-alpha levels and reactivity for GUR 1050 cross-linked polyethylene debris compared with an equivalent volume of noncross-linked GUR 1050 polyethylene. The use of cross-linked polyethylene in the knee reduces the volumetric wear rate. However, the clinical significance of reduced fracture toughness, elevated wear in abrasive conditions, and the elevated tumor necrosis factor-alpha release from smaller more reactive

  12. Antimicrobial activity of lactoperoxidase system incorporated into cross-linked alginate films.

    PubMed

    Yener, Fatih Y G; Korel, Figen; Yemenicioğlu, Ahmet

    2009-03-01

    In this study, the antimicrobial effect of lactoperoxidase (LPS) incorporated alginate films was investigated on Escherichia coli (NRRL B-3008), Listeria innocua (NRRL B-33314), and Pseudomonas fluorescens (NRRL B-253) in presence of different concentrations of H(2)O(2) (0.2, 0.4, and 0.8 mM) and KSCN (1, 2, and 4 mM). The incorporation of 70 nmol ABTS/min/cm(2) LPS into alginate films gave 0.66 to 0.85 nmol ABTS/min/cm(2) enzyme activity at 0.2 to 0.8 mM H(2)O(2) concentration range. The antimicrobial activity of LPS system on target bacteria changed according to the concentrations of KSCN and H(2)O(2). The growth of all tested bacteria was prevented for a 6-h period by applying LPS system in presence of 0.4 or 0.8 mM H(2)O(2) and 4 mM KSCN. At 0.8 mM H(2)O(2) and 4 mM KSCN, the LPS system also inhibited growth of L. innocua and P. fluorescens for a 24-h incubation period, whereas E. coli growth could not be inhibited for 24 h under these conditions. At 0.2 mM H(2)O(2) and 1 to 4 mM KSCN, a considerable inhibitory effect was obtained only on P. fluorescens. The decreasing order of the resistance of studied bacteria to LPS system is as follows: E. coli, L. innocua, and P. fluorescens. The developed antimicrobial system has a good potential for use in meat, poultry, and seafood since alginate coatings are already used in these products. Further studies are needed to test the LPS incorporated edible films in real food systems. PMID:19323761

  13. Cross-linked informofers.

    PubMed Central

    Prosvirnin, V V; Ruzidic, S; Samarina, O P

    1979-01-01

    The proteins of 30S RNP particles containing pre-mRNA (hnRNA) were cross-linked with bifunctional reagents (dimethyl-suberimidate and dimethyl-3,3'-dithiobispropionimidate). Further treatment with 1 or 2 M NaCl dissociates all RNA from protein. However, a significant part of protein particles--informofers being cross-linked survived high salt treatment. Their sedimentation coefficients were close to those of original particles. No RNA could be detected in the informofers even after labeling the cells with a precursor for a long period of time. Sodium dodecylsulfate or urea dissociated cross-linked informofers into oligomeric polypeptides. They could be dissociated by beta-mercaptoethanol treatment if a reversible cross-linked reagent had been used. The resulting polypeptides were represented by informatin. RNP particles (30S RNP or poly-particles) were reconstituted upon mixing of cross-linked informofers with pre-mRNA and removal of 2 M NaCl. PMID:503864

  14. Long-term Results of a First-Generation Annealed Highly Cross-Linked Polyethylene in Young, Active Patients.

    PubMed

    Ranawat, Chitranjan S; Ranawat, Amar S; Ramteke, Alankar A; Nawabi, Danyal; Meftah, Morteza

    2016-03-01

    The survivorship of total hip arthroplasty in younger patients is dependent on the wear characteristics of the bearing surfaces. Long-term results with conventional polyethylene in young patients show a high failure rate. This study assessed the long-term results of a first-generation annealed highly cross-linked polyethylene (HCLPE) in uncemented total hip arthroplasty in young, active patients. Between 1999 and 2003, 112 total hip arthroplasty procedures performed in 91 patients with an average University of California Los Angeles activity score of 8 and mean age of 53 years (range, 24-65 years) were included from a prospective database. In all patients, a 28-mm metal femoral head on annealed HCLPE (Crossfire; Stryker, Mahwah, New Jersey) was used. At minimum 10-year follow-up (11.5±0.94 years), Kaplan-Meier survivorship was 97% for all failures (1 periprosthetic infection and 1 late dislocation) and 100% for mechanical failure (no revisions for osteolysis or loosening). This study showed low revision rates for wear-related failure and superior survivorship in young, active patients. Oxidation causing failure of the locking mechanism has not been a problem with Crossfire for up to 10 years. [Orthopedics. 2016; 39(2):e225-e229.]. PMID:26811959

  15. Polyaniline shell cross-linked Fe3O4 magnetic nanoparticles for heat activated killing of cancer cells.

    PubMed

    Rana, Suman; Jadhav, Neena V; Barick, K C; Pandey, B N; Hassan, P A

    2014-08-28

    Superparamagnetic Fe3O4 nanoparticles are appealing materials for heat activated killing of cancer cells. Here, we report a novel method to enhance the heat activated killing of cancer cells under an AC magnetic field (AMF) by introducing a polyaniline impregnated shell onto the surface of Fe3O4 nanoparticles. These polyaniline shell cross-linked magnetic nanoparticles (PSMN) were prepared by in situ polymerization of aniline hydrochloride on the surface of carboxyl PEGylated Fe3O4 nanoparticles. XRD and TEM analyses revealed the formation of single phase inverse spinel Fe3O4 nanoparticles of a size of about 10 nm. The successful growth of the polyaniline shell on the surface of carboxyl PEGylated magnetic nanoparticles (CPMN) is evident from FTIR spectra, DLS, TGA, zeta-potential and magnetic measurements. Both CPMN and PSMN show good colloidal stability, superparamagnetic behavior at room temperature and excellent heating efficacy under AMF. It has been observed that the heating efficacy of PSMN under AMF was slightly reduced as compared to that of CPMN. The enhanced toxicity of PSMN to cancer cells under AMF suggests their strong potential for magnetic hyperthermia. Furthermore, PSMN shows high loading affinity for an anticancer drug (doxorubicin), its sustained release and substantial internalization in tumor cells. PMID:24948377

  16. Intramolecular cross-linking of domains at the active site links A1 and B subfragments of the Ca2+-ATPase of sarcoplasmic reticulum.

    PubMed

    Ross, D C; McIntosh, D B

    1987-02-15

    Glutaraldehyde treatment of sarcoplasmic reticulum vesicles results in formation of cross-linked Ca2+-ATPase oligomers. Under limiting reaction conditions, where minimal interpolypeptide cross-linking occurs, hydrodynamic properties of the monomer are altered, such that, on sodium dodecyl sulfate-polyacrylamide electrophoresis, the enzyme migrates with an apparent molecular weight of 125,000 (E(125], as compared to the native enzyme (E(110]. The E(125) species was also formed following reaction with other cross-linking bis-aldehydes, with formaldehyde and with a bissuccinimidyl ester. Derivitization resulted in inactivation of ATPase activity and of phosphoprotein formation from Pi. E(125) formation was inhibited by ATP, ADP, AMPPCP, and orthovanadate, and by specific modification of active site Lys-514 with fluorescein-5'-isothiocyanate. Tryptic cleavage patterns of the glutaraldehyde-modified enzyme were consistent with covalent linkage of A1 and B fragments that have been postulated to comprise the phosphorylation and nucleotide-binding domains (MacLennan, D. H., Brandt, C. J., Korczak, B., and Green, N. M. (1985) Nature 316, 696-700). The denaturing detergent, sodium dodecyl sulfate, prevented cross-link formation. Interdomain cross-linking was inhibited by prior modification with either 2,4,6-trinitrobenzene sulfonate, phenylglyoxal, or pyridoxal-5'-phosphate but was unaffected by thiol group modification with iodoacetate or N-ethylmaleimide, suggesting involvement of lysine residues. These findings indicate that intramolecular cross-linking at the active site of the Ca2+-ATPase involves phosphorylation- and ATP-binding domains that are widely separated in the linear sequence. PMID:2950084

  17. 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. PMID:27262203

  18. Effect of alpha-actinin on actin structure. Actin ATPase activity.

    PubMed

    Singh, I; Goll, D E; Robson, R M

    1981-08-28

    Alpha-Actinin increases the ATPase activity of actin by up to 84%, depending un pH, divalent cations present and the added Mg2+: ATP ratio. Dithiothreitol decreases actin ATPase activity approx. 20% but does not reduce the ability of alpha-actinin to increase actin ATP activity. Increasing amounts of added alpha-actinin up to 1 mos alpha-actinin to 49 mol actin cause in increasing increment in actin ATPase activity, but adding alpha-actinin beyond 1 mol alpha-actinin to 49 mol actin elicits only small additional increments in activity. Actin ATPase activity ranges from approx 100 nmol Pi/mg actin per h (4.3 mol Pi/mol actin per h) at high levels (10 mM) of ATP in the presence of lower amounts (1 mM) of added mg2+ to approx. 12.5 nmol Pi/mg actin per h (0.52 mol Pi/mol actin per h) at high pH (8.5) or at low levels (0.5-1.0 mM) of ATP in the presence of higher amounts (10 mM) of added Mg2+ ATp uncomplexed with Mg2+ inhibits the ability of alpha-actinin to increase F-actin ATPase activity. Activities with different divalent cations showed that the actin ATPase in these studies, which was 1/100 as great as Mg2+-modified actomyosin ATPase activity, was not due to trace amounts of myosin contaminating the actin preparations. The results are consistent with the concept that alpha-actinin can alter the structure of actin monomers. PMID:6456018

  19. 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. PMID:25659629

  20. Enhancement of mechanical properties, microstructure, and antimicrobial activities of zein films cross-linked using succinic anhydride, eugenol, and citric Acid.

    PubMed

    Khalil, Ashraf A; Deraz, Sahar F; Elrahman, Somia Abd; El-Fawal, Gomaa

    2015-08-18

    Zein constitutes about half of the endosperm proteins in corn. Recently, attempts have been made to utilize zein for food coatings and biodegradable materials, which require better physical properties, using chemical modification of zein. In this study, zein proteins were modified using citric acid, succinic anhydride, and eugenol as natural cross-linking agents in the wet state. The cross-linkers were added either separately or combined in increment concentrations (0.1, 0.2, 0.3, and 0.4%). The effects of those agents on the mechanical properties, microstructure, optical properties, infrared (IR) spectroscopy, and antibacterial activities of zein were investigated. The addition of cross-linking agents promoted changes in the arrangement of groups in zein film-forming particles. Regarding the film properties, incorporation of cross-linking agents into zein films prepared in ethanol resulted in two- to three-fold increases in tensile strength (TS) values. According to the Fourier-transform infrared (FTIR) spectra and Hunter parameters there were no remarkable changes in the structure and color of zein films. Transparency of zein films was decreased differentially according to the type and cross-linker concentration. The mechanical and optical properties of zein films were closely related to their microstructure. All cross-linked films showed remarkable antibacterial activities against Bacillus cereus ATCC 49064 and Salmonella enterica ATCC 25566. Food spoilage and pathogenic bacteria were affected in a film-dependent manner. Our experimental results show that even with partial cross-linking the mechanical properties and antipathogen activities of zein films were significantly improved, which would be useful for various industrial applications. PMID:25036665

  1. Effects of F/G-actin ratio and actin turn-over rate on NADPH oxidase activity in microglia

    PubMed Central

    2010-01-01

    Background Most in vivo studies that have addressed the role of actin dynamics in NADPH oxidase function in phagocytes have used toxins to modulate the polymerization state of actin and mostly effects on actin has been evaluated by end point measurements of filamentous actin, which says little about actin dynamics, and without consideration for the subcellular distribution of the perturbed actin cytoskeleton. Results Here, we in addition to toxins use conditional expression of the major actin regulatory protein LIM kinase-1 (LIMK1), and shRNA knock-down of cofilin to modulate the cellular F/G-actin ratio in the Ra2 microglia cell line, and we use Fluorescence Recovery after Photobleaching (FRAP) in β-actin-YFP-transduced cells to obtain a dynamic measure of actin recovery rates (actin turn-over rates) in different F/G-actin states of the actin cytoskeleton. Our data demonstrate that stimulated NADPH oxidase function was severely impaired only at extreme actin recovery rates and F/G-actin ratios, and surprisingly, that any moderate changes of these parameters of the actin cytoskeleton invariably resulted in an increased NADPH oxidase activity. Conclusion moderate actin polymerization and depolymerization both increase the FMLP and PMA-stimulated NADPH oxidase activity of microglia, which is directly correlated with neither actin recovery rate nor F/G- actin ratio. Our results indicate that NADPH oxidase functions in an enhanced state of activity in stimulated phagocytes despite widely different states of the actin cytoskeleton. PMID:20825680

  2. Actively-targeted polyion complex micelles stabilized by cholesterol and disulfide cross-linking for systemic delivery of siRNA to solid tumors.

    PubMed

    Oe, Yusuke; Christie, R James; Naito, Mitsuru; Low, Stewart A; Fukushima, Shigeto; Toh, Kazuko; Miura, Yutaka; Matsumoto, Yu; Nishiyama, Nobuhiro; Miyata, Kanjiro; Kataoka, Kazunori

    2014-09-01

    For small interfering RNA (siRNA)-based cancer therapies, we report an actively-targeted and stabilized polyion complex micelle designed to improve tumor accumulation and cancer cell uptake of siRNA following systemic administration. Improvement in micelle stability was achieved using two stabilization mechanisms; covalent disulfide cross-linking and non-covalent hydrophobic interactions. The polymer component was designed to provide disulfide cross-linking and cancer cell-targeting cyclic RGD peptide ligands, while cholesterol-modified siRNA (Chol-siRNA) provided additional hydrophobic stabilization to the micelle structure. Dynamic light scattering confirmed formation of nano-sized disulfide cross-linked micelles (<50 nm in diameter) with a narrow size distribution. Improved stability of Chol-siRNA-loaded micelles (Chol-siRNA micelles) was demonstrated by resistance to both the dilution in serum-containing medium and counter polyion exchange with dextran sulfate, compared to control micelles prepared with Chol-free siRNA (Chol-free micelles). Improved stability resulted in prolonged blood circulation time of Chol-siRNA micelles compared to Chol-free micelles. Furthermore, introduction of cRGD ligands onto Chol-siRNA micelles significantly facilitated accumulation of siRNA in a subcutaneous cervical cancer model following systemic administration. Ultimately, systemically administered cRGD/Chol-siRNA micelles exhibited significant gene silencing activity in the tumor, presumably due to their active targeting ability combined with the enhanced stability through both hydrophobic interactions of cholesterol and disulfide cross-linking. PMID:24930854

  3. Covalent cross-linking of the bovine somatotropin dimer. Effects on growth-promoting, receptor-binding and immunological activities and preliminary characterization of the self-association.

    PubMed Central

    Fernández, H N; Delfino, J M

    1983-01-01

    Bovine somatotropin, at pH 8.5 in 0.02 M-Bicine [NN-bis-(2-hydroxyethyl)glycine]/0.09M-NaCl, showed by frontal analysis the characteristics of a rapid monomer-dimer equilibrium whose dissociation constant was estimated to be 6.6 X 10(-6)M. Reaction of the hormone with dimethyl suberimidate lead to covalent cross-linking of the dimeric species. Under the conditions chosen (0.4 mg of bifunctional imidate and 1 mg of protein/ml at room temperature for 1 h) the cross-linked dimers accounted for 26% of the total protein, and these were isolated by molecular sieving in 0.29M-NH3/0.12M-NaCl. Covalent stabilization greatly diminished the growth-promoting activity and the ability to interact with somatogenic sites in both rat liver in vivo and rabbit liver microsomal fractions. Evidence indicating a non-critical role for amino groups involved in the covalent cross-linking was provided by a nearly equivalent derivative obtained after reaction with 3,3'-dithiobispropionimidate, which had substantial hormonal activity upon cleavage of the disulphide links. Conversely, immunological reactivity as demonstrated by radioimmunoassay was not affected by cross-linking. Details of the least-squares procedure employed to evaluate the self-association equilibrium constant has been deposited as Supplement SUP 50115 (7 pages) with the British Library Lending Division, Boston Spa, Wetherby, West Yorkshire LS23 7BQ, U.K., from whom copies may be obtained on the terms indicated in Biochem.J. (1981) 193,5. Images Fig. 1. PMID:6303298

  4. Tyrosine phosphorylation-dependent activation of phosphatidylinositide 3-kinase occurs upstream of Ca2+-signalling induced by Fcgamma receptor cross-linking in human neutrophils.

    PubMed Central

    Vossebeld, P J; Homburg, C H; Schweizer, R C; Ibarrola, I; Kessler, J; Koenderman, L; Roos, D; Verhoeven, A J

    1997-01-01

    The effect of wortmannin on IgG-receptor (FcgammaR)-mediated stimulation of human neutrophils was investigated. The Ca2+ influx induced by clustering of both Fcgamma receptors was inhibited by wortmannin, as was the release of Ca2+ from intracellular stores. Wortmannin also inhibited, with the same efficacy, the accumulation of Ins(1,4,5)P3 observed after FcgammaR stimulation, but did not affect the increase in Ins(1,4,5)P3 induced by the chemotactic peptide, formyl-methionine-leucine-phenylalanine. Because wortmannin is, in the concentrations used here, an inhibitor of PtdIns 3-kinase, these results suggested a role for PtdIns 3-kinase upstream of Ca2+ signalling, induced by FcgammaR cross-linking. Support for this notion was obtained by investigating the effect of another inhibitor of PtdIns 3-kinase, LY 294002, and by studying the kinetics of PtdIns 3-kinase activation. We found translocation of PtdIns 3-kinase to the plasma membrane and increased PtdIns 3-kinase activity in the membrane as soon as 5 s after FcgammaR cross-linking, even before the onset of the Ca2+ response. Moreover, the translocation of PtdIns 3-kinase to the plasma membrane was inhibited by co-cross-linking of either FcgammaRIIa and FcgammaRIIIb with the tyrosine phosphatase, CD45, indicating a requirement for protein tyrosine phosphorylation in the recruitment of PtdIns 3-kinase to the plasma membrane. Taken together, our results suggest a role for PtdIns 3-kinase in early signal transduction events after FcgammaR cross-linking in human neutrophils. PMID:9173906

  5. Site-Specific, Intramolecular Cross-Linking of Pin1 Active Site Residues by the Lipid Electrophile 4-Oxo-2-nonenal

    PubMed Central

    2016-01-01

    Products of oxidative damage to lipids include 4-hydroxy-2-nonenal (HNE) and 4-oxo-2-nonenal (ONE), both of which are cytotoxic electrophiles. ONE reacts more rapidly with nucleophilic amino acid side chains, resulting in covalent protein adducts, including residue–residue cross-links. Previously, we demonstrated that peptidylprolyl cis/trans isomerase A1 (Pin1) was highly susceptible to adduction by HNE and that the catalytic cysteine (Cys113) was the preferential site of modification. Here, we show that ONE also preferentially adducts Pin1 at the catalytic Cys but results in a profoundly different modification. Results from experiments using purified Pin1 incubated with ONE revealed the principal product to be a Cys-Lys pyrrole-containing cross-link between the side chains of Cys113 and Lys117. In vitro competition assays between HNE and ONE demonstrate that ONE reacts more rapidly than HNE with Cys113. Exposure of RKO cells to alkynyl-ONE (aONE) followed by copper-mediated click chemistry and streptavidin purification revealed that Pin1 is also modified by ONE in cells. Analysis of the Pin1 crystal structure reveals that Cys113 and Lys117 are oriented toward each other in the active site, facilitating formation of an ONE cross-link. PMID:25739016

  6. Glutamyl Phosphate Is an Activated Intermediate in Actin Crosslinking by Actin Crosslinking Domain (ACD) Toxin

    PubMed Central

    Kudryashova, Elena; Kalda, Caitlin; Kudryashov, Dmitri S.

    2012-01-01

    Actin Crosslinking Domain (ACD) is produced by several life-threatening Gram-negative pathogenic bacteria as part of larger toxins and delivered into the cytoplasm of eukaryotic host cells via Type I or Type VI secretion systems. Upon delivery, ACD disrupts the actin cytoskeleton by catalyzing intermolecular amide bond formation between E270 and K50 residues of actin, leading to the formation of polymerization-deficient actin oligomers. Ultimately, accumulation of the crosslinked oligomers results in structural and functional failure of the actin cytoskeleton in affected cells. In the present work, we advanced in our understanding of the ACD catalytic mechanism by discovering that the enzyme transfers the gamma-phosphoryl group of ATP to the E270 actin residue, resulting in the formation of an activated acyl phosphate intermediate. This intermediate is further hydrolyzed and the energy of hydrolysis is utilized for the formation of the amide bond between actin subunits. We also determined the pH optimum for the reaction and the kinetic parameters of ACD catalysis for its substrates, ATP and actin. ACD showed sigmoidal, non-Michaelis-Menten kinetics for actin (K0.5 = 30 µM) reflecting involvement of two actin molecules in a single crosslinking event. We established that ACD can also utilize Mg2+-GTP to support crosslinking, but the kinetic parameters (KM = 8 µM and 50 µM for ATP and GTP, respectively) suggest that ATP is the primary substrate of ACD in vivo. The optimal pH for ACD activity was in the range of 7.0–9.0. The elucidated kinetic mechanism of ACD toxicity adds to understanding of complex network of host-pathogen interactions. PMID:23029200

  7. 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.

  8. 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. PMID:24055882

  9. 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.

  10. Preferential nitration with tetranitromethane of a specific tyrosine residue in penicillinase from Staphylococcus aureus PCl. Evidence that the preferentially nitrated residue is not part of the active site but that loss of activity is due to intermolecular cross-linking.

    PubMed Central

    Bristow, A F; Virden, R

    1978-01-01

    1. Nitration of tyrosine residues of staphylococal penicillinase was accompanied by a partial loss of enzymic activity, which was not readily explained by nitration of a single residue. 2. Loss of activity correlated with low recovery of tyrosine plus nitrotyrosine, which was consistent with cross-linking. 3. The fraction of treated enzyme that was eluted from Sephadex G-75 earlier than native penicillinase was similar to the fraction of enzyme activity lost. Protein eluted in positions corresponding to monomer, dimer and higher oligomers respectively showed major bands in corresponding positions in sodium dodecyl sulphate/polyacrylamide-gel electrophoresis, indicating that the increase in molecular weight was due to intermolecular cross-linking. Monomeric enzyme containing up to 4 mol of nitrotyrosine/mol retained full catalytic activity. Dimeric enzyme retained 50% of normal activity, whereas higher oligomers retained an average of 8-15% of normal activity. 4. Monomeric enzyme isolated after treatment with equimolar tetranitromethane was nitrated predominantly at tyrosine-72.5. Reaction of reduced nitrated monomer with 1,5-difluoro-2,4-dinitrobenzene gave a monomeric, apparently cross-linked product with full catalytic activity. 6. It is concluded that tyrosine-72 plays no part in the active site. Its preferential nitration may be due to its being insufficiently exposed to be available for intermolecular cross-linking. This poperty may make it useful for attachment of a reporter group. PMID:629760

  11. The role of redox-active amino acids on compound I stability, substrate oxidation, and protein cross-linking in yeast cytochrome C peroxidase.

    PubMed

    Pfister, T D; Gengenbach, A J; Syn, S; Lu, Y

    2001-12-11

    The role of two tryptophans (Trp51 and Trp191) and six tyrosines (Tyr36, Tyr39, Tyr42, Tyr187, Tyr229, and Tyr236) in yeast cytochrome c peroxidase (CcP) has been probed by site-directed mutagenesis. A series of sequential mutations of these redox-active amino acid residues to the corresponding, less oxidizable residues in lignin peroxidase (LiP) resulted in an increasingly more stable compound I, with rate constants for compound I decay decreasing from 57 s(-1) for CcP(MI, W191F) to 7 s(-1) for CcP(MI, W191F,W51F,Y187F,Y229F,Y236F,Y36F,Y39E,Y42F). These results provide experimental support for the proposal that the stability of compound I depends on the number of endogenous oxidizable amino acids in proteins. The higher stability of compound I in the variant proteins also makes it possible to observe its visible absorption spectroscopic features more clearly. The effects of the mutations on oxidation of ferrocytochrome c and 2,6-dimethoxyphenol were also examined. Since the first mutation in the series involved the change of Trp191, a residue that plays a critical role in the electron transfer pathway between CcP and cyt c, the ability to oxidize cyt c was negligible for all mutant proteins. On the other hand, the W191F mutation had little effect on the proteins' ability to oxidize 2,6-dimethoxyphenol. Instead, the W51F mutation resulted in the largest increase in the k(cat)/K(M), from 2.1 x 10(2) to 5.0 x 10(3) M(-1) s(-1), yielding an efficiency that is comparable to that of manganese peroxidase (MnP). The effect in W51F mutation can be attributed to the residue's influence on the stability and thus reactivity of the ferryl oxygen of compound II, whose substrate oxidation is the rate-determining step in the reaction mechanism. Finally, out of all mutant proteins in this study, only the variant containing the Y36F, Y39E, and Y42F mutations was found to prevent covalent protein cross-links in the presence of excess hydrogen peroxide and in the absence of exogenous

  12. Nonlinear elasticity of cross-linked networks

    NASA Astrophysics Data System (ADS)

    John, Karin; Caillerie, Denis; Peyla, Philippe; Raoult, Annie; Misbah, Chaouqi

    2013-04-01

    Cross-linked semiflexible polymer networks are omnipresent in living cells. Typical examples are actin networks in the cytoplasm of eukaryotic cells, which play an essential role in cell motility, and the spectrin network, a key element in maintaining the integrity of erythrocytes in the blood circulatory system. We introduce a simple mechanical network model at the length scale of the typical mesh size and derive a continuous constitutive law relating the stress to deformation. The continuous constitutive law is found to be generically nonlinear even if the microscopic law at the scale of the mesh size is linear. The nonlinear bulk mechanical properties are in good agreement with the experimental data for semiflexible polymer networks, i.e., the network stiffens and exhibits a negative normal stress in response to a volume-conserving shear deformation, whereby the normal stress is of the same order as the shear stress. Furthermore, it shows a strain localization behavior in response to an uniaxial compression. Within the same model we find a hierarchy of constitutive laws depending on the degree of nonlinearities retained in the final equation. The presented theory provides a basis for the continuum description of polymer networks such as actin or spectrin in complex geometries and it can be easily coupled to growth problems, as they occur, for example, in modeling actin-driven motility.

  13. Cross-linked structure of network evolution

    NASA Astrophysics Data System (ADS)

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

    2014-03-01

    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. 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.

  15. 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

  16. Normal Activation of Discoidin Domain Receptor 1 Mutants with Disulfide Cross-links, Insertions, or Deletions in the Extracellular Juxtamembrane Region

    PubMed Central

    Xu, Huifang; Abe, Takemoto; Liu, Justin K. H.; Zalivina, Irina; Hohenester, Erhard; Leitinger, Birgit

    2014-01-01

    The discoidin domain receptors, DDR1 and DDR2, are receptor tyrosine kinases that are activated by collagen. DDR activation does not appear to occur by the common mechanism of ligand-induced receptor dimerization: the DDRs form stable noncovalent dimers in the absence of ligand, and ligand-induced autophosphorylation of cytoplasmic tyrosines is unusually slow and sustained. Here we sought to identify functionally important dimer contacts within the extracellular region of DDR1 by using cysteine-scanning mutagenesis. Cysteine substitutions close to the transmembrane domain resulted in receptors that formed covalent dimers with high efficiency, both in the absence and presence of collagen. Enforced covalent dimerization did not result in constitutive activation and did not affect the ability of collagen to induce receptor autophosphorylation. Cysteines farther away from the transmembrane domain were also cross-linked with high efficiency, but some of these mutants could no longer be activated. Furthermore, the extracellular juxtamembrane region of DDR1 tolerated large deletions as well as insertions of flexible segments, with no adverse effect on activation. These findings indicate that the extracellular juxtamembrane region of DDR1 is exceptionally flexible and does not constrain the basal or ligand-activated state of the receptor. DDR1 transmembrane signaling thus appears to occur without conformational coupling through the juxtamembrane region, but requires specific receptor interactions farther away from the cell membrane. A plausible mechanism to explain these findings is signaling by DDR1 clusters. PMID:24671415

  17. Characterization of actin filament deformation in response to actively driven microspheres propagated through entangled actin networks

    NASA Astrophysics Data System (ADS)

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

    2014-03-01

    The semi-flexible biopolymer actin is a ubiquitous component of nearly all biological organisms, playing an important role in many biological processes such as cell structure and motility, cancer invasion and metastasis, muscle contraction, and cell signaling. Concentrated actin networks possess unique viscoelastic properties that have been the subject of much theoretical and experimental work. However, much is still unknown regarding the correlation of the applied stress on the network to the induced filament strain at the molecular level. Here, we use dual optical traps alongside fluorescence microscopy to carry out active microrheology measurements that link mechanical stress to structural response at the micron scale. Specifically, we actively drive microspheres through entangled actin networks while simultaneously measuring the force the surrounding filaments exert on the sphere and visualizing the deformation and subsequent relaxation of fluorescent labeled filaments within the network. These measurements, which provide much needed insight into the link between stress and strain in actin networks, are critical for clarifying our theoretical understanding of the complex viscoelastic behavior exhibited in actin networks.

  18. 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

    2016-04-01

    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. PMID:26966832

  19. 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.

  20. Synthesis and antitumor activity evaluation of a novel combi-nitrosourea prodrug: Designed to release a DNA cross-linking agent and an inhibitor of O(6)-alkylguanine-DNA alkyltransferase.

    PubMed

    Sun, Guohui; Zhang, Na; Zhao, Lijiao; Fan, Tengjiao; Zhang, Shufen; Zhong, Rugang

    2016-05-01

    The drug resistance of CENUs induced by O(6)-alkylguanine-DNA alkyltransferase (AGT), which repairs the O(6)-alkylated guanine and subsequently inhibits the formation of dG-dC cross-links, hinders the application of CENU chemotherapies. Therefore, the discovery of CENU analogs with AGT inhibiting activity is a promising approach leading to novel CENU chemotherapies with high therapeutic index. In this study, a new combi-nitrosourea prodrug 3-(3-(((2-amino-9H-purin-6-yl)oxy)methyl)benzyl)-1-(2-chloroethyl)-1-nitrosourea (6), designed to release a DNA cross-linking agent and an inhibitor of AGT, was synthesized and evaluated for its antitumor activity and ability to induce DNA interstrand cross-links (ICLs). The results indicated that 6 exhibited higher cytotoxicity against mer(+) glioma cells compared with ACNU, BCNU, and their respective combinations with O(6)-benzylguanine (O(6)-BG). Quantifications of dG-dC cross-links induced by 6 were performed using HPLC-ESI-MS/MS. Higher levels of dG-dC cross-link were observed in 6-treated human glioma SF763 cells (mer(+)), whereas lower levels of dG-dC cross-link were observed in 6-treated calf thymus DNA, when compared with the groups treated with BCNU and ACNU. The results suggested that the superiority of 6 might result from the AGT inhibitory moiety, which specifically functions in cells with AGT activity. Molecular docking studies indicated that five hydrogen bonds were formed between the O(6)-BG analogs released from 6 and the five residues in the active pocket of AGT, which provided a reasonable explanation for the higher AGT-inhibitory activity of 6 than O(6)-BG. PMID:27041398

  1. Pathogenic microbes manipulate cofilin activity to subvert actin cytoskeleton.

    PubMed

    Zheng, Kai; Kitazato, Kaio; Wang, Yifei; He, Zhendan

    2016-09-01

    Actin-depolymerizing factor (ADF)/cofilin proteins are key players in controlling the temporal and spatial extent of actin dynamics, which is crucial for mediating host-pathogen interactions. Pathogenic microbes have evolved molecular mechanisms to manipulate cofilin activity to subvert the actin cytoskeletal system in host cells, promoting their internalization into the target cells, modifying the replication niche and facilitating their intracellular and intercellular dissemination. The study of how these pathogens exploit cofilin pathways is crucial for understanding infectious disease and providing potential targets for drug therapies. PMID:25853495

  2. Photomobile polymer materials: photoresponsive behavior of cross-linked liquid-crystalline polymers with mesomorphic diarylethenes.

    PubMed

    Mamiya, Jun-ichi; Kuriyama, Akito; Yokota, Naoki; Yamada, Munenori; Ikeda, Tomiki

    2015-02-16

    Cross-linked liquid-crystalline (LC) polymers with a mesomorphic diarylethene were prepared to demonstrate a versatile strategy for cross-linked photochromic LC polymers as photomobile materials. Upon exposure to UV light to cause photocyclization of the diarylethene chromophore, the cross-linked polymer films bend toward an actinic light source. By irradiation with visible light to cause a closed-ring to open-ring isomerization, the bent films revert to the initial flat state. Without visible-light irradiation, the bent films remain bent even at 120 °C, indicating high thermal stability of the cross-linked diarylethene LC polymers. PMID:25581255

  3. 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

  4. Influence of Unmodified and β-Glycerophosphate Cross-Linked Chitosan on Anti-Candida Activity of Clotrimazole in Semi-Solid Delivery Systems

    PubMed Central

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

    2014-01-01

    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. PMID:25272230

  5. 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

    2014-01-01

    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. PMID:25272230

  6. Test of hirudin activity by tracking the binding of hirudin to thrombin in the presence of BS3 cross-linking.

    PubMed

    Liu, Yanfang; Yang, Jian; Wang, Jiangmin; Huang, Qingmei; Yang, Xiaohong; Zhang, Jianhua

    2015-10-01

    Hirudin has a great potential in inhibiting thrombin, and its antithrombin activity has direct bearing on its clinical application. Using bovine alpha-thrombin and recombinant hirudin of Poecilobdella javanica purified from Phichia pastoris as materials, this study introduced a novel method to testing antithrombin activity of hirudin visually and dynamically by tracking the binding of hirudin to thrombin. After incubating the mixture of thrombin and hirudin at 37 °C for 5 min, the binding of hirudin to thrombin was cross-linked by bis[sulfosuccinimidyl] suberate for 30 min and visualized by SDS-polyacrylamide gel electrophoresis. With the aid of image analysis on the basis of INRA-Noésis E1D analysis software, antithrombin activity of hirudin was calculated through intensity variations of protein bands of either thrombin-hirudin compound, unbound thrombin, or unbound hirudin. In this regard, activity of the given hirudin was tested to be 5625 ATU/mg based on a single reaction, and 5675.3 ATU/mg based on a series of reactions in a stepwise manner, close to the result of 6000 ATU/mg concluded by titration method. The superiorities of the method include good accuracy (the minimum testable concentration of hirudin is 1.5 μg/ml) and little sample consumption (sample consumption of hirudin is generally 1-11.5 μl using the apparatus of Mini Protean 3 Cell). Easy operation, low input, and equipment requirement also grant it as an effective way. PMID:26332983

  7. Investigation and characterization of receptors for pituitary adenylate cyclase-activating polypeptide in human brain by radioligand binding and chemical cross-linking

    SciTech Connect

    Suda, K.; Smith, D.M.; Ghatei, M.A.; Murphy, J.K.; Bloom, S.R. )

    1991-05-01

    Pituitary adenylate cyclase-activating polypeptide (PACAP) is a novel peptide of hypothalamic origin which increases adenylate cyclase activity in rat anterior pituitary cell cultures. The 38-amino acid peptide shows a close sequence homology to vasoactive intestinal peptide (VIP). Binding sites for PACAP in membranes from postmortem human brain tissue were studied using ({sup 125}I)PACAP27 as the radioligand. High specific binding sites (amount of specific binding measured at 0.25 nM ({sup 125}I)PACAP27 in femtomoles per mg protein +/- SEM; n = 4) were present in hypothalamus (344.5 +/- 13.0), brain stem (343.0 +/- 29.3), cerebellum (292.0 +/- 21.1), cortex (259.6 +/- 19.8), and basal ganglia (259.2 +/- 50.3). Specific binding sites in pituitary, although present, were less abundant (35.0 +/- 8.9). Binding of ({sup 125}I)PACAP27 was reversible and time, pH, and temperature dependent. Despite the homology with VIP, VIP was a poor inhibitor of ({sup 125}I)PACAP27 binding (IC50, greater than 1 microM) compared with PACAP27 (IC50, 0.5-1.3 nM) and PACAP38 (IC50, 0.2-1.3 nM). Scatchard plots of ({sup 125}I)PACAP27 binding showed the presence of both high and lower affinity sites. Chemical cross-linking of PACAP-binding sites revealed that ({sup 125}I)PACAP27 was bound to polypeptide chains of 67,000 and 48,000 mol wt. Thus, we have demonstrated the presence of PACAP-specific receptors in human brain which are not VIP receptors. This opens the possibility of PACAP functioning as a novel neurotransmitter/neuromodulator in human brain.

  8. Contraction of cross-linked actomyosin bundles

    NASA Astrophysics Data System (ADS)

    Yoshinaga, Natsuhiko; Marcq, Philippe

    2012-08-01

    Cross-linked actomyosin bundles retract when severed in vivo by laser ablation, or when isolated from the cell and micromanipulated in vitro in the presence of ATP. We identify the timescale for contraction as a viscoelastic time τ, where the viscosity is due to (internal) protein friction. We obtain an estimate of the order of magnitude of the contraction time τ ≈ 10-100 s, consistent with available experimental data for circumferential microfilament bundles and stress fibers. Our results are supported by an exactly solvable, hydrodynamic model of a retracting bundle as a cylinder of isotropic, active matter, from which the order of magnitude of the active stress is estimated.

  9. 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...

  10. Activity of a gelsolin-like actin modulator in rat skeletal muscle under protein catabolic conditions.

    PubMed Central

    D'Haese, J; Rutschmann, M; Dahlmann, B; Hinssen, H

    1987-01-01

    A gelsolin-like actin-modulating protein was isolated from rat skeletal muscle and characterized with respect to its interaction with actin. The protein, with a molecular mass of approx. 85 kDa, forms a stoichiometric complex with two actin molecules and is activated by micromolar concentrations of Ca2+. It effectively severs actin filaments and promotes nucleation of actin polymerization. The activity of this protein is detectable already in crude extracts by its capability to reduce the steady state viscosity of actin. Actin-modulating activities were determined in muscle extracts of rats kept under protein catabolic conditions, i.e. as generated by corticosterone treatment and starvation. In both cases we found a marked increase of modulator activity. The possibility is discussed that the increased activity of actin modulator indicates a fragmentation of actin filaments prior to the proteolytic degradation of actin. Images Fig. 2. PMID:3435453

  11. 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.

  12. 3D Actin Network Centerline Extraction with Multiple Active Contours

    PubMed Central

    Xu, Ting; Vavylonis, Dimitrios; Huang, Xiaolei

    2013-01-01

    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. PMID:24316442

  13. Actin-binding proteins: the long road to understanding the dynamic landscape of cellular actin networks.

    PubMed

    Lappalainen, Pekka

    2016-08-15

    The actin cytoskeleton supports a vast number of cellular processes in nonmuscle cells. It is well established that the organization and dynamics of the actin cytoskeleton are controlled by a large array of actin-binding proteins. However, it was only 40 years ago that the first nonmuscle actin-binding protein, filamin, was identified and characterized. Filamin was shown to bind and cross-link actin filaments into higher-order structures and contribute to phagocytosis in macrophages. Subsequently many other nonmuscle actin-binding proteins were identified and characterized. These proteins regulate almost all steps of the actin filament assembly and disassembly cycles, as well as the arrangement of actin filaments into diverse three-dimensional structures. Although the individual biochemical activities of most actin-regulatory proteins are relatively well understood, knowledge of how these proteins function together in a common cytoplasm to control actin dynamics and architecture is only beginning to emerge. Furthermore, understanding how signaling pathways and mechanical cues control the activities of various actin-binding proteins in different cellular, developmental, and pathological processes will keep researchers busy for decades. PMID:27528696

  14. 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

  15. Wdr1-Dependent Actin Reorganization in Platelet Activation.

    PubMed

    Dasgupta, Swapan K; Le, Anhquyen; Da, Qi; Cruz, Miguel; Rumbaut, Rolando E; Thiagarajan, Perumal

    2016-01-01

    In resting platelets, the integrin αIIbβ3 is present in a low-affinity "bent" state. During platelet aggregation, intracytoplasmic signals induce conformational changes (inside-out signaling) that result in a "swung-out" conformation competent to bind ligands such as fibrinogen. The cytoskeleton plays an essential role in αIIbβ3 activation. We investigated the role of the actin interacting protein Wdr1 in αIIbβ3 activation. Wdr1-hypomorphic mice had a prolonged bleeding time (> 10 minutes) compared to that of wild-type mice (2.1 ± 0.7 minutes). Their platelets had impaired aggregation to collagen and thrombin. In a FeCl3 induced carotid artery thrombosis model, vessel occlusion in Wdr1-hypomorphic mice was prolonged significantly compared to wild-type mice (9.0 ± 10.5 minutes versus 5.8 ± 12.6 minutes (p = 0.041). Activation-induced binding of JON/A (a conformation-specific antibody to activated αIIbβ3) was significantly less in Wdr1-hypomorphic platelets at various concentrations of collagen, indicating impaired inside-out activation of αIIbβ3, despite a normal calcium response. Actin turnover, assessed by measuring F-actin and G-actin ratios during collagen- and thrombin-induced platelet aggregation, was highly impaired in Wdr1-hypomorphic platelets. Furthermore, talin failed to redistribute and translocate to the cytoskeleton following activation in Wdr1-hypomorphic platelets. These studies show that Wdr1 is essential for talin-induced activation of αIIbβ3 during platelet activation. PMID:27627652

  16. Piccolo Directs Activity Dependent F-Actin Assembly from Presynaptic Active Zones via Daam1

    PubMed Central

    Wagh, Dhananjay; Terry-Lorenzo, Ryan; Waites, Clarissa L.; Leal-Ortiz, Sergio A.; Maas, Christoph; Reimer, Richard J.; Garner, Craig C.

    2015-01-01

    The dynamic assembly of filamentous (F) actin plays essential roles in the assembly of presynaptic boutons, the fusion, mobilization and recycling of synaptic vesicles (SVs), and presynaptic forms of plasticity. However, the molecular mechanisms that regulate the temporal and spatial assembly of presynaptic F-actin remain largely unknown. Similar to other F-actin rich membrane specializations, presynaptic boutons contain a set of molecules that respond to cellular cues and trans-synaptic signals to facilitate activity-dependent assembly of F-actin. The presynaptic active zone (AZ) protein Piccolo has recently been identified as a key regulator of neurotransmitter release during SV cycling. It does so by coordinating the activity-dependent assembly of F-Actin and the dynamics of key plasticity molecules including Synapsin1, Profilin and CaMKII. The multidomain structure of Piccolo, its exquisite association with the AZ, and its ability to interact with a number of actin-associated proteins suggest that Piccolo may function as a platform to coordinate the spatial assembly of F-actin. Here we have identified Daam1, a Formin that functions with Profilin to drive F-actin assembly, as a novel Piccolo binding partner. We also found that within cells Daam1 activation promotes Piccolo binding, an interaction that can spatially direct the polymerization of F-Actin. Moreover, similar to Piccolo and Profilin, Daam1 loss of function impairs presynaptic-F-actin assembly in neurons. These data suggest a model in which Piccolo directs the assembly of presynaptic F-Actin from the AZ by scaffolding key actin regulatory proteins including Daam1. PMID:25897839

  17. Chemical cross-linking of Chlamydia trachomatis.

    PubMed Central

    Birkelund, S; Lundemose, A G; Christiansen, G

    1988-01-01

    Purified elementary bodies (EBs) of Chlamydia trachomatis serovar L2 were analyzed by chemical cross-linking with disuccinimidyl selenodipropionate. The effect of the cross-linking was analyzed by immunoblotting sodium dodecyl sulfate-polyacrylamide gel electrophoresis-separated components which were reacted with monoclonal antibodies against major outer membrane protein (MOMP) and lipopolysaccharide (LPS). It was shown that in EBs, MOMP was cross-linked to the LPS component of the outer membrane. Migration analysis of the cross-linked components showed that with extensive cross-linking, most of the MOMP became cross-linked to LPS, changing the migration rate from 40 to 42.5 kilodaltons. A small fraction of MOMP associated with LPS was shown to be present in bands with migration rates of 100 and 110 kilodaltons. No association of MOMP or LPS to other proteins, or to dimer or multimer forms of MOMP without LPS, was observed. A totally different membrane structure must be present in reticulate bodies, since there, MOMP was so heavily cross-linked that it did not enter the polyacrylamide gel and thus became impossible to analyze. Furthermore, the monoclonal antibody, which reacted with LPS associated with MOMP in the cross-linked EBs, did not react with reticulate bodies. Images PMID:2449399

  18. Actin-based endosome and phagosome rocketing in macrophages: activation by the secretagogue antagonists lanthanum and zinc.

    PubMed

    Southwick, Frederick S; Li, Wei; Zhang, Fangliang; Zeile, William L; Purich, Daniel L

    2003-01-01

    Although motile endocytic vesicles form actin-rich rocket tails [Merrifield et al., 1999: Nature Cell Biol 1:72-74], the mechanism of intracellular organelle locomotion remains poorly understood. We now demonstrate that bone marrow macrophages treated with lanthanum and zinc ions, well-known secretagogue antagonists, reliably exhibit vesicle motility. This treatment results in accentuated membrane ruffling and the formation of phagosomes and early endosomes that move rapidly through the cytoplasm by assembling actin filament rocket tails. Protein-specific immunolocalization demonstrated the presence of Arp2/3 complex in the polymerization zone and throughout the actin-rich tail, whereas N-WASP was most abundant in the polymerization zone. Although Arp2/3 and N-WASP play essential roles in nucleating filament assembly, other processes (i.e., elongation and filament cross-linking) are required to produce forces needed for motility. Efficient elongation was found to require zyxin, VASP, and profilin, proteins that interact by means of their ABM-1 and ABM-2 proline-rich motifs. The functional significance of these motifs was demonstrated by inhibition of vesicle motility by the motif-specific ABM-1 and ABM-2 analogues. Furthermore, lanthanum/zinc treatment also facilitated the early onset of actin-based vaccinia motility, a process that also utilizes Arp2/3 and N-WASP for nucleation and the zyxin-VASP-profilin complex for efficient elongation. Although earlier studies using cell extracts clouded the role of oligoproline sequences in activating the polymerization zone, our studies emphasize the importance of evaluating motility in living cells. PMID:12451594

  19. Quinone cross-linked polysaccharide hybrid fiber.

    PubMed

    Kuboe, Yoshiko; Tonegawa, Hitomi; Ohkawa, Kousaku; Yamamoto, Hiroyuki

    2004-01-01

    The present article describes the synthesis of the N-(Lys-Gly-Tyr-Gly)-chitosan using the water-soluble active ester method, the preparation of the N-(Lys-Gly-Tyr-Gly)-chitosan-gellan hybrid fibers, and the reinforcement of the hybrid fibers by enzymatic cross-linking between the N-grafted peptides chains of chitosan. The cationic polysaccharide chitosan was treated with Boc-Lys(Z)-Gly-Tyr(Bzl)-Gly (4-hydroxyphenyl)dimethylsulfonium methyl sulfate ester in DMF-0.15 M acetic acid to incorporate the peptides into the side chain amino groups of chitosan followed by the acidic removals of the Z and Bzl groups. The degrees of N substitution were estimated to be 2.0 and 10 molar % by changing the molar ratios of the amino groups of the parent chitosan and the active ester. The resulting cationic N-(Lys-Gly-Tyr-Gly)-chitosan was spun into the hybrid fibers with the anionic polysaccharide gellan in water. The tensile strengths of the N-(Lys-Gly-Tyr-Gly)-chitosan hybrid fibers were superior to those of the original chitosan-gellan fibers. The mechanical strengths of the hybrid fibers further increased upon enzymatic oxidation using tyrosinase. Based on these results, we concluded that the covalent cross-linking due to the enzyme oxidation between the grafted peptides significantly contributed to reinforcement of the polysaccharide hybrid fibers. The present results afford a new methodology for the reinforcement achieved by the polymer modification inspired by a biological process. PMID:15002994

  20. Histone cross-linking by transglutaminase.

    PubMed

    Kim, Jae-Hong; Nam, Kang Hoon; Kwon, Oh-Seok; Kim, In Gyu; Bustin, Michael; Choy, Hyon E; Park, Sang Chul

    2002-05-24

    Transglutaminases irreversibly catalyze covalent cross-linking of proteins by forming isopeptide bonds between peptide-bound glutamine and lysine residues. Among several transglutaminases, tissue-type transglutaminase (tTGase) is most ubiquitously found in every type of cells and tissues in animals, but its natural substrate has yet to be identified. In an attempt to identify the natural substrate for tTGase, we examined in vitro if core histones were subject to cross-linking by tTGase. We found core histone subunits, H2A and H2B, were specifically cross-linked by tTGase. The cross-linking was between either one or both glutamines at C-terminal end of H2A (-VTIAQ104 GGVLPNTQ112 SVLLPKKTESSKSK-C' end) and the first and/or third lysine from C-terminal end of H2B (-AVESEGK116 AVTKYTSSK125-C' end). The cross-linking occurred only when these subunits were released from nucleosome but not when these were organized in nucleosome. Most interestingly, in chicken erythrocyte the cross-linked H2A-H2B was present in a significant amount. From these results, it can be proposed that tTGase-mediated cross-linking is an another form of core histone modification and it may play a role of chromatin condensation during erythrocyte differentiation. PMID:12054678

  1. 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.

  2. Structural dynamics of an actin spring.

    PubMed

    Mahadevan, L; Riera, C S; Shin, Jennifer H

    2011-02-16

    Actin-based motility in cells is usually associated with either polymerization/depolymerization in the presence of cross-linkers or contractility in the presence of myosin motors. Here, we focus on a third distinct mechanism involving actin in motility, seen in the dynamics of an active actin spring that powers the acrosomal reaction of the horseshoe crab (Limulus polyphemus) sperm. During this process, a 60-μm bent and twisted bundle of cross-linked actin uncoils and becomes straight in a few seconds in the presence of Ca(2+). This straightening, which occurs at a constant velocity, allows the acrosome to forcefully penetrate the egg. Synthesizing ultrastructural information with the kinetics, energetics, and imaging of calcium binding allows us to construct a dynamical theory for this mechanochemical engine consistent with our experimental observations. It also illuminates the general mechanism by which energy may be stored in conformational changes and released cooperatively in ordered macromolecular assemblies. PMID:21320427

  3. Physicochemical properties and bioactivity of nisin-containing cross-linked hydroxypropylmethylcellulose films.

    PubMed

    Sebti, Issam; Delves-Broughton, John; Coma, Véronique

    2003-10-22

    Cross-linked hydroxypropylmethylcellulose (HPMC) cast films with citric acid as polycarboxylic cross-linker were elaborated to study the effect of cross-linking level on various properties. Increased amounts of cross-linking agent were not connected to statistically different tensile strength and Young's modulus. Whatever the cross-linking level of the film was, the ultimate elongation parameter decreased by approximately 60% compared to the HMPC control film. Moisture sorption isotherms and water contact angle meter showed that the effect of cross-linking degree tends to reduce the hygroscopic and hydrophilic characteristics of films. In addition, to control bacteria growth on food surfaces, the antimicrobial activity of both 98% cross-linked HPMC-nisin and control HPMC-nisin films was tested on Micrococcus luteus. Despite the incorporation of a significant content of nisin, cross-linked HPMC-nisin films were completely inactive on the microbial strain compared to the HPMC-nisin control films. Cross-linking conditions likely either denatured the nisin or irreversibly bound nisin to the cross-linked HPMC. However, nisin adsorbed into films made from previously cross-linked HPMC maintained its activity. PMID:14558764

  4. Loss of Complex I activity in the Escherichia coli enzyme results from truncating the C-terminus of subunit K, but not from cross-linking it to subunits N or L.

    PubMed

    Zhu, Shaotong; Canales, Alejandra; Bedair, Mai; Vik, Steven B

    2016-06-01

    Complex I is a multi-subunit enzyme of the respiratory chain with seven core subunits in its membrane arm (A, H, J, K, L, M, and N). In the enzyme from Escherichia coli the C-terminal ten amino acids of subunit K lie along the lateral helix of subunit L, and contribute to a junction of subunits K, L and N on the cytoplasmic surface. Using double cysteine mutagenesis, the cross-linking of subunit K (R99C) to either subunit L (K581C) or subunit N (T292C) was attempted. A partial yield of cross-linked product had no effect on the activity of the enzyme, or on proton translocation, suggesting that the C-terminus of subunit K has no dynamic role in function. To further elucidate the role of subunit K genetic deletions were constructed at the C-terminus. Upon the serial deletion of the last 4 residues of the C-terminus of subunit K, various results were obtained. Deletion of one amino acid had little effect on the activity of Complex I, but deletions of 2 or more amino acids led to total loss of enzyme activity and diminished levels of subunits L, M, and N in preparations of membrane vesicles. Together these results suggest that while the C-terminus of subunit K has no dynamic role in energy transduction by Complex I, it is vital for the correct assembly of the enzyme. PMID:26931547

  5. Carrier free co-immobilization of alpha amylase, glucoamylase and pullulanase as combined cross-linked enzyme aggregates (combi-CLEAs): a tri-enzyme biocatalyst with one pot starch hydrolytic activity.

    PubMed

    Talekar, Sachin; Pandharbale, Amol; Ladole, Mayur; Nadar, Shamraja; Mulla, Mosin; Japhalekar, Kshitija; Pattankude, Kishori; Arage, Devika

    2013-11-01

    A tri-enzyme biocatalyst "combi-CLEAs" with starch hydrolytic activity was prepared from commercially available alpha amylase, glucoamylase and pullulanase preparations by aggregating enzymes with ammonium sulphate followed by cross-linking formed aggregates for 4.5h with 40 mM glutaraldehyde. The effects of precipitant type and cross-linking were studied and the biocatalyst was characterized. Scanning electron microscopy analysis showed that tri-enzyme biocatalyst was of spherical structure. For one pot starch hydrolytic activity, shift in optimum pH from 6 to 7 and temperature from 65 to 75 °C were observed after co-immobilization of enzymes. After one pot starch hydrolysis reaction in batch mode, 100%, 60% and 40% conversions were obtained with combi-CLEAs, separate CLEAs mixture and free enzyme mixture, respectively. Co-immobilization also enhanced the thermal stability of enzymes. Finally, the catalytic activity of enzymes in combi-CLEAs during one pot starch hydrolysis was well maintained up to five cycles without performance changes. PMID:23999260

  6. 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.

  7. 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.

  8. Alpha-Smooth Muscle Actin Expression Upregulates Fibroblast Contractile Activity

    PubMed Central

    Hinz, Boris; Celetta, Giuseppe; Tomasek, James J.; Gabbiani, Giulio; Chaponnier, Christine

    2001-01-01

    To evaluate whether α-smooth muscle actin (α-SMA) plays a role in fibroblast contractility, we first compared the contractile activity of rat subcutaneous fibroblasts (SCFs), expressing low levels of α-SMA, with that of lung fibroblasts (LFs), expressing high levels of α-SMA, with the use of silicone substrates of different stiffness degrees. On medium stiffness substrates the percentage of cells producing wrinkles was similar to that of α-SMA–positive cells in each fibroblast population. On high stiffness substrates, wrinkle production was limited to a subpopulation of LFs very positive for α-SMA. In a second approach, we measured the isotonic contraction of SCF- and LF-populated attached collagen lattices. SCFs exhibited 41% diameter reduction compared with 63% by LFs. TGFβ1 increased α-SMA expression and lattice contraction by SCFs to the levels of LFs; TGFβ-antagonizing agents reduced α-SMA expression and lattice contraction by LFs to the level of SCFs. Finally, 3T3 fibroblasts transiently or permanently transfected with α-SMA cDNA exhibited a significantly higher lattice contraction compared with wild-type 3T3 fibroblasts or to fibroblasts transfected with α-cardiac and β- or γ-cytoplasmic actin. This took place in the absence of any change in smooth muscle or nonmuscle myosin heavy-chain expression. Our results indicate that an increased α-SMA expression is sufficient to enhance fibroblast contractile activity. PMID:11553712

  9. Cross-linking Chemistry of Squid Beak*

    PubMed Central

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

    2010-01-01

    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 (1H 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. PMID:20870720

  10. Cross-linking chemistry of squid beak.

    PubMed

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

    2010-12-01

    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. PMID:20870720

  11. Visible-Light Photocatalyzed Cross-Linking of Diacetylene Ligands by Quantum Dots to Improve Their Aqueous Colloidal Stability

    PubMed Central

    2015-01-01

    Ligand cross-linking is known to improve the colloidal stability of nanoparticles, particularly in aqueous solutions. However, most cross-linking is performed chemically, in which it is difficult to limit interparticle cross-linking, unless performed at low concentrations. Photochemical cross-linking is a promising approach but usually requires ultraviolet (UV) light to initiate. Using such high-energy photons can be harmful to systems in which the ligand–nanoparticle bond is fairly weak, as is the case for the commonly used semiconductor quantum dots (QDs). Here, we introduce a novel approach to cross-link thiolated ligands on QDs by utilizing the photocatalytic activity of QDs upon absorbing visible light. We show that using visible light leads to better ligand cross-linking by avoiding the problem of ligand dissociation that occurs upon UV light exposure. Once cross-linked, the ligands significantly enhance the colloidal stability of those same QDs that facilitated cross-linking. PMID:25036275

  12. Specific covalent immobilization of proteins through dityrosine cross-links.

    PubMed

    Endrizzi, Betsy J; Huang, Gang; Kiser, Patrick F; Stewart, Russell J

    2006-12-19

    Dityrosine cross-links are widely observed in nature in structural proteins such as elastin and silk. Natural oxidative cross-linking between tyrosine residues is catalyzed by a diverse group of metalloenzymes. Dityrosine formation is also catalyzed in vitro by metal-peptide complexes such as Gly-Gly-His-Ni(II). On the basis of these observations, a system was developed to specifically and covalently surface immobilize proteins through dityrosine cross-links. Methacrylate monomers of the catalytic peptide Gly-Gly-His-Tyr-OH (GGHY) and the Ni(II)-chelating group nitrilotriacetic acid (NTA) were copolymerized with acrylamide into microbeads. Green fluorescent protein (GFP), as a model protein, was genetically tagged with a tyrosine-modified His6 peptide on its carboxy terminus. GFP-YGH6, specifically associated with the NTA-Ni(II) groups, was covalently coupled to the bead surface through dityrosine bond formation catalyzed by the colocalized GGHY-Ni(II) complex. After extensive washing with EDTA to disrupt metal coordination bonds, we observed that up to 75% of the initially bound GFP-YGH6 remained covalently bound to the bead while retaining its structure and activity. Dityrosine cross-linking was confirmed by quenching the reaction with free tyrosine. The method may find particular utility in the construction and optimization of protein microarrays. PMID:17154619

  13. Hybridization triggered cross-linking of deoxyoligonucleotides.

    PubMed Central

    Webb, T R; Matteucci, M D

    1986-01-01

    This paper reports details of the synthesis of oligodeoxynucleotides containing the modified base 5-methyl-N4,N4-ethanocytosine (Ce). The 9-fluorenylmethoxycarbonyl group is used as a protecting group for the exocyclic amines of dA and dC. This group can be removed rapidly under very mild conditions. Oligomers containing the Ce base form a cross-link when hybridized to their complementary deoxyoligonucleotides. Some of the scope and limitations of these cross-link forming oligonucleotides are reported. Images PMID:3774542

  14. VASP Activation via the Gα13/RhoA/PKA Pathway Mediates Cucurbitacin-B-Induced Actin Aggregation and Cofilin-Actin Rod Formation

    PubMed Central

    Zhang, Yan-Ting; Xu, Li-Hui; Lu, Qun; Liu, Kun-Peng; Liu, Pei-Yan; Ji, Fang; Liu, Xiao-Ming; Ouyang, Dong-Yun; He, Xian-Hui

    2014-01-01

    Cucurbitacin B (CuB), a potent antineoplastic agent of cucurbitacin triterpenoids, induces rapid disruption of actin cytoskeleton and aberrant cell cycle inhibiting carcinogenesis. However, the underlying molecular mechanism of such anticancer effects remains incompletely understood. In this study, we showed that CuB treatment rapidly induced vasodilator-stimulated phosphoprotein (VASP) phosphorylation (i.e. activation) at the Ser157 residue and generated VASP clumps which were co-localized with amorphous actin aggregates prior to the formation of highly-ordered cofilin-actin rods in melanoma cells. Knockdown of VASP or inhibition of VASP activation using PKA-specific inhibitor H89 suppressed CuB-induced VASP activation, actin aggregation and cofilin-actin rod formation. The VASP activation was mediated by cAMP-independent PKA activation as CuB decreased the levels of cAMP while MDL12330A, an inhibitor of adenylyl cyclase, had weak effect on VASP activation. Knockdown of either Gα13 or RhoA not only suppressed VASP activation, but also ameliorated CuB-induced actin aggregation and abrogated cofilin-actin rod formation. Collectively, our studies highlighted that the CuB-induced actin aggregation and cofilin-actin rod formation was mediated via the Gα13/RhoA/PKA/VASP pathway. PMID:24691407

  15. Covalent cross-links in polyampholytic chitosan fibers enhances bone regeneration in a rabbit model.

    PubMed

    Ghosh, Paulomi; Rameshbabu, Arun Prabhu; Das, Dipankar; Francis, Nimmy K; Pawar, Harpreet Singh; Subramanian, Bhuvaneshwaran; Pal, Sagar; Dhara, Santanu

    2015-01-01

    Chitosan fibers were prepared in citric acid bath, pH 7.4 and NaOH solution at pH 13, to form ionotropically cross-linked and uncross-linked fibers, respectively. The fibers formed in citric acid bath were further cross-linked via carbodiimide chemistry; wherein the pendant carboxyl moieties of citric acid were used for new amide bond formation. Moreover, upon covalent cross-linking in the ionically gelled citrate-chitosan fibers, incomplete conversion of the ion pairs to amide linkages took place resulting in the formation of a dual network structure. The dual cross-linked fibers displayed improved mechanical property, higher stability against enzymatic degradation, hydrophobicity and superior bio-mineralization compared to the uncross-linked and native citrate cross-linked fibers. Additionally, upon cyclic loading, the ion pairs in the dual cross-linked fibers dissociated by dissipating energy and reformed during the relaxation period. The twin property of elasticity and energy dissipation mechanism makes the dual cross-linked fiber unique under dynamic mechanical conditions. The differences in the physico-chemical characteristics were reflected in protein adsorption, which in turn influenced the cellular activities on the fibers. Compared to the uncross-linked and ionotropically cross-linked fibers, the dual cross-linked fibers demonstrated higher proliferation and osteogenic differentiation of the MSCs in vitro as well as better osseous tissue regeneration in a rabbit model. PMID:25483844

  16. Stretching and bending in cross-linked biopolymer networks

    NASA Astrophysics Data System (ADS)

    Heussinger, Claus; Frey, Erwin

    2007-03-01

    The elastic response of cross-linked biopolymer networks is usually interpreted in terms of affine stretching models, adopted from the theory of rubber-elasticity valid for flexible polymer gels. Unlike flexible polymers, however, stiff polymers have a highly anisotropic elastic response, where the low-energy elastic excitations are actually of bending nature. As a consequence, similar to springs connected in series, one would expect the softer bending mode to dominate the elastic energy rather than the stiff stretching mode. We propose a theory that, unlike recent affine models, properly accounts for the soft bending response of stiff polymers. It allows calculating the macroscopic elastic moduli starting from a microscopic characterization of the (non-affine) deformation field. The calculated scaling properties for the shear modulus are in excellent agreement with the results of recent simulations obtained in simple two-dimensional model networks, and can also be applied to rationalize bulk rheological data in reconstituted actin networks.

  17. TAGLN2 regulates T cell activation by stabilizing the actin cytoskeleton at the immunological synapse

    PubMed Central

    Na, Bo-Ra; Kim, Hye-Ran; Piragyte, Indre; Oh, Hyun-Mee; Kwon, Min-Sung; Akber, Uroos; Lee, Hyun-Su; Park, Do-Sim; Song, Woo Keun; Park, Zee-Yong; Im, Sin-Hyeog; Rho, Mun-Chual; Hyun, Young-Min; Kim, Minsoo

    2015-01-01

    The formation of an immunological synapse (IS) requires tight regulation of actin dynamics by many actin polymerizing/depolymerizing proteins. However, the significance of actin stabilization at the IS remains largely unknown. In this paper, we identify a novel function of TAGLN2—an actin-binding protein predominantly expressed in T cells—in stabilizing cortical F-actin, thereby maintaining F-actin contents at the IS and acquiring LFA-1 (leukocyte function-associated antigen-1) activation after T cell receptor stimulation. TAGLN2 blocks actin depolymerization and competes with cofilin both in vitro and in vivo. Knockout of TAGLN2 (TAGLN2−/−) reduced F-actin content and destabilized F-actin ring formation, resulting in decreased cell adhesion and spreading. TAGLN2−/− T cells displayed weakened cytokine production and cytotoxic effector function. These findings reveal a novel function of TAGLN2 in enhancing T cell responses by controlling actin stability at the IS. PMID:25869671

  18. 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

  19. 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.

  20. 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

  1. Fullerenol Nanoparticles with Structural Activity Induce Variable Intracellular Actin Filament Morphologies.

    PubMed

    Jin, Junjiang; Dong, Ying; Wang, Ying; Xia, Lin; Gu, Weihong; Bai, Xue; Chang, Yanan; Zhang, Mingyi; Chen, Kui; Li, Juan; Zhao, Lina; Xing, Gengmei

    2016-06-01

    Fullerenol nanoparticles are promising for various biological applications; many studies have shown that they induce variable and diverse biological effects including side effects. Separation and purification of two fractions of fullerenols has demonstrated that they have varied chemical structures on the surfaces of their carbon cages. Actin is an important structural protein that is able to transform functional structures under varied physiological conditions. We assessed the abilities of the two fractions of fullerenols to attach to actin and induce variable morphological features in actin filament structures. Specifically the fullerenol fraction with a surface electric charge of -1.913 ± 0.008q (x10(-6) C) has percentages of C-OH and C=O on the carbon cage of 16.14 ± 0.60 and 17.55 ± 0.69. These features allow it to form intermolecular hydrogen bonds with actin at a stoichiometric ratio of four fullerenols per actin subunit. Molecular simulations revealed these specific binding sites and binding modes in atomic details in the interaction between the active fullerenol and actin filament. Conversely, these interactions were not possible for the other fraction of fullerenol with that percentages of C-OH and C=O on the carbon cage were 15.59 ± 0.01 and 1.94 ± 0.11. Neither sample induced appreciable cytotoxicity or acute cell death. After entering cells, active fullerenol binding to actin induces variable morphological features and may transform ATP-actin to ADP-actin. These changes facilitate the binding of ADF/cofilin, allowing cofilin to sever actin filaments to form cofilin/actin/fullerenol rods. Our findings suggest that fullerenol with structural activity binding disturbs actin filament structure, which may inhibit locomotion of cell or induce chronic side effects in to cells. PMID:27319217

  2. 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. PMID:26777253

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

    PubMed Central

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

    2015-01-01

    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. PMID:25697790

  4. 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.

  5. The catalytic domain of inositol-1,4,5-trisphosphate 3-kinase-a contributes to ITPKA-induced modulation of F-actin.

    PubMed

    Ashour, Dina Julia; Pelka, Benjamin; Jaaks, Patricia; Wundenberg, Torsten; Blechner, Christine; Zobiak, Bernd; Failla, Antonio Virgilio; Windhorst, Sabine

    2015-02-01

    Inositol-1,4,5-trisphosphate-3-kinase-A (ITPKA) has been considered as an actin bundling protein because its N-terminal actin binding domain (ABD) induces formation of linear actin bundles. Since in many cancer cell lines ITPKA is essential for formation of lamellipodia, which consist of cross-linked actin filaments, here we analyzed if full length-ITPKA may induce formation of more complex actin structures. Indeed, we found that incubation of F-actin with ITPKA resulted in formation of dense, branched actin networks. Based on our result that ITPKA does not exhibit an additional C-terminal ABD, we exclude that ITPKA cross-links actin filaments by simultaneous F-actin binding with two different ABDs. Instead, stimulated-emission-depletion-microscopy and measurement of InsP3 Kinase activity give evidence that that N-terminal ABD-homodimers of ITPKA bind to F-actin while the monomeric C-termini insert between adjacent actin filaments. Thereby, they prevent formation of thick actin bundles but induce formation of thin branched actin structures. Interestingly, when embedded in this dense actin network, InsP3 Kinase activity is doubled and the product of InsP3 Kinase activity, Ins(1,3,4,5)P4 , inhibits spontaneous actin polymerization which may reflect a local negative feedback regulation of InsP3 Kinase activity. In conclusion, we demonstrate that not only the ABD of ITPKA modulates actin dynamics but reveal that the InsP3 Kinase domain substantially contributes to this process. PMID:25620569

  6. Development of a 3'-amino linker with high conjugation activity and its application to conveniently cross-link blunt ends of a duplex.

    PubMed

    Kowata, Keiko; Kojima, Naoshi; Komatsu, Yasuo

    2016-05-01

    The 2-aminoethyl carbamate linker (ssH linker) exhibits high activity in modifying the 5'-termini of oligonucleotides; however, the ssH linker is not appropriate for 3'-terminal modification because it undergoes intramolecular trans-acylation under heat-aqueous ammonia conditions. We developed an N-(2-aminoethyl)carbamate linker (revH linker), in which the carbamate is oriented in the reverse direction relative to that in 2-aminoethyl carbamate. The revH linker was tolerant to heat-alkaline conditions and retained its high reactivity in conjugation with exogenous molecules. The 3'-revH linker was efficiently linked with the 5'-ssH linker at the termini of complementary double strands with a bifunctional molecule, producing a synthetic loop structure. An anti-microRNA oligonucleotide (AMO) was prepared from the chemical ligation of three-stranded 2'-O-methyl RNAs, and the AMO with two alkyl loops exhibited high inhibition activity toward miRNA function. The revH linker is not only useful for 3'-terminal modification of oligonucleotides but also expands the utility range in combination with the 5'-ssH linker. PMID:27041396

  7. Photoinduced DNA Interstrand Cross-Link Formation by Naphthalene Boronates via a Carbocation.

    PubMed

    Wang, Yibin; Lin, Zechao; Fan, Heli; Peng, Xiaohua

    2016-07-18

    Most photoinduced DNA cross-link formation by a bifunctional aryl derivative is through a bisquinone methide. DNA cross-linking via a bisarylcarbocation remains a less explored area. We designed and synthesized a series of naphthalene boronates that produce DNA interstrand cross-links via a carbocation upon UV irradiation. A free radical was generated from the naphthalene boronates with 350 nm irradiation and further converted to a carbocation by electron transfer. The activation mechanism was determined using the orthogonal traps, 2,2,6,6-tetramethylpiperidin-1-oxyl (TEMPO) and methoxyamine that react with either the free radical or the carbocation but not both. This represents a novel example of photoinduced DNA cross-link formation via carbocations generated from a bisaryl derivative. This work provides information useful for the design of novel photoactivated DNA cross-linking agents. PMID:27189512

  8. 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.

  9. 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

  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. Force Feedback Controls Motor Activity and Mechanical Properties of Self-Assembling Branched Actin Networks.

    PubMed

    Bieling, Peter; Li, Tai-De; Weichsel, Julian; McGorty, Ryan; Jreij, Pamela; Huang, Bo; Fletcher, Daniel A; Mullins, R Dyche

    2016-01-14

    Branched actin networks--created by the Arp2/3 complex, capping protein, and a nucleation promoting factor--generate and transmit forces required for many cellular processes, but their response to force is poorly understood. To address this, we assembled branched actin networks in vitro from purified components and used simultaneous fluorescence and atomic force microscopy to quantify their molecular composition and material properties under various forces. Remarkably, mechanical loading of these self-assembling materials increases their density, power, and efficiency. Microscopically, increased density reflects increased filament number and altered geometry but no change in average length. Macroscopically, increased density enhances network stiffness and resistance to mechanical failure beyond those of isotropic actin networks. These effects endow branched actin networks with memory of their mechanical history that shapes their material properties and motor activity. This work reveals intrinsic force feedback mechanisms by which mechanical resistance makes self-assembling actin networks stiffer, stronger, and more powerful. PMID:26771487

  12. Rapid Glucose Depletion Immobilizes Active Myosin-V on Stabilized Actin Cables

    PubMed Central

    Xu, Li; Bretscher, Anthony

    2014-01-01

    Summary Polarization of eukaryotic cells requires organelles and protein complexes to be transported to their proper destinations along the cytoskeleton [1]. When nutrients are abundant, budding yeast grows rapidly transporting secretory vesicles for localized growth and actively segregating organelles [2, 3]. This is mediated by myosin-Vs transporting cargos along F-actin bundles known as actin cables [4]. Actin cables are dynamic structures regulated by assembly, stabilization and disassembly [5]. Polarized growth and actin filament dynamics consume energy. For most organisms, glucose is the preferred energy source and generally represses alternative carbon source usage [6]. Thus upon abrupt glucose depletion, yeast shuts down pathways consuming large amounts of energy, including the vacuolar-ATPase [7, 8], translation [9] and phosphoinositide metabolism [10]. Here we show that glucose withdrawal rapidly (<1 min) depletes ATP levels and the yeast myosin V, Myo2, responds by relocalizing to actin cables, making it the fastest response documented. Myo2 immobilized on cables releases its secretory cargo, defining a new rigor-like state of a myosin-V in vivo. Only actively transporting Myo2 can be converted to the rigor-like state. Glucose depletion has differential effects on the actin cytoskeleton resulting in disassembly of actin patches with concomitant inhibition of endocytosis, and strong stabilization of actin cables, thereby revealing a selective and previously unappreciated ATP requirement for actin cable disassembly. A similar response is seen in HeLa cells to ATP depletion. These findings reveal a new fast-acting energy conservation strategy halting growth by immobilizing myosin-V in a newly described state on selectively stabilized actin cables. PMID:25308080

  13. Cross-linked Bioreducible Layer-by-layer Films for Increased Cell Adhesion and Transgene Expression

    PubMed Central

    Blacklock, Jenifer; Sievers, Torsten K.; Handa, Hitesh; You, Ye-Zi; Oupický, David; Mao, Guangzhao; Möhwald, Helmuth

    2010-01-01

    The effect of cross-linking layer-by-layer (LbL) films consisting of bioreducible poly(2-dimethylaminoethyl methacrylate) (rPDMAEMA) and DNA is examined with regards to rigidity, biodegradability, cell adhesion, and transfection activity using 1,5-diiodopentane (DIP) cross-linker. DIP chemically reacts with the tertiary amines of rPDMAEMA, altering the chemical composition of these LbL films. The result is a change in surface morphology, film swelling behavior and film rigidity, measured with AFM and ellipsometry. It is found that the apparent Young’s modulus is increased more than four times its original value upon cross-linking. Cross-linking mass is additionally confirmed with quartz crystal microbalance with dissipation (QCM-D). Comprehensive analyses of these experimental values were investigated to calculate the degree of cross-linking using the rubber elasticity theory and the Flory-Rehner theory. Additionally, the Flory-Huggins parameter, χ, was calculated. Good agreement in the two methods yields a cross-linking density of ~0.82 mmol/cm3. The Flory-Huggins parameter increased upon cross-linking from 1.07 to 1.2, indicating increased hydrophobicity of the network and formation of bulk water droplets within the films. In addition, the effects of cross-linking on film disassembly by 1,4-dithiothreitol (DTT) is found to be insignificant despite the alteration in film rigidity. Mouse fibroblast cells and smooth muscle cells are used to study the effect of cross-linking on cell adhesion and cell transfection activity. In vitro transfection activity up to seven days is quantified using secreted alkaline phosphatase (SEAP) DNA. Film cross-linking is found to enhance cell adhesion and prolong the duration of cellular transfection. These results contribute to the development of bioreducible polymer coatings for localized gene delivery. PMID:20369813

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

    PubMed

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

    2015-08-01

    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

  15. 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

  16. 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. PMID:25000866

  17. 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

  18. 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

  19. 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

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

    2012-10-12

    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(+)Actin(high). In contrast, CD45RA T cells are phenotypically CD45RA(+)Actin(low). 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

  20. Regulation of water flow by actin-binding protein-induced actin gelatin.

    PubMed Central

    Ito, T; Suzuki, A; Stossel, T P

    1992-01-01

    Actin filaments inhibit osmotically driven water flow (Ito, T., K.S. Zaner, and T.P. Stossel. 1987. Biophys. J. 51: 745-753). Here we show that the actin gelation protein, actin-binding protein (ABP), impedes both osmotic shrinkage and swelling of an actin filament solution and reduces markedly the concentration of actin filaments required for this inhibition. These effects depend on actin filament immobilization, because the ABP concentration that causes initial impairment of water flow by actin filaments corresponds to the gel point measured viscometrically and because gelsolin, which noncovalently severs actin filaments, solates actin gels and restores water flow in a solution of actin cross-linked by ABP. Since ABP gels actin filaments in the periphery of many eukaryotic cells, such actin networks may contribute to physiological cell volume regulation. PMID:1318095

  1. Dephosphorylated synapsin I anchors synaptic vesicles to actin cytoskeleton: an analysis by videomicroscopy.

    PubMed

    Ceccaldi, P E; Grohovaz, F; Benfenati, F; Chieregatti, E; Greengard, P; Valtorta, F

    1995-03-01

    Synapsin I is a synaptic vesicle-associated protein which inhibits neurotransmitter release, an effect which is abolished upon its phosphorylation by Ca2+/calmodulin-dependent protein kinase II (CaM kinase II). Based on indirect evidence, it was suggested that this effect on neurotransmitter release may be achieved by the reversible anchoring of synaptic vesicles to the actin cytoskeleton of the nerve terminal. Using video-enhanced microscopy, we have now obtained experimental evidence in support of this model: the presence of dephosphorylated synapsin I is necessary for synaptic vesicles to bind actin; synapsin I is able to promote actin polymerization and bundling of actin filaments in the presence of synaptic vesicles; the ability to cross-link synaptic vesicles and actin is specific for synapsin I and is not shared by other basic proteins; the cross-linking between synaptic vesicles and actin is specific for the membrane of synaptic vesicles and does not reflect either a non-specific binding of membranes to the highly surface active synapsin I molecule or trapping of vesicles within the thick bundles of actin filaments; the formation of the ternary complex is virtually abolished when synapsin I is phosphorylated by CaM kinase II. The data indicate that synapsin I markedly affects synaptic vesicle traffic and cytoskeleton assembly in the nerve terminal and provide a molecular basis for the ability of synapsin I to regulate the availability of synaptic vesicles for exocytosis and thereby the efficiency of neurotransmitter release. PMID:7876313

  2. 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

  3. Riboflavin for corneal cross-linking.

    PubMed

    O'Brart, D P S

    2016-06-01

    Corneal collagen cross-linking (CXL) with riboflavin and ultraviolet A (UVA) radiation is the first therapeutic modality that appears to arrest the progression of keratoconus and other corneal ectasias. Riboflavin is central to the process, acting as a photosensitizer for the production of oxygen singlets and riboflavin triplets. These free radicals drive the CXL process within the proteins of the corneal stroma, altering its biomechanical properties. Riboflavin also absorbs the majority of the UVA radiation, which is potentially cytotoxic and mutagenic, within the anterior stroma, preventing damage to internal ocular structures, such as the corneal endothelium, lens and retina. Clinical studies report cessation of ectatic progression in over 90% of cases and the majority document significant improvements in visual, keratometric and topographic parameters. Clinical follow-up is limited to 5-10 years, but suggests sustained stability and enhancement in corneal shape. Sight-threatening complications are rare. The optimal stromal riboflavin dosage for CXL is as yet undetermined. PMID:27458610

  4. 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

  5. Chloride channel activity of ClC-2 is modified by the actin cytoskeleton.

    PubMed Central

    Ahmed, N; Ramjeesingh, M; Wong, S; Varga, A; Garami, E; Bear, C E

    2000-01-01

    The chloride channel ClC-2 has been implicated in essential physiological functions, including cell-volume regulation and fluid secretion by specific epithelial tissues. Although ClC-2 is known to be activated by hyperpolarization and hypo-osmotic shock, the molecular basis for the regulation of this channel remains unclear. Here we show in the Xenopus oocyte expression system that the chloride-channel activity of ClC-2 is enhanced after treatment with the actin-disrupting agents cytochalasin and latrunkulin. These findings suggest that the actin cytoskeleton normally exerts an inhibitory effect on ClC-2 activity. An inhibitory domain was previously defined in the N-terminus of ClC-2, so we sought to determine whether this domain might interact directly with actin in binding assays in vitro. We found that a glutathione S-transferase fusion protein containing the inhibitory domain was capable of binding actin in overlay and co-sedimentation assays. Further, the binding of actin to this relatively basic peptide (pI 8.4) might be mediated through electrostatic interactions because binding was inhibited at high concentrations of NaCl with a half-maximal decrease in signal at 180 mM NaCl. This work suggests that electrostatic interactions between the N-terminus of ClC-2 and the actin cytoskeleton might have a role in the regulation of this channel. PMID:11104687

  6. Differential Actin-regulatory Activities of Tropomodulin1 and Tropomodulin3 with Diverse Tropomyosin and Actin Isoforms*

    PubMed Central

    Yamashiro, Sawako; Gokhin, David S.; Sui, Zhenhua; Bergeron, Sarah E.; Rubenstein, Peter A.; Fowler, Velia M.

    2014-01-01

    Tropomodulins (Tmods) are F-actin pointed end capping proteins that interact with tropomyosins (TMs) and cap TM-coated filaments with higher affinity than TM-free filaments. Here, we tested whether differences in recognition of TM or actin isoforms by Tmod1 and Tmod3 contribute to the distinct cellular functions of these Tmods. We found that Tmod3 bound ∼5-fold more weakly than Tmod1 to α/βTM, TM5b, and TM5NM1. However, surprisingly, Tmod3 was as effective as Tmod1 at capping pointed ends of skeletal muscle α-actin (αsk-actin) filaments coated with α/βTM, TM5b, or TM5NM1. Tmod3 only capped TM-coated αsk-actin filaments more weakly than Tmod1 in the presence of recombinant αTM2, which is unacetylated at its NH2 terminus, binds F-actin weakly, and has a disabled Tmod-binding site. Moreover, both Tmod1 and Tmod3 were similarly effective at capping pointed ends of platelet β/cytoplasmic γ (γcyto)-actin filaments coated with TM5NM1. In the absence of TMs, both Tmod1 and Tmod3 had similarly weak abilities to nucleate β/γcyto-actin filament assembly, but only Tmod3 could sequester cytoplasmic β- and γcyto-actin (but not αsk-actin) monomers and prevent polymerization under physiological conditions. Thus, differences in TM binding by Tmod1 and Tmod3 do not appear to regulate the abilities of these Tmods to cap TM-αsk-actin or TM-β/γcyto-actin pointed ends and, thus, are unlikely to determine selective co-assembly of Tmod, TM, and actin isoforms in different cell types and cytoskeletal structures. The ability of Tmod3 to sequester β- and γcyto-actin (but not αsk-actin) monomers in the absence of TMs suggests a novel function for Tmod3 in regulating actin remodeling or turnover in cells. PMID:24644292

  7. Curcumin cross-linked collagen aerogels with controlled anti-proteolytic and pro-angiogenic efficacy.

    PubMed

    Dharunya, G; Duraipandy, N; Lakra, Rachita; Korapatti, Purna Sai; Jayavel, R; Kiran, Manikantan Syamala

    2016-01-01

    This paper elucidates the development of a curcumin cross-linked collagen aerogel system with controlled anti-proteolytic activity and pro-angiogenic efficacy. The results of this study showed that in situ cross-linking of curcumin with collagen leads to the development of aerogels with enhanced physical and mechanical properties. The integrity of collagen after cross-linking with curcumin was studied via FTIR spectroscopy. The results confirmed that the cross-linking with curcumin did not induce any structural changes in the collagen. The curcumin cross-linked collagen aerogels exhibited potent anti-proteolytic and anti-microbial activity. Scanning electron and atomic force microscopic analysis of curcumin cross-linked collagen aerogels showed a 3D microstructure that enhanced the adhesion and proliferation of cells. The highly organized geometry of collagen-curcumin aerogels enhanced the permeability and water-retaining ability required for the diffusion of nutrients that aid cellular growth. The pro-angiogenic properties of collagen-curcumin aerogels were ascribed to the cumulative effect of the nutraceutical and the collagen molecule, which augmented the restoration of damaged tissue. Further, these aerogels exhibited controlled anti-proteolytic activity, which makes them suitable 3D scaffolds for biomedical applications. This study provides scope for the development of biocompatible and bioresorbable collagen aerogel systems that use a nutraceutical as a cross-linker for biomedical applications. PMID:27509047

  8. 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

  9. Steric Effects Induce Geometric Remodeling of Actin Bundles in Filopodia.

    PubMed

    Dobramysl, Ulrich; Papoian, Garegin A; Erban, Radek

    2016-05-10

    Filopodia are ubiquitous fingerlike protrusions, spawned by many eukaryotic cells, to probe and interact with their environments. Polymerization dynamics of actin filaments, comprising the structural core of filopodia, largely determine their instantaneous lengths and overall lifetimes. The polymerization reactions at the filopodial tip require transport of G-actin, which enter the filopodial tube from the filopodial base and diffuse toward the filament barbed ends near the tip. Actin filaments are mechanically coupled into a tight bundle by cross-linker proteins. Interestingly, many of these proteins are relatively short, restricting the free diffusion of cytosolic G-actin throughout the bundle and, in particular, its penetration into the bundle core. To investigate the effect of steric restrictions on G-actin diffusion by the porous structure of filopodial actin filament bundle, we used a particle-based stochastic simulation approach. We discovered that excluded volume interactions result in partial and then full collapse of central filaments in the bundle, leading to a hollowed-out structure. The latter may further collapse radially due to the activity of cross-linking proteins, hence producing conical-shaped filament bundles. Interestingly, electron microscopy experiments on mature filopodia indeed frequently reveal actin bundles that are narrow at the tip and wider at the base. Overall, our work demonstrates that excluded volume effects in the context of reaction-diffusion processes in porous networks may lead to unexpected geometric growth patterns and complicated, history-dependent dynamics of intermediate metastable configurations. PMID:27166814

  10. EXTRACTION AND ANALYSIS OF ACTIN NETWORKS BASED ON OPEN ACTIVE CONTOUR MODELS

    PubMed Central

    Xu, Ting; Li, Hongsheng; Shen, Tian; Ojkic, Nikola; Vavylonis, Dimitrios; Huang, Xiaolei

    2011-01-01

    Network structures formed by actin filaments are present in many kinds of fluorescence microscopy images. In order to quantify the conformations and dynamics of such actin filaments, we propose a fully automated method to extract actin networks from images and analyze network topology. The method handles well intersecting filaments and, to some extent, overlapping filaments. First we automatically initialize a large number of Stretching Open Active Contours (SOACs) from ridge points detected by searching for plus-to-minus sign changes in the gradient map of the image. These initial SOACs then elongate simultaneously along the bright center-lines of filaments by minimizing an energy function. During their evolution, they may merge or stop growing, thus forming a network that represents the topology of the filament ensemble. We further detect junction points in the network and break the SOACs at junctions to obtain “SOAC segments”. These segments are then re-grouped using a graph-cut spectral clustering method to represent the configuration of actin filaments. The proposed approach is generally applicable to extracting intersecting curvilinear structures in noisy images. We demonstrate its potential using two kinds of data: (1) actin filaments imaged by Total Internal Reflection Fluorescence Microscopy (TIRFM) in vitro; (2) actin cytoskeleton networks in fission yeast imaged by spinning disk confocal microscopy. PMID:21822463

  11. EXTRACTION AND ANALYSIS OF ACTIN NETWORKS BASED ON OPEN ACTIVE CONTOUR MODELS.

    PubMed

    Xu, Ting; Li, Hongsheng; Shen, Tian; Ojkic, Nikola; Vavylonis, Dimitrios; Huang, Xiaolei

    2011-03-30

    Network structures formed by actin filaments are present in many kinds of fluorescence microscopy images. In order to quantify the conformations and dynamics of such actin filaments, we propose a fully automated method to extract actin networks from images and analyze network topology. The method handles well intersecting filaments and, to some extent, overlapping filaments. First we automatically initialize a large number of Stretching Open Active Contours (SOACs) from ridge points detected by searching for plus-to-minus sign changes in the gradient map of the image. These initial SOACs then elongate simultaneously along the bright center-lines of filaments by minimizing an energy function. During their evolution, they may merge or stop growing, thus forming a network that represents the topology of the filament ensemble. We further detect junction points in the network and break the SOACs at junctions to obtain "SOAC segments". These segments are then re-grouped using a graph-cut spectral clustering method to represent the configuration of actin filaments. The proposed approach is generally applicable to extracting intersecting curvilinear structures in noisy images. We demonstrate its potential using two kinds of data: (1) actin filaments imaged by Total Internal Reflection Fluorescence Microscopy (TIRFM) in vitro; (2) actin cytoskeleton networks in fission yeast imaged by spinning disk confocal microscopy. PMID:21822463

  12. F-actin dampens NLRP3 inflammasome activity via Flightless-I and LRRFIP2

    PubMed Central

    Burger, Danielle; Fickentscher, Céline; de Moerloose, Philippe; Brandt, Karim J.

    2016-01-01

    NLRP3 and ASC are able to form a large multimeric complex called inflammasome in response to a number danger signals. The NLRP3 inflammasome is required for the activation of caspase-1 and subsequent maturation of pro-IL-1β into active IL-1β. Although the mechanisms regulating the formation and activity of NLRP3 inflammasome are yet not fully elucidated, data suggest that the assembly of NLRP3 inflammasome requires microtubules to induce the proximity of ASC and NLRP3. In this study we show that microfilaments (F-actin) inhibit NLRP3 inflammasome activity and interact with NLRP3 and ASC. We demonstrate that the inhibition depends on the actin polymerization state but not on the active polymerization process. In ATP- or nigericin-activated macrophages, our data further indicate that Flightless-I (FliI) and leucine-rich repeat FliI-interaction protein 2 (LRRFIP2) are required for the co-localization of NLRP3, ASC and F-actin. We also established that the ability of Ca2+ to accentuate the activity of NLRP3 inflammasome is abrogated in FliI- and LRRFIP2-knockdown macrophages, suggesting that Ca2+ signaling requires the presence of FliI and LRRFIP2. Accordingly, we observed that Ca2+/FliI-dependent severing of F-actin suppresses F-actin/FliI/LRRFIP2-dependent NLRP3 inflammasome inhibition leading to increase IL-1β production. Altogether, our results unveil a new function of F-actin in the regulation of NLRP3 inflammasome activity strengthening the importance of cytoskeleton in the regulation of inflammation. PMID:27431477

  13. 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. PMID:26836479

  14. Dual-Cross-Linked Methacrylated Alginate Sub-Microspheres for Intracellular Chemotherapeutic Delivery.

    PubMed

    Fenn, Spencer L; Miao, Tianxin; Scherrer, Ryan M; Oldinski, Rachael A

    2016-07-20

    Intracellular delivery vehicles comprised of methacrylated alginate (Alg-MA) were developed for the internalization and release of doxorubicin hydrochloride (DOX). Alg-MA was synthesized via an anhydrous reaction, and a mixture of Alg-MA and DOX was formed into sub-microspheres using a water/oil emulsion. Covalently cross-linked sub-microspheres were formed via exposure to green light, in order to investigate effects of cross-linking on drug release and cell internalization, compared to traditional techniques, such as ultraviolet (UV) light irradiation. Cross-linking was performed using light exposure alone or in combination with ionic cross-linking using calcium chloride (CaCl2). Alg-MA sub-microsphere diameters were between 88 and 617 nm, and ζ-potentials were between -20 and -37 mV. Using human lung epithelial carcinoma cells (A549) as a model, cellular internalization was confirmed using flow cytometry; different sub-microsphere formulations varied the efficiency of internalization, with UV-cross-linked sub-microspheres achieving the highest internalization percentages. While blank (nonloaded) Alg-MA submicrospheres were noncytotoxic to A549 cells, DOX-loaded sub-microspheres significantly reduced mitochondrial activity after 5 days of culture. Photo-cross-linked Alg-MA sub-microspheres may be a potential chemotherapeutic delivery system for cancer treatment. PMID:27378419

  15. Collagen cross-linking and resorption: effect of glutaraldehyde concentration.

    PubMed

    Roe, S C; Milthorpe, B K; Schindhelm, K

    1990-12-01

    Cross-linked collagen bioprostheses usually are designed to be inert and nonresorbable, resulting in fatigue and wear failure in high-stress environments. Eventual replacement of the implant, although minimizing strength loss during resorption, would result in a graft with reparative ability. Kangaroo tail tendon (KTT) partially cross-linked with glutaraldehyde (GA) was evaluated in vitro for resistance to bacterial collagenase digestion and in vivo for biocompatibility and resorbability in an intramuscular implant assay. Cross-linking was quantified by thermal denaturation studies. Incomplete cross-linking was achieved with concentrations of GA less than 0.1% (w/v). KTT cross-linked in greater than or equal to 0.05% GA were collagenase resistant being incompletely digested after 240 h. Cross-linking of KTT with low concentrations of GA resulted in partial collagenase resistance and slowed resorption. PMID:2126427

  16. Corneal Cross-Linking and Safety Issues

    PubMed Central

    Spoerl, Eberhard; Hoyer, Anne; Pillunat, Lutz E; Raiskup, Frederik

    2011-01-01

    Purpose: To compile the safety aspects of the corneal collagen cross-linking (CXL) by means of the riboflavin/UVA (370 nm) approach. Materials and Methodology: Analysis of the current treatment protocol with respect to safety during CXL. Results: The currently used UVA dose density of 5.4 J/cm2 and the corresponding irradiance of 3 mW/cm2 are below the known damage thresholds of UVA for the corneal endothelium, lens, and retina. Regarding the photochemical damages due to the free radicals the damage threshold for endothelial cells is 0.35 mW/cm2. In a 400μm thick corneal stroma saturated with riboflavin, the irradiance at the endothelial level is about 0.18 mW/cm2, which is a factor of 2 smaller than the damage threshold. Conclusion: As long as the corneal stroma treated has a minimal thickness of 400 microns (as recommended), neither corneal endothelium nor deeper structures such as lens and retina will suffer any damages. The light source should provide a homogenous irradiance avoiding hot spots. PMID:21399770

  17. Tea derived galloylated polyphenols cross-link purified gastrointestinal mucins.

    PubMed

    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

  18. Structure, Subunit Topology, and Actin-binding Activity of the Arp2/3 Complex from Acanthamoeba

    PubMed Central

    Mullins, R. Dyche; Stafford, Walter F.; Pollard, Thomas D.

    1997-01-01

    The Arp2/3 complex, first isolated from Acanthamoeba castellani by affinity chromatography on profilin, consists of seven polypeptides; two actinrelated proteins, Arp2 and Arp3; and five apparently novel proteins, p40, p35, p19, p18, and p14 (Machesky et al., 1994). The complex is homogeneous by hydrodynamic criteria with a Stokes' radius of 5.3 nm by gel filtration, sedimentation coefficient of 8.7 S, and molecular mass of 197 kD by analytical ultracentrifugation. The stoichiometry of the subunits is 1:1:1:1:1:1:1, indicating the purified complex contains one copy each of seven polypeptides. In electron micrographs, the complex has a bilobed or horseshoe shape with outer dimensions of ∼13 × 10 nm, and mathematical models of such a shape and size are consistent with the measured hydrodynamic properties. Chemical cross-linking with a battery of cross-linkers of different spacer arm lengths and chemical reactivities identify the following nearest neighbors within the complex: Arp2 and p40; Arp2 and p35; Arp3 and p35; Arp3 and either p18 or p19; and p19 and p14. By fluorescent antibody staining with anti-p40 and -p35, the complex is concentrated in the cortex of the ameba, especially in linear structures, possibly actin filament bundles, that lie perpendicular to the leading edge. Purified Arp2/3 complex binds actin filaments with a Kd of 2.3 μM and a stoichiometry of approximately one complex molecule per actin monomer. In electron micrographs of negatively stained samples, Arp2/3 complex decorates the sides of actin filaments. EDC/NHS cross-links actin to Arp3, p35, and a low molecular weight subunit, p19, p18, or p14. We propose structural and topological models for the Arp2/3 complex and suggest that affinity for actin filaments accounts for the localization of complex subunits to actinrich regions of Acanthamoeba. PMID:9015304

  19. Intra-molecular cross-linking of acidic residues for protein structure studies.

    PubMed

    Novak, Petr; Kruppa, Gary H

    2008-01-01

    Intra-molecular cross-linking has been suggested as a method of obtaining distance constraints that would help to develop structural models of proteins. Recent work published on intra-molecular cross-linking for protein structural studies has employed commercially available primary amine (lysine, the amino terminus) selective reagents. 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 side chains 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 side chains can react to form "zero-length" cross-links with nearby primary amine containing residues, lysines (K) and the amino terminus (X), via the formation of a new amide bond. We also show that the EDC-activated DEO side chains can be cross-linked to each other using dihydrazides, two hydrazide moieties connected by an alkyl cross-linker arm 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 two 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 A. These results show that additional structural information can be obtained by exploiting new cross-linker chemistry

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

    PubMed

    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

    2016-09-01

    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

  1. Activation of Protein Tyrosine Kinases by Coxiella burnetii: Role in Actin Cytoskeleton Reorganization and Bacterial Phagocytosis

    PubMed Central

    Meconi, Sonia; Capo, Christian; Remacle-Bonnet, Maryse; Pommier, Gilbert; Raoult, Didier; Mege, Jean-Louis

    2001-01-01

    Coxiella burnetii, the agent of Q fever, is an obligate intracellular microorganism that grows in monocytes/macrophages. The internalization of virulent organisms by monocytes is lower than that of avirulent variants and is associated with actin cytoskeleton reorganization. We studied the activation of protein tyrosine kinases (PTKs) by C. burnetii in THP-1 monocytes. Virulent organisms induced early PTK activation and the tyrosine phosphorylation of several endogenous substrates, including Hck and Lyn, two Src-related kinases. PTK activation reflects C. burnetii virulence since avirulent variants were unable to stimulate PTK. We also investigated the role of PTK activation in C. burnetii-stimulated F-actin reorganization. Tyrosine-phosphorylated proteins were colocalized with F-actin inside cell protrusions induced by C. burnetii, and PTK activity was increased in Triton X-100-insoluble fractions. In addition, lavendustin A, a PTK inhibitor, and PP1, a Src kinase inhibitor, prevented C. burnetii-induced cell protrusions and F-actin reorganization. We finally assessed the role of PTK activation in bacterial phagocytosis. Pretreatment of THP-1 cells with lavendustin A and PP1 upregulated the uptake of virulent C. burnetii but had no effect on the phagocytosis of avirulent organisms. Thus, it is likely that PTK activation by C. burnetii negatively regulates bacterial uptake by interfering with cytoskeleton organization. PMID:11254615

  2. 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.

  3. Activation of a muscle-specific actin gene promoter in serum-stimulated fibroblasts.

    PubMed Central

    Stoflet, E S; Schmidt, L J; Elder, P K; Korf, G M; Foster, D N; Strauch, A R; Getz, M J

    1992-01-01

    Treatment of AKR-2B mouse fibroblasts with serum growth factors or inhibitors of protein synthesis, such as cycloheximide, results in a stimulation of cytoskeletal beta-actin transcription but has no effect on transcription of muscle-specific isotypes, such as the vascular smooth muscle (VSM) alpha-actin gene. Deletion mapping and site-specific mutagenesis studies demonstrated that a single "CArG" element of the general form CC(A/T)6GG was necessary and possibly sufficient to impart serum and cycloheximide-inducibility to the beta-actin promoter. Although the VSM alpha-actin promoter exhibits at least three similar sequence elements, it remained refractory to serum and cycloheximide induction. However, deletion of a 33 base pair sequence between -191 and -224 relative to the transcription start site resulted in the transcriptional activation of this muscle-specific promoter in rapidly growing or serum-stimulated fibroblasts. Although the activity of this truncated promoter was potentiated by cycloheximide in a manner indistinguishable from that of the beta-actin promoter, this was dependent on a more complex array of interacting elements. These included at least one CArG box and a putative upstream activating element closely associated with the -191 to -224 inhibitory sequences. These results demonstrate that the expression of a muscle-specific actin gene in fibroblasts is suppressed by a cis-acting negative control element and that in the absence of this element, the promoter is responsive to growth factor-induced signal transduction pathways. Images PMID:1421567

  4. Cross-Linking Mast Cell Specific Gangliosides Stimulates the Release of Newly Formed Lipid Mediators and Newly Synthesized Cytokines.

    PubMed

    Filho, Edismauro Garcia Freitas; da Silva, Elaine Zayas Marcelino; Zanotto, Camila Ziliotto; Oliver, Constance; Jamur, Maria Célia

    2016-01-01

    Mast cells are immunoregulatory cells that participate in inflammatory processes. Cross-linking mast cell specific GD1b derived gangliosides by mAbAA4 results in partial activation of mast cells without the release of preformed mediators. The present study examines the release of newly formed and newly synthesized mediators following ganglioside cross-linking. Cross-linking the gangliosides with mAbAA4 released the newly formed lipid mediators, prostaglandins D2 and E2, without release of leukotrienes B4 and C4. The effect of cross-linking these gangliosides on the activation of enzymes in the arachidonate cascade was then investigated. Ganglioside cross-linking resulted in phosphorylation of cytosolic phospholipase A2 and increased expression of cyclooxygenase-2. Translocation of 5-lipoxygenase from the cytosol to the nucleus was not induced by ganglioside cross-linking. Cross-linking of GD1b derived gangliosides also resulted in the release of the newly synthesized mediators, interleukin-4, interleukin-6, and TNF-α. The effect of cross-linking the gangliosides on the MAP kinase pathway was then investigated. Cross-linking the gangliosides induced the phosphorylation of ERK1/2, JNK1/2, and p38 as well as activating both NFκB and NFAT in a Syk-dependent manner. Therefore, cross-linking the mast cell specific GD1b derived gangliosides results in the activation of signaling pathways that culminate with the release of newly formed and newly synthesized mediators. PMID:27578923

  5. Cross-Linking Mast Cell Specific Gangliosides Stimulates the Release of Newly Formed Lipid Mediators and Newly Synthesized Cytokines

    PubMed Central

    Zanotto, Camila Ziliotto

    2016-01-01

    Mast cells are immunoregulatory cells that participate in inflammatory processes. Cross-linking mast cell specific GD1b derived gangliosides by mAbAA4 results in partial activation of mast cells without the release of preformed mediators. The present study examines the release of newly formed and newly synthesized mediators following ganglioside cross-linking. Cross-linking the gangliosides with mAbAA4 released the newly formed lipid mediators, prostaglandins D2 and E2, without release of leukotrienes B4 and C4. The effect of cross-linking these gangliosides on the activation of enzymes in the arachidonate cascade was then investigated. Ganglioside cross-linking resulted in phosphorylation of cytosolic phospholipase A2 and increased expression of cyclooxygenase-2. Translocation of 5-lipoxygenase from the cytosol to the nucleus was not induced by ganglioside cross-linking. Cross-linking of GD1b derived gangliosides also resulted in the release of the newly synthesized mediators, interleukin-4, interleukin-6, and TNF-α. The effect of cross-linking the gangliosides on the MAP kinase pathway was then investigated. Cross-linking the gangliosides induced the phosphorylation of ERK1/2, JNK1/2, and p38 as well as activating both NFκB and NFAT in a Syk-dependent manner. Therefore, cross-linking the mast cell specific GD1b derived gangliosides results in the activation of signaling pathways that culminate with the release of newly formed and newly synthesized mediators. PMID:27578923

  6. Actin and DNA Protect Histones from Degradation by Bacterial Proteases but Inhibit Their Antimicrobial Activity

    PubMed Central

    Sol, Asaf; Skvirsky, Yaniv; Blotnick, Edna; Bachrach, Gilad; Muhlrad, Andras

    2016-01-01

    Histones are small polycationic proteins located in the cell nucleus. Together, DNA and histones are integral constituents of the nucleosomes. Upon apoptosis, necrosis, and infection – induced cell death, histones are released from the cell. The extracellular histones have strong antimicrobial activity but are also cytotoxic and thought as mediators of cell death in sepsis. The antimicrobial activity of the cationic extracellular histones is inhibited by the polyanionic DNA and F-actin, which also become extracellular upon cell death. DNA and F-actin protect histones from degradation by the proteases of Pseudomonas aeruginosa and Porphyromonas gingivalis. However, though the integrity of the histones is protected, the activity of histones as antibacterial agents is lost. The inhibition of the histone’s antibacterial activity and their protection from proteolysis by DNA and F-actin indicate a tight electrostatic interaction between the positively charged histones and negatively charged DNA and F-actin, which may have physiological significance in maintaining the equilibrium between the beneficial antimicrobial activity of extracellular histones and their cytotoxic effects. PMID:27555840

  7. A photolithographic approach to spatially resolved cross-linked nanolayers.

    PubMed

    Fuchise, Keita; Lindemann, Peter; Heißler, Stefan; Gliemann, Hartmut; Trouillet, Vanessa; Welle, Alexander; Berson, Jonathan; Walheim, Stefan; Schimmel, Thomas; Meier, Michael A R; Barner-Kowollik, Christopher

    2015-03-17

    The preparation of cross-linked nanosheets with 1-2 nm thickness and predefined shape was achieved by lithographic immobilization of trimethacryloyl thioalkanoates onto the surface of Si wafers, which were functionalized with 2-(phenacylthio)acetamido groups via a photoinduced reaction. Subsequent cross-linking via free radical polymerization as well as a phototriggered Diels-Alder reaction under mild conditions on the surface led to the desired nanosheets. Electrospray ionization mass spectrometry (ESI-MS), X-ray photoelectron spectroscopy (XPS), time-of-flight secondary ion mass spectrometry (ToF-SIMS), as well as infrared reflection-absorption spectroscopy (IRRAS) confirmed the success of individual surface-modification and cross-linking reactions. The thickness and lateral size of the cross-linked structures were determined by atomic force microscopy (AFM) for samples prepared on Si wafers functionalized with a self-assembled monolayer of 1H,1H,2H,2H-perfluorodecyl groups bearing circular pores obtained via a polymer blend lithographic approach, which led to the cross-linking reactions occurring in circular nanoareas (diameter of 50-640 nm) yielding an average thickness of 1.2 nm (radical cross-linking), 1.8 nm (radical cross-linking in the presence of 2,2,2-trifluoroethyl methacrylate as a comonomer), and 1.1 nm (photochemical cross-linking) of the nanosheets. PMID:25705846

  8. 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.

  9. Theoretical models for cooperative steady-state ATPase activity of myosin subfragment-1 on regulated actin.

    PubMed Central

    Hill, T L; Eisenberg, E; Chalovich, J M

    1981-01-01

    Recent theoretical work on the cooperative equilibrium binding of myosin subfragment-1-ADP to regulated actin, as influenced by Ca2+, is extended here to the cooperative steady-state ATPase activity of myosin subfragment-1 on regulated actin. Exact solution of the general steady-state problem will require Monte Carlo calculations. Three interrelated special cases are discussed in some detail and sample computer (not Monte Carlo) solutions are given. The eventual objective is to apply these considerations to in vitro experimental data and to in vivo muscle models. PMID:6455170

  10. 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.

  11. 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.

  12. 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.

  13. Microfluidic Investigation Reveals Distinct Roles for Actin Cytoskeleton and Myosin II Activity in Capillary Leukocyte Trafficking

    PubMed Central

    Gabriele, Sylvain; Benoliel, Anne-Marie; Bongrand, Pierre; Théodoly, Olivier

    2009-01-01

    Circulating leukocyte sequestration in pulmonary capillaries is arguably the initiating event of lung injury in acute respiratory distress syndrome. We present a microfluidic investigation of the roles of actin organization and myosin II activity during the different stages of leukocyte trafficking through narrow capillaries (entry, transit and shape relaxation) using specific drugs (latrunculin A, jasplakinolide, and blebbistatin). The deformation rate during entry reveals that cell stiffness depends strongly on F-actin organization and hardly on myosin II activity, supporting a microfilament role in leukocyte sequestration. In the transit stage, cell friction is influenced by stiffness, demonstrating that the actin network is not completely broken after a forced entry into a capillary. Conversely, membrane unfolding was independent of leukocyte stiffness. The surface area of sequestered leukocytes increased by up to 160% in the absence of myosin II activity, showing the major role of molecular motors in microvilli wrinkling and zipping. Finally, cell shape relaxation was largely independent of both actin organization and myosin II activity, whereas a deformed state was required for normal trafficking through capillary segments. PMID:19450501

  14. 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. PMID:25131196

  15. Mass spectrometry evidence for cisplatin as a protein cross-linking reagent

    PubMed Central

    Li, Huilin; Zhao, Yao; Phillips, Hazel I. A.; Qi, Yulin; Lin, Tzu-Yung; Sadler, Peter J.; O’Connor, Peter B.

    2011-01-01

    Cisplatin is a potent anti-cancer drug, which functions by cross-linking adjacent DNA guanine residues. However within one day of injection, 65~98% of the platinum in the blood plasma is protein-bound. It is generally accepted that cisplatin binds to methionine and histidine residues, but what is often underappreciated is that platinum from cisplatin has a 2+ charge and can form up to four bonds. Thus, it has the potential to function as a cross-linker. In this report, the cross-linking ability of cisplatin is demonstrated by Fourier transform ion cyclotron resonance (FTICR) mass spectrometry (MS) with the use of standard peptides, the 16.8 kDa protein calmodulin (CaM), but was unsuccessful for the 64 kDa protein hemoglobin. The high resolution and mass accuracy of FTICR MS along with the high degree of fragmentation of large peptides afforded by collisionally activated dissociation (CAD) and electron capture dissociation (ECD) are shown to be a valuable means of characterizing cross-linking sites. Cisplatin is different from current cross-linking reagents by targeting new functional groups, thioethers, and imidazoles groups, which provides complementarity with existing cross-linkers. In addition, platinum(II) inherently has two positive charges which enhance the detection of cross-linked products. Higher charge states not only promote the detection of cross-linking products with less purification, but result in more comprehensive MS/MS fragmentation and can assist the assignment of modification sites. Moreover, the unique isotopic pattern of platinum flags cross-linking products and modification sites by mass spectrometry. PMID:21591778

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

    NASA Astrophysics Data System (ADS)

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

    2009-06-01

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

  17. A 133Cs nuclear magnetic resonance study of endothelial Na(+)-K(+)-ATPase activity: can actin regulate its activity?

    PubMed Central

    Gruwel, M L; Culíc, O; Schrader, J

    1997-01-01

    Using (133)Cs+ NMR, we developed a technique to repetitively measure, in vivo, Na(+)-K(+)-ATPase activity in endothelial cells. The measurements were made without the use of an exogenous shift reagent, because of the large chemical shift of 1.36 +/- 0.13 ppm between intra- and extracellular Cs+. Intracellularly we obtained a spin lattice relaxation time (T1) of 2.0 +/- 0.3 s, and extracellular T1 was 7.9 +/- 0.4 s. Na(+)-K+ pump activity in endothelial cells was determined at 12 +/- 3 nmol Cs+ x min(-1) x (mg Prot)[-1] under control conditions. When intracellular ATP was depleted by the addition of 5 mM 2-deoxy-D-glucose (DOG) and NaCN to about 5% of control, the pump rate decreased by 33%. After 80 min of perfusion with 5 mM DOG and NaCN, reperfusion with control medium rapidly reestablished the endothelial membrane Cs+ gradient. Using (133)Cs+ NMR as a convenient tool, we further addressed the proposed role of actin as a regulator of Na(+)-K+ pump activity in intact cells. Two models of actin rearrangement were tested. DOG caused a rearrangement of F-actin and an increase in G-actin, with a simultaneous decrease in ATP concentration. Cytochalasin D, however, caused an F-actin rearrangement different from that observed for DOG and an increase in G-actin, and cellular ATP levels remained unchanged. In both models, the Na(+)-K(+)-pump activity remained unchanged, as measured with (133)Cs NMR. Our results demonstrate that (133)Cs NMR can be used to repetitively measure Na(+)-K(+)-ATPase activity in endothelial cells. No evidence for a regulatory role of actin on Na(+)-K(+)-ATPase was found. Images FIGURE 6 PMID:9168052

  18. Peptidoglycan cross-linking in glycopeptide-resistant Actinomycetales.

    PubMed

    Hugonnet, Jean-Emmanuel; Haddache, Nabila; Veckerlé, Carole; Dubost, Lionel; Marie, Arul; Shikura, Noriyasu; Mainardi, Jean-Luc; Rice, Louis B; Arthur, Michel

    2014-01-01

    Synthesis of peptidoglycan precursors ending in D-lactate (D-Lac) is thought to be responsible for glycopeptide resistance in members of the order Actinomycetales that produce these drugs and in related soil bacteria. More recently, the peptidoglycan of several members of the order Actinomycetales was shown to be cross-linked by L,D-transpeptidases that use tetrapeptide acyl donors devoid of the target of glycopeptides. To evaluate the contribution of these resistance mechanisms, we have determined the peptidoglycan structure of Streptomyces coelicolor A(3)2, which harbors a vanHAX gene cluster for the production of precursors ending in D-Lac, and Nonomuraea sp. strain ATCC 39727, which is devoid of vanHAX and produces the glycopeptide A40296. Vancomycin retained residual activity against S. coelicolor A(3)2 despite efficient incorporation of D-Lac into cytoplasmic precursors. This was due to a D,D-transpeptidase-catalyzed reaction that generated a stem pentapeptide recognized by glycopeptides by the exchange of D-Lac for D-Ala and Gly. The contribution of L,D-transpeptidases to resistance was limited by the supply of tetrapeptide acyl donors, which are essential for the formation of peptidoglycan cross-links by these enzymes. In the absence of a cytoplasmic metallo-D,D-carboxypeptidase, the tetrapeptide substrate was generated by hydrolysis of the C-terminal D-Lac residue of the stem pentadepsipeptide in the periplasm in competition with the exchange reaction catalyzed by D,D-transpeptidases. In Nonomuraea sp. strain ATCC 39727, the contribution of L,D-transpeptidases to glycopeptide resistance was limited by the incomplete conversion of pentapeptides into tetrapeptides despite the production of a cytoplasmic metallo-D,D-carboxypeptidase. Since the level of drug production exceeds the level of resistance, we propose that L,D-transpeptidases merely act as a tolerance mechanism in this bacterium. PMID:24395229

  19. Comparison of cross-linked polyethylene materials for orthopaedic applications.

    PubMed

    Collier, John P; Currier, Barbara H; Kennedy, Francis E; Currier, John H; Timmins, Graham S; Jackson, Simon K; Brewer, Robin L

    2003-09-01

    Cross-linked polyethylenes are being marketed by orthopaedic manufacturers to address the problem of osteolysis caused by polyethylene particulate wear debris. Wear testing of these cross-linked polyethylenes in hip simulators has shown dramatic reduction in wear rate compared with standard ultrahigh molecular weight polyethylene, either gamma irradiated in air or nitrogen - or ethylene oxide-sterilized. However, this reduction in wear rate is not without cost. The cross-linking processes can result in materials with lower mechanical properties than standard ultrahigh molecular weight polyethylene. To evaluate the effect of the various cross-linking processes on physical and mechanical properties of ultrahigh molecular weight polyethylene, commercially available cross-linked polyethylenes from six orthopaedic manufacturers were tested. This study was the culmination of collaboration with these manufacturers, who provided cross-linked polyethylene for this study, wear characteristics of the material they provided, and review of the physical and mechanical properties measure for their polyethylene. Cross-linked materials were evaluated as received and after an accelerated aging protocol. Free radical identity and concentration, oxidation, crystallinity, melt temperature, ultimate tensile strength, elongation at break, tensile stress at yield, and toughness are reported for each material. By comparing these physical and mechanical properties, surgeons can evaluate the trade-off that results from developing materials with substantially lower wear rates. PMID:12966304

  20. Preparation and characterization of cross-linked collagen-phospholipid polymer hybrid gels.

    PubMed

    Nam, Kwangwoo; Kimura, Tsuyoshi; Kishida, Akio

    2007-01-01

    2-methacryloyloxyethyl phosphorylcholine (MPC)-immobilized collagen gel was developed. Using 1-ethyl-3-(3-dimethyl aminopropyl)-1-carbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS), we cross-linked a collagen film in 2-morpholinoethane sulfonic acid (MES) buffer (EN gel). EN gel was prepared under both pH 4.5 and pH 9.0 in order to observe changes in cross-linking ability. To cross-link MPC to collagen gel, poly(MPC-co-methacrylic acid) (PMA) having a carboxyl group side chain was chosen. E/N gel was added to the MES buffer having pre-NHS activated PMA to make MPC-immobilized collagen gel (MiC gel). MiC gel was prepared under both acidic and alkaline conditions to observe the changes in the cross-linking ability of PMA. X-ray photoelectron spectroscopy showed that the PMA was cross-linked with collagen under both acidic and alkaline conditions. Differential scanning calorimetry (DSC) results showed that the shrinkage temperature increased for the MiC gels and that the increase would be greater for the MiC gel prepared under alkaline conditions. The data showed that swelling would be less when the MiC gel was prepared under alkaline conditions. The biodegradation caused by collagenase was suppressed for the MiC gel prepared under alkaline conditions due to stable inter- and intrahelical networks. PMID:16959313

  1. 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

  2. Phospholipase Cη2 Activation Redirects Vesicle Trafficking by Regulating F-actin*

    PubMed Central

    Yamaga, Masaki; Kielar-Grevstad, D. Michelle; Martin, Thomas F. J.

    2015-01-01

    PI(4,5)P2 localizes to sites of dense core vesicle exocytosis in neuroendocrine cells and is required for Ca2+-triggered vesicle exocytosis, but the impact of local PI(4,5)P2 hydrolysis on exocytosis is poorly understood. Previously, we reported that Ca2+-dependent activation of phospholipase Cη2 (PLCη2) catalyzes PI(4,5)P2 hydrolysis, which affected vesicle exocytosis by regulating the activities of the lipid-dependent priming factors CAPS (also known as CADPS) and ubiquitous Munc13-2 in PC12 cells. Here we describe an additional role for PLCη2 in vesicle exocytosis as a Ca2+-dependent regulator of the actin cytoskeleton. Depolarization of neuroendocrine PC12 cells with 56 or 95 mm KCl buffers increased peak Ca2+ levels to ∼400 or ∼800 nm, respectively, but elicited similar numbers of vesicle exocytic events. However, 56 mm K+ preferentially elicited the exocytosis of plasma membrane-resident vesicles, whereas 95 mm K+ preferentially elicited the exocytosis of cytoplasmic vesicles arriving during stimulation. Depolarization with 95 mm K+ but not with 56 mm K+ activated PLCη2 to catalyze PI(4,5)P2 hydrolysis. The decrease in PI(4,5)P2 promoted F-actin disassembly, which increased exocytosis of newly arriving vesicles. Consistent with its role as a Ca2+-dependent regulator of the cortical actin cytoskeleton, PLCη2 localized with F-actin filaments. The results highlight the importance of PI(4,5)P2 for coordinating cytoskeletal dynamics with vesicle exocytosis and reveal a new role for PLCη2 as a Ca2+-dependent regulator of F-actin dynamics and vesicle trafficking. PMID:26432644

  3. Redox-Responsive, Core Cross-Linked Polyester Micelles

    PubMed Central

    Zhang, Zhonghai; Yin, Lichen; Tu, Chunlai; Song, Ziyuan; Zhang, Yanfeng; Xu, Yunxiang; Tong, Rong; Zhou, Qin; Ren, Jie; Cheng, Jianjun

    2013-01-01

    Monomethoxy poly(ethylene glycol)-b-poly(Tyr(alkynyl)-OCA), a biodegradable amphiphilic block copolymer, was synthesized by means of ring-opening polymerization of 5-(4-(prop-2-yn-1-yloxy)benzyl)-1,3-dioxolane-2,4-dione (Tyr(alkynyl)-OCA) and used to prepare core cross-linked polyester micelles via click chemistry. Core cross-linking not only improved the structural stability of the micelles but also allowed controlled release of cargo molecules in response to the reducing reagent. This new class of core cross-linked micelles can potentially be used in controlled release and drug delivery applications. PMID:23536920

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

    PubMed

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

    2016-06-01

    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

  5. 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

  6. Chemical cross-linking of mitochondrial NADH dehydrogenase from bovine heart.

    PubMed Central

    Cleeter, M W; Banister, S H; Ragan, C I

    1985-01-01

    The structure of bovine heart mitochondrial NADH dehydrogenase was investigated by using two cleavable cross-linking agents, disuccinimidyl tartrate and (ethylene glycol)yl bis-(succinimidyl succinate). Cross-linking was analysed primarily by immunoblotting to detect products containing subunits of the iron-protein fraction from chaotropic resolution of the enzyme, namely those of 75, 49, 30 and 13 kDa. By using both the isolated iron-protein fraction and the intact dehydrogenase, cross-links were identified between these four subunits, from these subunits to the largest subunit of the flavoprotein fraction, which contains the active site for NADH, and from these subunits to polypeptides in the hydrophobic shell, which surrounds the hydrophilic iron-protein and flavoprotein fractions. Images Fig. 1. Fig. 2. Fig. 3. Fig. 4. Fig. 5. PMID:4004775

  7. 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.

  8. Study of the effect of mixing approach on cross-linking efficiency of hyaluronic acid-based hydrogel cross-linked with 1,4-butanediol diglycidyl ether.

    PubMed

    Al-Sibani, Mohammed; Al-Harrasi, Ahmed; Neubert, Reinhard H H

    2016-08-25

    Regardless of various strategies reported for cross-linking hyaluronic acid (HA) with 1,4-butanediol diglycidyl ether (BDDE), seeking new strategies that enhance cross-linking efficiency with a low level of cross-linker is essential. In this work, we studied the influence of mixing approach on two cross-linked BDDE-HA hydrogels prepared by two different mixing approaches; the large-batch mixing approach in which the hydrogel quantities were all mixed as a single lump in one container (hydrogel 1), and the small-batches mixing approach in which the hydrogel quantities were divided into smaller batches, mixed separately at various HA/BDDE ratios then combined in one reaction mixture (hydrogel 2). The result showed that the cross-linking reaction was mixing process-dependent. Degradation tests proved that, in relation to hydrogel 1, hydrogel 2 was more stable, and exhibited a higher resistance towards hyaluronidase activity. The swelling ratio of hydrogel 1 was significantly higher than that of hydrogel 2 in distilled water; however, in phosphate buffer saline, both hydrogels showed no significant difference. SEM images demonstrated that hydrogel 2 composite showed a denser network structure and smaller pore-size than hydrogel 1. In comparison to native HA, the occurrence of chemical modification in the cross-linked hydrogels was confirmed by FTIR and NMR distinctive peaks. These peaks also provided evidence that hydrogel 2 exhibited a higher degree of modification than hydrogel 1. In conclusion, the small-batches mixing approach proved to be more effective than large-batch mixing in promoting HA-HA entanglement and increasing the probability of BDDE molecules for binding with HA chains. PMID:27312477

  9. 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. PMID:26327373

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

    SciTech Connect

    Miranda, Lisa; Carpentier, Sarah; Platek, Anna; Hussain, Nusrat; Gueuning, Marie-Agnes; 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{sup 2+}-dependent AMPK activation via calmodulin-dependent protein kinase kinase-{beta}(CaMKK{beta}), a known upstream kinase of AMPK. Indeed, hypertonicity-induced AMPK activation was markedly reduced by the STO-609 CaMKK{beta} inhibitor, as was the increase in MLC and cofilin phosphorylation. We suggest that AMPK links osmotic stress to the reorganization of the actin cytoskeleton.

  11. Chemical Cross-linking of Neighboring Thylakoid Membrane Polypeptides 12

    PubMed Central

    Novak-Hofer, Ilse; Siegenthaler, Paul-Andre

    1978-01-01

    Cross-linking between protein components of whole spinach (Spinacia oleracea var. Nobel) thylakoids and of photosystem I- and II-enriched thylakoid fractions has been produced by reaction with the bifunctional imidoester dimethyl-3,3′-dithiobispropionimidate dihydrochloride as well as by the oxidation of intrinsic sulfydryl groups with an orthophenanthrolinecupric ion complex. The mixture of membrane proteins and their cross-linked products has been analyzed by two-dimensional sodium dodecyl sulfate electrophoresis, with a reductive cleavage step of the cross-linkages before the second dimension. Cross-linked aggregates up to a molecular weight of about 130 kilodaltons (kD) were analyzed, and it was inferred that the polypeptides appearing together in the same aggregates were neighbors within the membrane. In thylakoids as well as in isolated photosystem fractions, oligomers were formed by cross-linking polypeptides of the 60 to 90 kD range, among them the polypeptides of the chlorophyll-protein complex I. Polypeptides of 46, 19, and 12 kD were cross-linked to these complexes. Polypeptides of 25 and 22 kD, which are related to the chlorophyll-protein complex II, were cross-linked in thylakoids as well as in photosystem II fractions, suggesting that in the membrane these molecules are close together. In photosystem II fractions an oligomer having a molecular weight of about 60 kD was formed by cross-linking several polypeptides of different molecular weights: 40, 25, and 22 kD. Our cross-linking experiments show that protein interactions in the thylakoid membrane occurred mainly among the polypeptides of the two chlorophyll-protein complexes, thus suggesting an oligomeric nature of these apoproteins. ImagesFig. 1Fig. 2Fig. 3 PMID:16660519

  12. 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

  13. 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

    2016-03-01

    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. PMID:26862769

  14. 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.

  15. Efficient Gene Transfection into Mammalian Cells Mediated by Cross-linked Polyethylenimine

    PubMed Central

    Dong, Wei; Li, Shufeng; Jin, Guanghui; Sun, Qiming; Ma, Dingyuan; Hua, Zichun

    2007-01-01

    25 kDa branched polyethylenimine (PEI) has successfully been used for in vitro and in vivo gene delivery approaches, but it is cytotoxic. Smaller PEIs are usually non-cytotoxic but less efficient. In order to enhance the gene delivery efficiency and minimize cytotoxicity of PEI, we explored to synthesize cross-linked PEIs with degradable bonds by reacting amines of small branched 2000 Da PEI with small diacrylate (1,4-butanediol diacrylate or ethyleneglycol dimethacrylate) for 2–6 hours. The efficiency of the cross-linked PEIs during in vitro delivering plasmid containing enhanced green fluorescent protein (EGFP) gene reporter and their cytotoxicity were assessed in melanoma B16F10 cell and other cell lines. In vivo gene delivery efficiency was evaluated by direct injection delivery of the EGFP plasmid/cross-linked PEI complexes into mice and by estimating the EGFP expression in animal muscles. Compared to commercially available 25-kDa branched PEI, the cross-linked PEIs reported here could mediate more efficient expression of reporter gene than the 25-kDa PEI control, 19-fold more efficiently in B16F10 cells, 17-fold in 293T cells, 2.3-fold in 3T3 cells, and they exhibited essentially nontoxic at their optimized condition for gene delivery. Furthermore the transfection activity of polyplexs was preserved in the presence of serum proteins. The muscle transfected with the cross-linked PEI prepared here exhibited normal morphology and excellent gene expression. The cross-linked PEIs reported here were evidently more efficient than the commercial 25-kD PEI control and had less cytotoxicity in gene delivery in vitro and in vivo.

  16. Actin-dependent intranuclear repositioning of an active gene locus in vivo

    PubMed Central

    Dundr, Miroslav; Ospina, Jason K.; Sung, Myong-Hee; John, Sam; Upender, Madhvi; Ried, Thomas; Hager, Gordon L.; Matera, A. Gregory

    2007-01-01

    Although bulk chromatin is thought to have limited mobility within the interphase eukaryotic nucleus, directed long-distance chromosome movements are not unknown. Cajal bodies (CBs) are nuclear suborganelles that nonrandomly associate with small nuclear RNA (snRNA) and histone gene loci in human cells during interphase. However, the mechanism responsible for this association is uncertain. In this study, we present an experimental system to probe the dynamic interplay of CBs with a U2 snRNA target gene locus during transcriptional activation in living cells. Simultaneous four-dimensional tracking of CBs and U2 genes reveals that target loci are recruited toward relatively stably positioned CBs by long-range chromosomal motion. In the presence of a dominant-negative mutant of β-actin, the repositioning of activated U2 genes is markedly inhibited. This supports a model in which nuclear actin is required for these rapid, long-range chromosomal movements. PMID:18070915

  17. 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.

  18. 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

  19. Activator-inhibitor coupling between Rho signalling and actin assembly makes the cell cortex an excitable medium.

    PubMed

    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

    2015-11-01

    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, although Rho activity elicits F-actin assembly, F-actin subsequently inactivates Rho. Experimental and modelling 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

  20. The actin-bundling protein L-plastin supports T-cell motility and activation

    PubMed Central

    Morley, Sharon Celeste

    2013-01-01

    Summary Tight regulation of actin dynamics is essential for T-cell trafficking and activation. Recent studies in human and murine T cells reveal that T-cell motility and full T-cell activation require the hematopoietic-specific, actin-bundling protein L-plastin. T cells lacking L-plastin do not form fully mature synapses and thus demonstrate reduced cytokine production and proliferation. Reduction or loss of L-plastin expression also reduces the velocity of T cells and impairs thymic egress and intranodal motility. While dispensable for proximal T-cell receptor and chemokine receptor signaling, L-plastin is critical to the later stages of synapse maturation and cellular polarization. Serine phosphorylation, calcium, and calmodulin binding regulate the bundling activity and localization of LPL following T-cell receptor and chemokine receptor engagement. However, the interaction between these regulatory domains and resulting changes in local control of actin cytoskeletal structures has not been fully elucidated. Circumstantial evidence suggests a function for L-plastin in either the formation or maintenance of integrin-associated adhesion structures. As L-plastin may be a target of the commonly used immunosuppressive agent dexamethasone, full elucidation of the regulation and function of L-plastin in T-cell biology may illuminate new pathways for clinically useful immunotherapeutics. PMID:24117812

  1. Actinic Keratosis

    MedlinePlus

    ... rashes clinical tools newsletter | contact Share | Actinic Keratosis (Solar Keratosis) Information for adults A A A Actinic ... the touch. Overview Actinic keratoses, also known as solar keratoses, are small rough or scaly areas of ...

  2. Inhibiting Hexamer Disassembly of Human UDP-Glucose Dehydrogenase by Photoactivated Amino Acid Cross-Linking.

    PubMed

    Grady, George; Thelen, Ashley; Albers, Jaleen; Ju, Tong; Guo, Jiantao; Barycki, Joseph J; Simpson, Melanie A

    2016-06-01

    The enzyme UDP-glucose dehydrogenase (UGDH) catalyzes the reaction of UDP-glucose to UDP-glucuronate through two successive NAD(+)-dependent oxidation steps. Human UGDH apoprotein is purified as a mixture of dimeric and hexameric species. Addition of substrate and cofactor stabilizes the oligomeric state to primarily the hexameric form. To determine if the dynamic conformations of hUGDH are required for catalytic activity, we used site-specific unnatural amino acid incorporation to facilitate cross-linking of monomeric subunits into predominantly obligate oligomeric species. Optimal cross-linking was achieved by encoding p-benzoyl-l-phenylalanine at position 458, normally a glutamine located within the dimer-dimer interface, and exposing the enzyme to long wavelength ultraviolet (UV) radiation in the presence of substrate and cofactor. Hexameric complexes were purified by gel filtration chromatography and found to contain significant fractions of dimer and trimer (approximately 50%) along with another 10% higher-molecular mass species. The activity of the cross-linked enzyme was reduced by almost 60% relative to that of the un-cross-linked UGDH mutant, and UV exposure had no effect on the activity of the wild-type enzyme. These results support a model for catalysis in which the ability to dissociate the dimer-dimer interface is as important for maximal enzyme function as has been previously shown for the formation of the hexamer. PMID:27198584

  3. Role and structural mechanism of WASP-triggered conformational changes in branched actin filament nucleation by Arp2/3 complex.

    PubMed

    Rodnick-Smith, Max; Luan, Qing; Liu, Su-Ling; Nolen, Brad J

    2016-07-01

    The Arp2/3 (Actin-related proteins 2/3) complex is activated by WASP (Wiskott-Aldrich syndrome protein) family proteins to nucleate branched actin filaments that are important for cellular motility. WASP recruits actin monomers to the complex and stimulates movement of Arp2 and Arp3 into a "short-pitch" conformation that mimics the arrangement of actin subunits within filaments. The relative contribution of these functions in Arp2/3 complex activation and the mechanism by which WASP stimulates the conformational change have been unknown. We purified budding yeast Arp2/3 complex held in or near the short-pitch conformation by an engineered covalent cross-link to determine if the WASP-induced conformational change is sufficient for activity. Remarkably, cross-linked Arp2/3 complex bypasses the need for WASP in activation and is more active than WASP-activated Arp2/3 complex. These data indicate that stimulation of the short-pitch conformation is the critical activating function of WASP and that monomer delivery is not a fundamental requirement for nucleation but is a specific requirement for WASP-mediated activation. During activation, WASP limits nucleation rates by releasing slowly from nascent branches. The cross-linked complex is inhibited by WASP's CA region, even though CA potently stimulates cross-linking, suggesting that slow WASP detachment masks the activating potential of the short-pitch conformational switch. We use structure-based mutations and WASP-Arp fusion chimeras to determine how WASP stimulates movement toward the short-pitch conformation. Our data indicate that WASP displaces the autoinhibitory Arp3 C-terminal tail from a hydrophobic groove at Arp3's barbed end to destabilize the inactive state, providing a mechanism by which WASP stimulates the short-pitch conformation and activates Arp2/3 complex. PMID:27325766

  4. Enzymatic cross-linking of carboxymethylpullulan grafted with ferulic acid.

    PubMed

    Dulong, Virginie; Hadrich, Ahdi; Picton, Luc; Le Cerf, Didier

    2016-10-20

    Carboxymethylpullulan (CMP) has been modified in a two-step grafting reaction of ferulic acid (FA). Acid adipic dihydrazyde (ADH) was first reacted with FA activated with 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC). Then the product of this first reaction was reacted with CMP (activated with EDC). Grafted polysaccharides structure was confirmed by FTIR and (1)H NMR spectroscopy. Analyses by size-exclusion chromatography (SEC) coupling on-line with a multi-angle light scattering detector (MALS), a viscometer and a differential refractive index detector (DRI) (SEC/MALS/DRI/Visco) showed that associations between FA moieties occurred due to hydrophobic interactions. The grafting rates of FA were determined by the Folin-Ciocalteu method and were found between 1.0% and 11.2% (mol/mol anhydroglucose unit). The CMP-FA were then enzymatically cross-linked with laccase from Pleurotus ostreatus. The crosslinking reactions were followed by rheological measurements, demonstrating the influence of laccase concentration on kinetics. Elastic modulus and swelling rates of hydrogels depends on FA content only for low values. PMID:27474545

  5. Activation of the cAMP Pathway Induces RACK1-Dependent Binding of β-Actin to BDNF Promoter

    PubMed Central

    Neasta, Jeremie; Fiorenza, Anna; He, Dao-Yao; Phamluong, Khanhky; Kiely, Patrick A.; Ron, Dorit

    2016-01-01

    RACK1 is a scaffolding protein that contributes to the specificity and propagation of several signaling cascades including the cAMP pathway. As such, RACK1 participates in numerous cellular functions ranging from cell migration and morphology to gene transcription. To obtain further insights on the mechanisms whereby RACK1 regulates cAMP-dependent processes, we set out to identify new binding partners of RACK1 during activation of the cAMP signaling using a proteomics strategy. We identified β-actin as a direct RACK1 binding partner and found that the association between β-actin and RACK1 is increased in response to the activation of the cAMP pathway. Furthermore, we show that cAMP-dependent increase in BDNF expression requires filamentous actin. We further report that β-actin associates with the BDNF promoter IV upon the activation of the cAMP pathway and present data to suggest that the association of β-actin with BDNF promoter IV is RACK1-dependent. Taken together, our data suggest that β-actin is a new RACK1 binding partner and that the RACK1 and β-actin association participate in the cAMP-dependent regulation of BDNF transcription. PMID:27505161

  6. VAMP-7 links granule exocytosis to actin reorganization during platelet activation.

    PubMed

    Koseoglu, Secil; Peters, Christian G; Fitch-Tewfik, Jennifer L; Aisiku, Omozuanvbo; Danglot, Lydia; Galli, Thierry; Flaumenhaft, Robert

    2015-07-30

    Platelet activation results in profound morphologic changes accompanied by release of granule contents. Recent evidence indicates that fusion of granules with the plasma membrane during activation provides auxiliary membrane to cover growing actin structures. Yet little is known about how membrane fusion is coupled with actin reorganization. Vesicle-associated membrane protein (VAMP)-7 is found on platelet vesicles and possesses an N-terminal longin domain capable of linking exocytosis to cytoskeletal remodeling. We have evaluated platelets from VAMP-7(-/-) mice to determine whether this VAMP isoform contributes to granule release and platelet spreading. VAMP-7(-/-) platelets demonstrated a partial defect in dense granule exocytosis and impaired aggregation. α Granule exocytosis from VAMP-7(-/-) platelets was diminished both in vitro and in vivo during thrombus formation. Consistent with a role of VAMP-7 in cytoskeletal remodeling, spreading on matrices was decreased in VAMP-7(-/-) platelets compared to wild-type controls. Immunoprecipitation of VAMP-7 revealed an association with VPS9-domain ankyrin repeat protein (VARP), an adaptor protein that interacts with both membrane-bound and cytoskeleton proteins and with Arp2/3. VAMP-7, VARP, and Arp2/3 localized to the platelet periphery during spreading. These studies demonstrate that VAMP-7 participates in both platelet granule secretion and spreading and suggest a mechanism whereby VAMP-7 links granule exocytosis with actin reorganization. PMID:25999457

  7. 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

  8. 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. PMID:24998319

  9. The Tyrosine Kinase Activity of c-Src Regulates Actin Dynamics and Organization of Podosomes in Osteoclasts

    PubMed Central

    Destaing, Olivier; Sanjay, Archana; Itzstein, Cecile; Horne, William C.; Toomre, Derek

    2008-01-01

    Podosomes are dynamic actin-rich structures composed of a dense F-actin core surrounded by a cloud of more diffuse F-actin. Src performs one or more unique functions in osteoclasts (OCLs), and podosome belts and bone resorption are impaired in the absence of Src. Using Src−/− OCLs, we investigated the specific functions of Src in the organization and dynamics of podosomes. We found that podosome number and the podosome-associated actin cloud were decreased in Src−/− OCLs. Videomicroscopy and fluorescence recovery after photobleaching analysis revealed that the life span of Src−/− podosomes was increased fourfold and that the rate of actin flux in the core was decreased by 40%. Thus, Src regulates the formation, structure, life span, and rate of actin polymerization in podosomes and in the actin cloud. Rescue of Src−/− OCLs with Src mutants showed that both the kinase activity and either the SH2 or the SH3 binding domain are required for Src to restore normal podosome organization and dynamics. Moreover, inhibition of Src family kinase activities in Src−/− OCLs by Src inhibitors or by expressing dominant-negative SrcK295M induced the formation of abnormal podosomes. Thus, Src is an essential regulator of podosome structure, dynamics and organization. PMID:17978100

  10. Collagen/elastin hydrogels cross-linked by squaric acid.

    PubMed

    Skopinska-Wisniewska, J; Kuderko, J; Bajek, A; Maj, M; Sionkowska, A; Ziegler-Borowska, M

    2016-03-01

    Hydrogels based on collagen and elastin are very valuable materials for medicine and tissue engineering. They are biocompatible; however their mechanical properties and resistance for enzymatic degradation need to be improved by cross-linking. Up to this point many reagents have been tested but more secure reactants are still sought. Squaric acid (SqAc), 3,4-dihydroxy 3-cyclobutene 1,2-dione, is a strong, cyclic acid, which reacts easily with amine groups. The properties of hydrogels based on collagen/elastin mixtures (95/5, 90/10) containing 5%, 10% and 20% of SqAc and neutralized via dialysis against deionized water were tested. Cross-linked, 3-D, transparent hydrogels were created. The cross-linked materials are stiffer and more resistant to enzymatic degradation than those that are unmodified. The pore size, swelling ability and surface polarity are reduced due to 5% and 10% of SqAc addition. At the same time, the cellular response is not significantly affected by the cross-linking. Therefore, squaric acid would be regarded as a safe, effective cross-linking agent. PMID:26706512

  11. 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.

  12. 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.

  13. Tumor Suppressor Activity of Profilin Requires a Functional Actin Binding Site

    PubMed Central

    Wittenmayer, Nina; Jandrig, Burkhard; Rothkegel, Martin; Schlüter, Kathrin; Arnold, Wolfgang; Haensch, Wolfgang; Scherneck, Siegfried; Jockusch, Brigitte M.

    2004-01-01

    Profilin 1 (PFN1) is a regulator of the microfilament system and is involved in various signaling pathways. It interacts with many cytoplasmic and nuclear ligands. The importance of PFN1 for human tissue differentiation has been demonstrated by the findings that human cancer cells, expressing conspicuously low PFN1 levels, adopt a nontumorigenic phenotype upon raising their PFN1 level. In the present study, we characterize the ligand binding site crucial for profilin's tumor suppressor activity. Starting with CAL51, a human breast cancer cell line highly tumorigenic in nude mice, we established stable clones that express PFN1 mutants differentially defective in ligand binding. Clones expressing PFN1 mutants with reduced binding to either poly-proline-stretch ligands or phosphatidyl-inositol-4,5-bisphosphate, but with a functional actin binding site, were normal in growth, adhesion, and anchorage dependence, with only a weak tendency to elicit tumors in nude mice, similar to controls expressing wild-type PFN1. In contrast, clones expressing a mutant with severely reduced capacity to bind actin still behaved like the parental CAL51 and were highly tumorigenic. We conclude that the actin binding site on profilin is instrumental for normal differentiation of human epithelia and the tumor suppressor function of PFN1. PMID:14767055

  14. Cdc42-dependent actin dynamics controls maturation and secretory activity of dendritic cells

    PubMed Central

    Schulz, Anna M.; Stutte, Susanne; Hogl, Sebastian; Luckashenak, Nancy; Dudziak, Diana; Leroy, Céline; Forné, Ignasi; Imhof, Axel; Müller, Stephan A.; Brakebusch, Cord H.; Lichtenthaler, Stefan F.

    2015-01-01

    Cell division cycle 42 (Cdc42) is a member of the Rho guanosine triphosphatase family and has pivotal functions in actin organization, cell migration, and proliferation. To further study the molecular mechanisms of dendritic cell (DC) regulation by Cdc42, we used Cdc42-deficient DCs. Cdc42 deficiency renders DCs phenotypically mature as they up-regulate the co-stimulatory molecule CD86 from intracellular storages to the cell surface. Cdc42 knockout DCs also accumulate high amounts of invariant chain–major histocompatibility complex (MHC) class II complexes at the cell surface, which cannot efficiently present peptide antigens (Ag’s) for priming of Ag-specific CD4 T cells. Proteome analyses showed a significant reduction in lysosomal MHC class II–processing proteins, such as cathepsins, which are lost from DCs by enhanced secretion. As these effects on DCs can be mimicked by chemical actin disruption, our results propose that Cdc42 control of actin dynamics keeps DCs in an immature state, and cessation of Cdc42 activity during DC maturation facilitates secretion as well as rapid up-regulation of intracellular molecules to the cell surface. PMID:26553928

  15. The actin binding site of thymosin beta 4 mapped by mutational analysis.

    PubMed Central

    Van Troys, M; Dewitte, D; Goethals, M; Carlier, M F; Vandekerckhove, J; Ampe, C

    1996-01-01

    We characterized in detail the actin binding site of the small actin-sequestering protein thymosin beta 4 (T beta 4) using chemically synthesized full-length T beta 4 variants. The N-terminal part (residues 1-16) and a hexapeptide motif (residues 17-22) form separate structural entities. In both, we identified charged and hydrophobic residues that participate in the actin interaction using chemical cross-linking, complex formation in native gels and actin-sequestering experiments. Quantitative data on the activity of the variants and circular dichroism experiments allow to present a model in which the N-terminal part needs to adopt an alpha-helix for actin binding and interacts through a patch of hydrophobic residues (6M-I-F12) on one side of this helix. Also, electrostatic contacts between actin and lysine residues 18, in the motif, and 14, in the N-terminal alpha-helix, appear important for binding. The residues critical for contacting actin are conserved throughout the beta-thymosin family and in addition to this we identify a similar pattern in the C-terminal headpiece of villin and dematin. Images PMID:8617195

  16. Activation of F-Actin Binding Capacity of Ezrin: Synergism of PIP2 Interaction and Phosphorylation

    PubMed Central

    Bosk, Sabine; Braunger, Julia A.; Gerke, Volker; Steinem, Claudia

    2011-01-01

    Ezrin is a membrane-cytoskeleton linker protein that can bind F-actin in its active conformation. Several means of regulation of ezrin's activity have been described including phosphorylation of Thr-567 and binding of L-α-phosphatidylinositol-4,5-bisphosphate (PIP2). However, the relative contributions of these events toward activation of the protein and their potential interdependence are not known. We developed an assay based on solid-supported membranes, to which different ezrin mutants (ezrin T567A (inactive mutant), wild-type, and T567D (active pseudophosphorylated mutant)) were bound, that enabled us to analyze the influence of phosphorylation and PIP2 binding on ezrin's activation state in vitro. The lipid bilayers employed contained either DOGS-NTA-Ni to bind the proteins via an N-terminal His-tag, or PIP2, to which ezrin binds via specific binding sites located in the N-terminal region of the protein. Quantitative analysis of the binding behavior of all three proteins to the two different receptor lipids revealed that all three bind with high affinity and specificity to the two receptor lipids. Fluorescence microscopy on ezrin-decorated solid-supported membranes showed that, dependent on the mode of binding and the phosphorylation state, ezrin is capable of binding actin filaments. A clear synergism between phosphorylation and the receptor lipid PIP2 was observed, suggesting a conformational switch from the dormant to the active, F-actin binding state by recognition of PIP2, which is enhanced by the phosphorylation. PMID:21463584

  17. 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

  18. 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.

  19. 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. PMID:27048259

  20. Mutant huntingtin causes defective actin remodeling during stress: defining a new role for transglutaminase 2 in neurodegenerative disease

    PubMed Central

    Munsie, Lise; Caron, Nicholas; Atwal, Randy Singh; Marsden, Ian; Wild, Edward J.; Bamburg, James R.; Tabrizi, Sarah J.; Truant, Ray

    2011-01-01

    Huntington's disease (HD) is caused by an expanded CAG tract in the Interesting transcript 15 (IT15) gene encoding the 350 kDa huntingtin protein. Cellular stresses can trigger the release of huntingtin from the endoplasmic reticulum, allowing huntingtin nuclear entry. Here, we show that endogenous, full-length huntingtin localizes to nuclear cofilin–actin rods during stress and is required for the proper stress response involving actin remodeling. Mutant huntingtin induces a dominant, persistent nuclear rod phenotype similar to that described in Alzheimer's disease for cytoplasmic cofilin–actin rods. Using live cell temporal studies, we show that this stress response is similarly impaired when mutant huntingtin is present, or when normal huntingtin levels are reduced. In clinical lymphocyte samples from HD patients, we have quantitatively detected cross-linked complexes of actin and cofilin with complex formation varying in correlation with disease progression. By live cell fluorescence lifetime imaging measurement–Förster resonant energy transfer studies and western blot assays, we quantitatively observed that stress-activated tissue transglutaminase 2 (TG2) is responsible for the actin–cofilin covalent cross-linking observed in HD. These data support a direct role for huntingtin in nuclear actin re-organization, and describe a new pathogenic mechanism for aberrant TG2 enzymatic hyperactivity in neurodegenerative diseases. PMID:21355047

  1. Cross-Linked Nanotube Materials with Variable Stiffness Tethers

    NASA Technical Reports Server (NTRS)

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

    2004-01-01

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

  2. Transglutaminases: Widespread Cross-linking Enzymes in Plants

    PubMed Central

    Serafini-Fracassini, Donatella; Del Duca, Stefano

    2008-01-01

    Background Transglutaminases have been studied in plants since 1987 in investigations aimed at interpreting some of the molecular mechanisms by which polyamines affect growth and differentiation. Transglutaminases are a widely distributed enzyme family catalysing a myriad of biological reactions in animals. In plants, the post-translational modification of proteins by polyamines forming inter- or intra-molecular cross-links has been the main transglutaminase reaction studied. Characteristics of Plant Transglutaminases The few plant transglutaminases sequenced so far have little sequence homology with the best-known animal enzymes, except for the catalytic triad; however, they share a possible structural homology. Proofs of their catalytic activity are: (a) their ability to produce glutamyl-polyamine derivatives; (b) their recognition by animal transglutaminase antibodies; and (c) biochemical features such as calcium-dependency, etc. However, many of their fundamental biochemical and physiological properties still remain elusive. Transglutaminase Activity is Ubiquitous It has been detected in algae and in angiosperms in different organs and sub-cellular compartments, chloroplasts being the best-studied organelles. Possible Roles Possible roles concern the structural modification of specific protein substrates. In chloroplasts, transglutaminases appear to stabilize the photosynthetic complexes and Rubisco, being regulated by light and other factors, and possibly exerting a positive effect on photosynthesis and photo-protection. In the cytosol, they modify cytoskeletal proteins. Preliminary reports suggest an involvement in the cell wall construction/organization. Other roles appear to be related to fertilization, abiotic and biotic stresses, senescence and programmed cell death, including the hypersensitive reaction. Conclusions The widespread occurrence of transglutaminases activity in all organs and cell compartments studied suggests a relevance for their still

  3. Gcn1 and actin binding to Yih1: implications for activation of the eIF2 kinase GCN2.

    PubMed

    Sattlegger, Evelyn; Barbosa, João A R G; Moraes, Maria Carolina S; Martins, Rafael M; Hinnebusch, Alan G; Castilho, Beatriz A

    2011-03-25

    Yeast Yih1 protein and its mammalian ortholog IMPACT, abundant in neurons, are inhibitors of Gcn2, a kinase involved in amino acid homeostasis, stress response, and memory formation. Like Gcn2, Yih1/IMPACT harbors an N-terminal RWD domain that mediates binding to the Gcn2 activator Gcn1. Yih1 competes with Gcn2 for Gcn1 binding, thus inhibiting Gcn2. Yih1 also binds G-actin. Here, we show that Yih1-actin interaction is independent of Gcn1 and that Yih1-Gcn1 binding does not require actin. The Yih1 RWD (residues 1-132) was sufficient for Gcn2 inhibition and Gcn1 binding, but not for actin binding, showing that actin binding is dispensable for inhibiting Gcn2. Actin binding required Yih1 residues 68-258, encompassing part of the RWD and the C-terminal "ancient domain"; however, residues Asp-102 and Glu-106 in helix3 of the RWD were essential for Gcn1 binding and Gcn2 inhibition but dispensable for actin binding. Thus, the Gcn1- and actin-binding sites overlap in the RWD but have distinct binding determinants. Unexpectedly, Yih1 segment 68-258 was defective for inhibiting Gcn2 even though it binds Gcn1 at higher levels than does full-length Yih1. This and other results suggest that Yih1 binds with different requirements to distinct populations of Gcn1 molecules, and its ability to disrupt Gcn1-Gcn2 complexes is dependent on a complete RWD and hindered by actin binding. Modeling of the ancient domain on the bacterial protein YigZ showed peculiarities to the eukaryotic and prokaryotic lineages, suggesting binding sites for conserved cellular components. Our results support a role for Yih1 in a cross-talk between the cytoskeleton and translation. PMID:21239490

  4. Nuclear actin polymerization from faster growing ends in the initial activation of Hox gene transcription are nuclear speckles involved?

    PubMed

    Naum-Onganía, Gabriela; Díaz, Víctor M; Blasi, Francesco; Rivera-Pomar, Rolando

    2013-01-01

    The HoxB cluster expression is activated by retinoic acid and transcribed in a collinear manner. The DNA-binding Pknox1-Pbx1 complex modulates Hox protein activity. Here, NT2-D1 teratocarcinoma cells -a model of Hox gene expression- were used to show that upon retinoic acid induction, Pknox1 co-localizes with polymeric nuclear actin. We have found that globular actin aggregates, polymeric actin, the elongating RNA polymerase II and THOC match euchromatic regions corresponding to nuclear speckles. Moreover, RNA polymerase II, N-WASP, and transcription/splicing factors p54(nrb) and PSF were validated as Pknox1 interactors by tandem affinity purification. PSF pulled down with THOC and nuclear actin, both of which co-localize in nuclear speckles. Although latrunculin A slightly decreases the general level of HoxB gene expression, inhibition of nuclear actin polymerization by cytochalasin D blocks the expression of HoxB transcripts in a collinear manner. Thus, our results support the hypothesis that nuclear actin polymerization is involved in the activation of HoxB gene expression by means of nuclear speckles. PMID:24406343

  5. 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.

  6. p70 S6 kinase and actin dynamics

    PubMed Central

    Ip, Carman K.M.; Wong, Alice S.T.

    2012-01-01

    p70 S6 kinase (p70S6K), a member of the AGC serine/threonine kinase family, was initially identified as a key player, together with its downstream effector S6, in the regulation of cellular growth and survival. The p70S6K protein has emerged in recent years as a multifunctional protein which also regulates the actin cytoskeleton and thus plays a role in cell migration. This new function is through two important activities of p70S6K, namely actin cross-linking and Rac1 and Cdc42 activation. The testis is critically dependent on an intricate balance of fundamental cellular processes such as adhesion, migration, and differentiation. It is increasingly evident that Rho GTPases and actin binding proteins play fundamental roles in regulating spermatogenesis within the testis. In this review, we will discuss current findings of p70S6K in the control of actin cytoskeleton dynamics. In addition, the potential role of p70S6K in spermatogenesis and testicular function will be highlighted. PMID:22553489

  7. AFAP120 regulates actin organization during neuronal differentiation

    PubMed Central

    Xu, Xiaohua; Harder, Jennifer; Flynn, Daniel C.; Lanier, Lorene M.

    2008-01-01

    During development, dynamic changes in the actin cytoskeleton determine both cell motility and morphological differentiation. In most mature tissues, cells are generally minimally motile and have morphologies specialized to their functions. In metastatic cancer, cells generally loose their specialized morphology and become motile. Therefore, proteins that regulate the transition between the motile and morphologically differentiated states can play important roles in determining cancer outcomes. AFAP120 is a neuronal specific protein that binds Src Kinase and Protein Kinase C (PKC) and cross-links actin filaments. Here we report that expression and tyrosine phosphorylation of AFAP120 are developmentally regulated in the cerebellum. In cerebellar cultures, PKC activation induces Src-kinase dependent phosphorylation of AFAP120, indicating that AFAP120 may be a downstream effector of Src. In neuroblastoma cells induced to differentiate by treatment with a PKC activator, tyrosine phosphorylation of AFAP120 appears to regulate the formation of the lamellar actin structures and subsequent neurite initiation. Together, these results indicate that AFAP120 plays a role in organizing dynamic actin structures during neuronal differentiation and suggest that AFAP120 may help regulate the transition from motile precursor to morphologically differentiated neurons. PMID:19281763

  8. 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.

  9. Activation of myosin V-based motility and F-actin-dependent network formation of endoplasmic reticulum during mitosis.

    PubMed

    Wollert, Torsten; Weiss, Dieter G; Gerdes, Hans-Hermann; Kuznetsov, Sergei A

    2002-11-25

    It is widely believed that microtubule- and F-actin-based transport of cytoplasmic organelles and membrane fusion is down-regulated during mitosis. Here we show that during the transition of Xenopus egg extracts from interphase to metaphase myosin V-driven movement of small globular vesicles along F-actin is strongly inhibited. In contrast, the movement of ER and ER network formation on F-actin is up-regulated in metaphase extracts. Our data demonstrate that myosin V-driven motility of distinct organelles is differently controlled during the cell cycle and suggest an active role of F-actin in partitioning, positioning, and membrane fusion of the ER during cell division. PMID:12438410

  10. Actin filament tracking based on particle filters and stretching open active contour models.

    PubMed

    Li, Hongsheng; Shen, Tian; Vavylonis, Dimitrios; Huang, Xiaolei

    2009-01-01

    We introduce a novel algorithm for actin filament tracking and elongation measurement. Particle Filters (PF) and Stretching Open Active Contours (SOAC) work cooperatively to simplify the modeling of PF in a one-dimensional state space while naturally integrating filament body constraints to tip estimation. Our algorithm reduces the PF state spaces to one-dimensional spaces by tracking filament bodies using SOAC and probabilistically estimating tip locations along the curve length of SOACs. Experimental evaluation on TIRFM image sequences with very low SNRs demonstrates the accuracy and robustness of this approach. PMID:20426170

  11. Load transfer mechanisms in cross-linked DWNT fibers

    NASA Astrophysics Data System (ADS)

    Filleter, T.; Naraghi, M.; Moravsky, A.; Bernal, R.; Loutfy, R. O.; Espinosa, H. D.

    2011-03-01

    The application of carbon nanotubes (CNT) to macroscopic composite fibers has been limited by weak shear interfaces between adjacent CNT shells and composite matrix elements. A fundamental understanding of load transfer at multiple length-scales is needed to identify how the exceptional mechanical properties of CNTs can be scaled to produce high-performance fibers. Through in-situ electron microscopy tensile testing we have elucidated load transfer mechanisms across multiple scales of cross-linked double-walled nanotube (DWNT) fibers. A low density of polymer cross-links is found to increase the total energy dissipated at failure and ductility of fibers by 5 and 10X, respectively, without reducing strength. This mutiscale approach has identified a need to enhance shear interactions between individual DWNTs within the hierarchical DWNT fiber structures. Through in-situ TEM electron irradiation studies we have shown that load can be effectively transferred to inner DWNTs within bundles by covalently cross-linking the interfaces of adjacent DWNTs and shells. We have observed order of magnitude increases in strength and modulus and identified their dependence on irradiation dose. In future a combined approach of irradiation induced covalent and polymer cross-linking may lead to high-performance DWNT-based fibers and composites with tunable mechanical properties.

  12. Glutaraldehyde-cross-linked meniscal allografts: mechanical properties.

    PubMed

    Wisnewski, P J; Powers, D L; Kennedy, J M

    1988-01-01

    Removal of a severely damaged medial meniscus has been shown to lead to degradation of the articular cartilage and formation of degenerative arthritis. To counter this degenerative effect, meniscal prostheses, including glutaraldehyde-cross-linked allografts, have been evaluated in dogs. The purpose of this research was to quantify the mechanical properties of both fresh and glutaraldehyde-cross-linked canine medial menisci. Mechanical properties quantified were tensile strength, tensile modulus, and compressive stiffness. In addition, water content of compressive test samples was measured. Analysis of variance showed significantly lower tensile strength and tensile modulus and significantly higher compressive stiffness for the glutaraldehyde-cross-linked menisci, as compared to fresh specimens. Measurement of the weight percentage of water in fresh and cross-linked samples revealed no significant differences in water content. When implanted into a joint, the increased compressive stiffness could increase the peripheral tensile load. Due to the decreased tensile strength in this region, the prosthetic meniscus could be susceptible to peripheral tears. PMID:3155295

  13. 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. PMID:26652360

  14. 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...

  15. 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...

  16. 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...

  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, 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...

  19. 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.

  20. 21 CFR 177.2420 - Polyester resins, cross-linked.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... 21 Food and Drugs 3 2012-04-01 2012-04-01 false Polyester resins, cross-linked. 177.2420 Section 177.2420 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) FOOD FOR HUMAN CONSUMPTION (CONTINUED) INDIRECT FOOD ADDITIVES: POLYMERS Substances for Use Only as Components of Articles Intended for...

  1. 21 CFR 177.2420 - Polyester resins, cross-linked.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... 21 Food and Drugs 3 2010-04-01 2009-04-01 true Polyester resins, cross-linked. 177.2420 Section 177.2420 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) FOOD FOR HUMAN CONSUMPTION (CONTINUED) INDIRECT FOOD ADDITIVES: POLYMERS Substances for Use Only as Components of Articles Intended for...

  2. 21 CFR 177.2420 - Polyester resins, cross-linked.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... 21 Food and Drugs 3 2014-04-01 2014-04-01 false Polyester resins, cross-linked. 177.2420 Section 177.2420 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) INDIRECT FOOD ADDITIVES: POLYMERS Substances for Use Only as Components of Articles Intended for Repeated Use § 177.2420 Polyester...

  3. 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.

  4. 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.

  5. 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.

  6. Spectral Library Searching To Identify Cross-Linked Peptides.

    PubMed

    Schweppe, Devin K; Chavez, Juan D; Navare, Arti T; Wu, Xia; Ruiz, Bianca; Eng, Jimmy K; Lam, Henry; Bruce, James E

    2016-05-01

    Methods harnessing protein cross-linking and mass spectrometry (XL-MS) offer high-throughput means to identify protein-protein interactions (PPIs) and structural interfaces of protein complexes. Yet, specialized data dependent methods and search algorithms are often required to confidently assign peptide identifications to spectra. To improve the efficiency of matching high confidence spectra, we developed a spectral library based approach to search cross-linked peptide data derived from Protein Interaction Reporter (PIR) methods using the spectral library search algorithm, SpectraST. Spectral library matching of cross-linked peptide data from query spectra increased the absolute number of confident peptide relationships matched to spectra and thereby the number of PPIs identified. By matching library spectra from bona fide, previously established PIR-cross-linked peptide relationships, spectral library searching reduces the need for continued, complex mass spectrometric methods to identify peptide relationships, increases coverage of relationship identifications, and improves the accessibility of XL-MS technologies. PMID:27089058

  7. Synthesis of water-soluble, nonimmunogenic polyamide cross-linking agents.

    PubMed

    Hai, T T; Pereira, D E; Nelson, D J

    1998-01-01

    Novel polyamides were developed that can be used as cross-linking agents for proteins such as hemoglobin. Water-soluble, nonimmunogenic polyamides containing oxygen and sulfur atoms in the backbone were prepared by the polycondensation of the diacids bis(carboxymethyloxyacetyl)-1,4-diaminobutane (1a) or 3, 3'-thiodipropionic acid (1b) with diethylene glycol bis(3-aminopropyl) ether (2). The resulting alpha,omega-diacids were converted to the corresponding activated esters using any of a variety of carboxylic acid activating reagents including the novel reagent diphenyl(1-methylimidazol-2-thiyl)phosphonate (9). The resulting polyamides could be activated with a broad spectrum of groups that allow for the cross-linking and surface modification of proteins. PMID:9815156

  8. Processing of targeted psoralen cross-links in Xenopus oocytes.

    PubMed Central

    Segal, D J; Faruqi, A F; Glazer, P M; Carroll, D

    1997-01-01

    Psoralen cross-links have been shown to be both mutagenic and recombinagenic in bacterial, yeast, and mammalian cells. Double-strand breaks (DSBs) have been implicated as intermediates in the removal of psoralen cross-links. Recent work has suggested that site-specific mutagenesis and recombination might be achieved through the use of targeted psoralen adducts. The fate of plasmids containing psoralen adducts was evaluated in Xenopus oocytes, an experimental system that has well-characterized recombination capabilities and advantages in the analysis of intermediates in DNA metabolism. Psoralen adducts were delivered to a specific site by a triplex-forming oligonucleotide. These lesions are clearly recognized and processed in oocytes, since mutagenesis was observed at the target site. The spectrum of induced mutations was compared with that found in similar studies in mammalian cells. Plasmids carrying multiple random adducts were preferentially degraded, perhaps due to the introduction of DSBs. However, when DNAs carrying site-specific adducts were examined, no plasmid loss was observed and removal of cross-links was found to be very slow. Sensitive assays for DSB-dependent homologous recombination were performed with substrates with one or two cross-link sites. No adduct-stimulated recombination was observed with a single lesion, and only very low levels were observed with paired lesions, even when a large proportion of the cross-links was removed by the oocytes. We conclude that DSBs or other recombinagenic structures are not efficiently formed at psoralen adducts in Xenopus oocytes. While psoralen is not a promising reagent for stimulating site-specific recombination, it is effective in inducing targeted mutations. PMID:9343428

  9. Encapsulation of volatiles by homogenized partially-cross linked alginates.

    PubMed

    Inguva, Pavan K; Ooi, Shing Ming; Desai, Parind M; Heng, Paul W S

    2015-12-30

    Cross-linked calcium alginate gels are too viscous to be efficaciously incorporated into spray dried formulations. Thus, viscosity reduction is essential to ensure the processability of calcium alginate gels to be sprayed. Viscosity reduction by high pressure homogenization can open new formulation possibilities. Presently, testing of microcapsule integrity is also limited because either single particle tests neglect collective particle behaviours in bulk or bulk testing methods are often associated with single compressions which may not fully characterize individual particle strengths. The aim of this study was sub-divided into three objectives. First objective was to evaluate the impact of high pressure homogenization on gel viscosity. Second objective was to explore the use of the homogenized gels with modified starch for microencapsulation by spray drying. The final objective was to develop a stamping system as microcapsule strength tester that can assess microcapsules in bulk and evaluate the impact of multiple compressions. Collectively, this study would lead towards developing a pressure-activated patch of microcapsules with encapsulated volatiles and the method to assess the patch efficacy. The alginate gels largely experienced an exponential decay in viscosity when homogenized. Furthermore, the homogenized gels were successfully incorporated in spray drying formulations for microencapsulation. The custom-designed microcapsule strength tester was successfully used and shown to possess the required sensitivity to discern batches of microcapsules containing volatiles to have different release profiles. Addition of homogenized gels strengthened the microcapsules only at high wall to core ratios with low mass-load alginate gels. High mass-load gels weaken the microcapsules, exhibiting a higher release at low stamping pressures and wrinkling on the microcapsules surface. PMID:26581772

  10. 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

  11. Tandem Gramicidin Channels Cross-linked by Streptavidin

    PubMed Central

    Rokitskaya, Tatyana I.; Kotova, Elena A.; Antonenko, Yuri N.

    2003-01-01

    The interaction of biotin-binding proteins with biotinylated gramicidin (gA5XB) was studied by monitoring single-channel activity and sensitized photoinactivation kinetics. It was discovered that the addition of streptavidin or avidin to the bathing solutions of a bilayer lipid membrane (BLM) with incorporated gA5XB induced the opening of a channel characterized by approximately doubled single-channel conductance and extremely long open-state duration. We believe that the deceleration of the photoinactivation kinetics observed here with streptavidin and previously (Rokitskaya, T.I., Y.N. Antonenko, E.A. Kotova, A. Anastasiadis, and F. Separovic. 2000. Biochemistry. 39:13053–13058) with avidin reflects the formation of long-lived channels of this type. Both opening and closing of the double-conductance channels occurred via a transient sub-state of the conductance coinciding with that of the usual single-channel transition. The appearance of the double-conductance channels after the addition of streptavidin was preceded by bursts of fast fluctuations of the current with the open state duration of the individual events of 60 ms. The streptavidin-induced double-conductance channels appeared to be inherent only to the gramicidin analogue with a biotin group linked to the COOH terminus through a long linker arm. Including biotinylated phosphatidylethanolamine into the BLM prevented the formation of the double-conductance channels even with the excess streptavidin. In view of the results obtained here, it is suggested that the double-conductance channel represents a tandem of two neighboring gA5XB channels with their COOH termini being cross-linked by the bound streptavidin at both sides of the BLM. The finding that streptavidin induces the formation of the tandem gramicidin channel comprising two channels functioning in concert is considered to be relevant to the physiologically important phenomenon of ligand-induced receptor oligomerization. PMID:12719486

  12. Actin from Saccharomyces cerevisiae.

    PubMed Central

    Greer, C; Schekman, R

    1982-01-01

    Inhibition of DNase I activity has been used as an assay to purify actin from Saccharomyces cerevisiae (yeast actin). The final fraction, obtained after a 300-fold purification, is approximately 97% pure as judged by sodium dodecyl sulfate-gel electrophoresis. Like rabbit skeletal muscle actin, yeast actin has a molecular weight of about 43,000, forms 7-nm-diameter filaments when polymerization is induced by KCl or Mg2+, and can be decorated with a proteolytic fragment of muscle myosin (heavy meromyosin). Although heavy meromyosin ATPase activity is stimulated by rabbit muscle and yeast actins to approximately the same Vmax (2 mmol of Pi per min per mumol of heavy meromyosin), half-maximal activation (Kapp) is obtained with 14 micro M muscle actin, but requires approximately 135 micro M yeast actin. This difference suggests a low affinity of yeast actin for muscle myosin. Yeast and muscle filamentous actin respond similarly to cytochalasin and phalloidin, although the drugs have no effect on S. cerevisiae cell growth. Images PMID:6217414

  13. Ligand-induced activation of a formin–NPF pair leads to collaborative actin nucleation

    PubMed Central

    Graziano, Brian R.; Jonasson, Erin M.; Pullen, Jessica G.; Gould, Christopher J.

    2013-01-01

    Formins associate with other nucleators and nucleation-promoting factors (NPFs) to stimulate collaborative actin assembly, but the mechanisms regulating these interactions have been unclear. Yeast Bud6 has an established role as an NPF for the formin Bni1, but whether it also directly regulates the formin Bnr1 has remained enigmatic. In this paper, we analyzed NPF-impaired alleles of bud6 in a bni1Δ background and found that Bud6 stimulated Bnr1 activity in vivo. Furthermore, Bud6 bound directly to Bnr1, but its NPF effects were masked by a short regulatory sequence, suggesting that additional factors may be required for activation. We isolated a novel in vivo binding partner of Bud6, Yor304c-a/Bil1, which colocalized with Bud6 and functioned in the Bnr1 pathway for actin assembly. Purified Bil1 bound to the regulatory sequence in Bud6 and triggered NPF effects on Bnr1. These observations define a new mode of formin regulation, which has important implications for understanding NPF-nucleator pairs in diverse systems. PMID:23671312

  14. Modest Interference with Actin Dynamics in Primary T Cell Activation by Antigen Presenting Cells Preferentially Affects Lamellal Signaling

    PubMed Central

    Roybal, Kole T.; Mace, Emily M.; Clark, Danielle J.; Leard, Alan D.; Herman, Andrew; Verkade, Paul; Orange, Jordan S.; Wülfing, Christoph

    2015-01-01

    Dynamic subcellular distributions of signaling system components are critical regulators of cellular signal transduction through their control of molecular interactions. Understanding how signaling activity depends on such distributions and the cellular structures driving them is required for comprehensive insight into signal transduction. In the activation of primary murine T cells by antigen presenting cells (APC) signaling intermediates associate with various subcellular structures, prominently a transient, wide, and actin-associated lamellum extending from an interdigitated T cell:APC interface several micrometers into the T cell. While actin dynamics are well established as general regulators of cellular organization, their role in controlling signaling organization in primary T cell:APC couples and the specific cellular structures driving it is unresolved. Using modest interference with actin dynamics with a low concentration of Jasplakinolide as corroborated by costimulation blockade we show that T cell actin preferentially controls lamellal signaling localization and activity leading downstream to calcium signaling. Lamellal localization repeatedly related to efficient T cell function. This suggests that the transient lamellal actin matrix regulates T cell signaling associations that facilitate T cell activation. PMID:26237588

  15. Actin Rings of Power.

    PubMed

    Schwayer, Cornelia; Sikora, Mateusz; Slováková, Jana; Kardos, Roland; Heisenberg, Carl-Philipp

    2016-06-20

    Circular or ring-like actin structures play important roles in various developmental and physiological processes. Commonly, these rings are composed of actin filaments and myosin motors (actomyosin) that, upon activation, trigger ring constriction. Actomyosin ring constriction, in turn, has been implicated in key cellular processes ranging from cytokinesis to wound closure. Non-constricting actin ring-like structures also form at cell-cell contacts, where they exert a stabilizing function. Here, we review recent studies on the formation and function of actin ring-like structures in various morphogenetic processes, shedding light on how those different rings have been adapted to fulfill their specific roles. PMID:27326928

  16. Actinic keratosis

    MedlinePlus

    Solar keratosis; Sun-induced skin changes - keratosis; Keratosis - actinic (solar) ... Some actinic keratoses become squamous cell skin cancer . Have your health care provider look at all skin growths as soon as you find them. Your provider will ...

  17. Immobilization of cross-linked phenylalanine ammonia lyase aggregates in microporous silica gel.

    PubMed

    Cui, Jian Dong; Li, Lian Lian; Bian, Hong Jie

    2013-01-01

    A separable and highly-stable enzyme system was developed by adsorption of phenylalanine ammonia lyase (PAL) from Rhodotorula glutinis in amino-functionalized macroporous silica gel and subsequent enzyme crosslinking. This resulted in the formation of cross-linked enzyme aggregates (PAL-CLEAs) into macroporous silica gel (MSG-CLEAs). The effect of adsorptive conditions, type of aggregating agent, its concentration as well as that of cross-linking agent was studied. MSG-CLEAs production was most effective using ammonium sulfate (40%-saturation), followed by cross-linking for 1 h with 1.5% (v/v) glutaraldehyde. The resulting MSG-CLEAs extended the optimal temperature and pH range compared to free PAL and PAL-CLEAs. Moreover, MSG-CLEAs exhibited the excellent stability of the enzyme against various deactivating conditions such as temperature and denaturants, and showed higher storage stability compared to the free PAL and the conventional PAL-CLEAs. Such as, after 6 h incubation at 60°C, the MSG-CLEAs still retained more than 47% of the initial activity whereas PAL-CLEAs only retained 7% of the initial activity. Especially, the MSG-CLEAs exhibited good reusability due to its suitable size and active properties. These results indicated that PAL-CLEAs on MSG might be used as a feasible and efficient solution for improving properties of immobilized enzyme in industrial application. PMID:24260425

  18. Well-defined, Reversible Disulfide Cross-linked Micelles for On-demand Paclitaxel Delivery

    PubMed Central

    Li, Yuanpei; Xiao, Kai; Luo, Juntao; Xiao, Wenwu; Lee, Joyce S.; Gonik, Abby M.; Kato, Jason; Dong, Tiffany; Lam, Kit S.

    2011-01-01

    To minimize premature release of drugs from their carriers during circulation in the blood stream, we have recently developed reversible disulfide cross-linked micelles (DCMs) that can be triggered to release drug at the tumor site or in cancer cells. We designed and synthesized thiolated linear-dendritic polymers (telodendrimers) by introducing cysteines to the dendritic oligo-lysine backbone of our previously reported telodendrimers comprised of linear polyethylene glycol (PEG) and a dendritic cluster of cholic acids. Reversibly cross-linked micelles were then prepared by the oxidization of thiol groups to disulfide bond in the core of micelles after the self-assembly of thiolated telodendrimers. The DCMs were spherical with a uniform size of 28 nm, and were able to load paclitaxel (PTX) in the core with superior loading capacity up to 35.5% (w/w, drug/micelle). Cross-linking of the micelles within the core reduced their apparent critical micelle concentration and greatly enhanced their stability in non-reductive physiological conditions as well as severe micelle-disrupting conditions. The release of PTX from the DCMs was significantly slower than that from non-cross-linked micelles (NCMs), but can be gradually facilitated by increasing the concentration of reducing agent (glutathione) to an intracellular reductive level. The DCMs demonstrated a longer in vivo blood circulation time, less hemolytic activities, and superior toxicity profiles in nude mice, when compared to NCMs. DCMs were found to be able to preferentially accumulate at the tumor site in nude mice bearing SKOV-3 ovarian cancer xenograft. We also demonstrated that the disulfide cross-linked micellar formulation of PTX (PTX-DCMs) was more efficacious than both free drug and the non-cross-linked formulation of PTX at equivalent doses of PTX in the ovarian cancer xenograft mouse model. The anti-tumor effect of PTX-DCMs can be further enhanced by triggering the release of PTX on-demand by the

  19. Nanofilm biomaterials: localized cross-linking to optimize mechanical rigidity and bioactivity.

    PubMed

    Phelps, Jennifer A; Morisse, Samuel; Hindié, Mathilde; Degat, Marie-Christelle; Pauthe, Emmanuel; Van Tassel, Paul R

    2011-02-01

    Nanofilm biomaterials, formed by the layer-by-layer assembly of charged macromolecules, are important systems for a variety of cell-contacting biomedical and biotechnological applications. Mechanical rigidity and bioactivity are two key film properties influencing the behavior of contacting cells. Increased rigidity tends to improve cells attachment, and films may be rendered bioactive through the incorporation of proteins, peptides, or drugs. A key challenge is to realize films that are simultaneously rigid and bioactive. Chemical cross-linking of the polymer framework--the standard means of increasing a film's rigidity--can diminish bioactivity through deactivation or isolation of embedded biomolecules or inhibition of film biodegradation. We present here a strategy to decouple mechanical rigidity and bioactivity, potentially enabling nanofilm biomaterials that are both mechanically rigid and bioactive. Our idea is to selectively cross-link the outer region of the film, resulting in a rigid outer skin to promote cell attachment, while leaving the film interior (with any embedded bioactive species) unaffected. We propose an approach whereby an N-hydroxysulfosuccinimide (sulfo-NHS) activated poly(L-glutamic acid) is added as the terminal layer of a multilayer film and forms (covalent) amide bonds with amino groups of poly(L-lysine) placed previously within the film. We characterize film assembly and cross-linking extent via quartz crystal microbalance with dissipation monitoring (QCMD), Fourier transform infrared spectroscopy in attenuated total reflection mode (FTIR-ATR), and laser scanning confocal microscopy (LSCM) and measure the attachment and metabolic activity of preosteoblastic MC3T3-E1 cells. We show cross-linking to occur primarily at the film surface and the subsequent cell attachment and metabolic activity to be enhanced compared to native films. Our method appears promising as a means to realize films that are simultaneously mechanically rigid and

  20. Activation of exocytosis by cross-linking of the IgE receptor is dependent on ADP-ribosylation factor 1-regulated phospholipase D in RBL-2H3 mast cells: evidence that the mechanism of activation is via regulation of phosphatidylinositol 4,5-bisphosphate synthesis.

    PubMed Central

    Way, G; O'luanaigh, N; Cockcroft, S

    2000-01-01

    The physiological stimulus to exocytosis in mast cells is the cross-linking of the high-affinity IgE receptor, FcepsilonR1, with antigen. We demonstrate a novel function for ADP-ribosylation factor 1 (ARF1) in the regulation of antigen-stimulated secretion using cytosol-depleted RBL-2H3 mast cells for reconstitution of secretory responses. When antigen is used as the stimulus, ARF1 also reconstitutes phospholipase D activation. Using ethanol to divert the phosphatidic acid (the product of phospholipase D activity) to phosphatidylethanol causes inhibition of ARF1-reconstituted secretion. In addition. ARF1 causes an increase in phosphatidylinositol 4,5-bisphosphate (PIP(2)) levels at the expense of phosphatidylinositol 4-monophosphate. The requirement for PIP(2) in exocytosis was confirmed by using phosphatidylinositol transfer protein (PITPalpha) to increase PIP(2) levels. Exocytosis, restored by either ARF1 or PITPalpha, was inhibited when PIP(2) levels were depleted by phospholipase Cdelta1. We conclude that the function of ARF1 and PITPalpha is to increase the local synthesis of PIP(2), the function of which in exocytosis is likely to be linked to lipid-protein interactions, whereby recruitment of key components of the exocytotic machinery are targeted to the appropriate membrane compartment. PMID:10657240

  1. Syntenin-1 and Ezrin Proteins Link Activated Leukocyte Cell Adhesion Molecule to the Actin Cytoskeleton*

    PubMed Central

    Tudor, Cicerone; te Riet, Joost; Eich, Christina; Harkes, Rolf; Smisdom, Nick; Bouhuijzen Wenger, Jessica; Ameloot, Marcel; Holt, Matthew; Kanger, Johannes S.; Figdor, Carl G.; Cambi, Alessandra; Subramaniam, Vinod

    2014-01-01

    Activated leukocyte cell adhesion molecule (ALCAM) is a type I transmembrane protein member of the immunoglobulin superfamily of cell adhesion molecules. Involved in important pathophysiological processes such as the immune response, cancer metastasis, and neuronal development, ALCAM undergoes both homotypic interactions with other ALCAM molecules and heterotypic interactions with the surface receptor CD6 expressed at the T cell surface. Despite biochemical and biophysical evidence of a dynamic association between ALCAM and the actin cytoskeleton, no detailed information is available about how this association occurs at the molecular level. Here, we exploit a combination of complementary microscopy techniques, including FRET detected by fluorescence lifetime imaging microscopy and single-cell force spectroscopy, and we demonstrate the existence of a preformed ligand-independent supramolecular complex where ALCAM stably interacts with actin by binding to syntenin-1 and ezrin. Interaction with the ligand CD6 further enhances these multiple interactions. Altogether, our results propose a novel biophysical framework to understand the stabilizing role of the ALCAM supramolecular complex engaged to CD6 during dendritic cell-T cell interactions and provide novel information on the molecular players involved in the formation and signaling of the immunological synapse at the dendritic cell side. PMID:24662291

  2. Actin foci facilitate activation of the phospholipase C-γ in primary T lymphocytes via the WASP pathway

    PubMed Central

    Kumari, Sudha; Depoil, David; Martinelli, Roberta; Judokusumo, Edward; Carmona, Guillaume; Gertler, Frank B; Kam, Lance C; Carman, Christopher V; Burkhardt, Janis K; Irvine, Darrell J; Dustin, Michael L

    2015-01-01

    Wiscott Aldrich Syndrome protein (WASP) deficiency results in defects in calcium ion signaling, cytoskeletal regulation, gene transcription and overall T cell activation. The activation of WASP constitutes a key pathway for actin filament nucleation. Yet, when WASP function is eliminated there is negligible effect on actin polymerization at the immunological synapse, leading to gaps in our understanding of the events connecting WASP and calcium ion signaling. Here, we identify a fraction of total synaptic F-actin selectively generated by WASP in the form of distinct F-actin ‘foci’. These foci are polymerized de novo as a result of the T cell receptor (TCR) proximal tyrosine kinase cascade, and facilitate distal signaling events including PLCγ1 activation and subsequent cytoplasmic calcium ion elevation. We conclude that WASP generates a dynamic F-actin architecture in the context of the immunological synapse, which then amplifies the downstream signals required for an optimal immune response. DOI: http://dx.doi.org/10.7554/eLife.04953.001 PMID:25758716

  3. Arabidopsis VILLIN1 and VILLIN3 have overlapping and distinct activities in actin bundle formation and turnover

    SciTech Connect

    Khurana, P; Henty, Jessica L; Huang, S; Staiger, A M; Blanchoin, L; Staiger, Chris J

    2010-09-28

    Actin filament bundles are higher-order cytoskeletal structures that are crucial for the maintenance of cellular architecture and cell expansion. They are generated from individual actin filaments by the actions of bundling proteins like fimbrins, LIMs, and villins. However, the molecular mechanisms of dynamic bundle formation and turnover are largely unknown. Villins belong to the villin/gelsolin/fragmin superfamily and comprise at least five isovariants in Arabidopsis thaliana. Different combinations of villin isovariants are coexpressed in various tissues and cells. It is not clear whether these isovariants function together and act redundantly or whether they have unique activities. VILLIN1 (VLN1) is a simple filament-bundling protein and is Ca{sup 2+} insensitive. Based on phylogenetic analyses and conservation of Ca{sup 2+} binding sites, we predict that VLN3 is a Ca{sup 2+}-regulated villin capable of severing actin filaments and contributing to bundle turnover. The bundling activity of both isovariants was observed directly with time-lapse imaging and total internal reflection fluorescence (TIRF) microscopy in vitro, and the mechanism mimics the “catch and zipper” action observed in vivo. Using time-lapse TIRF microscopy, we observed and quantified the severing of individual actin filaments by VLN3 at physiological calcium concentrations. Moreover, VLN3 can sever actin filament bundles in the presence of VLN1 when calcium is elevated to micromolar levels. Collectively, these results demonstrate that two villin isovariants have overlapping and distinct activities.

  4. Characterization of porous collagen/hyaluronic acid scaffold modified by 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide cross-linking.

    PubMed

    Park, Si-Nae; Park, Jong-Chul; Kim, Hea Ok; Song, Min Jung; Suh, Hwal

    2002-02-01

    In order to develop a scaffolding material for tissue regeneration, porous matrices containing collagen and hyaluronic acid were fabricated by freeze drying at -20 degrees C, -70 degrees C or -196 degrees C. The fabricated porous membranes were cross-linked using 1-ethyl-3-(3-dimethyl aminopropyl) carbodiimide (EDC) in a range of 1-100 mM concentrations for enhancing mechanical stability of the composite matrix. Scanning electron microscope (SEM) views of the matrices demonstrated that the matrices obtained before cross-linking process had interconnected pores with mean diameters of 40, 90 or 230 microm and porosity of 58-66% according to the freezing temperature, and also the porous structures after cross-linking process were retained. The swelling test and IR spectroscopic measurement of different cross-linked membranes were carried out as a measure of the extent of cross-linking. The swelling behavior of cross-linked membranes showed no significant differences as cross-linking degree increased. FT-IR spectra showed the increase of the intensity of the absorbencies at amide bonds (1655, 1546, 1458 cm(-1)) compared to that of CH bond (2930 cm(-1)). In enzymatic degradation test, EDC treated membranes showed significant enhancement of the resistance to collagenase activity in comparison with 0.625% glutaraldehyde treated membranes. In cytotoxicity test using L929 fibroblastic cells, the EDC-cross-linked membranes demonstrated no significant toxicity. PMID:11791924

  5. Cross-linking Measurements of In Vivo Protein Complex Topologies*

    PubMed Central

    Zheng, Chunxiang; Yang, Li; Hoopmann, Michael R.; Eng, Jimmy K.; Tang, Xiaoting; Weisbrod, Chad R.; Bruce, James E.

    2011-01-01

    Identification and measurement of in vivo protein interactions pose critical challenges in the goal to understand biological systems. The measurement of structures and topologies of proteins and protein complexes as they exist in cells is particularly challenging, yet critically important to improve understanding of biological function because proteins exert their intended function only through the structures and interactions they exhibit in vivo. In the present study, protein interactions in E. coli cells were identified in our unbiased cross-linking approach, yielding the first in vivo topological data on many interactions and the largest set of identified in vivo cross-linked peptides produced to date. These data show excellent agreement with protein and complex crystal structures where available. Furthermore, our unbiased data provide novel in vivo topological information that can impact understanding of biological function, even for cases where high resolution structures are not yet available. PMID:21697552

  6. Hydration and swelling of amorphous cross-linked starch microspheres.

    PubMed

    Wojtasz, Joanna; Carlstedt, Jonas; Fyhr, Peter; Kocherbitov, Vitaly

    2016-01-01

    Hydration of cross-linked starch microspheres, commercially available as a medical device, was investigated using a multi-method approach. We found that the uptake of water is accompanied by substantial swelling and changes of the polymer structure. Sorption calorimetry provided information about thermodynamics of water sorption, revealed presence of isothermal glass transition and absence of hydration-induced crystallization, observed in non-cross linked starch material. The changes in the surface and bulk properties of microspheres at different water-starch concentrations were investigated using synchrotron radiation X-ray scattering and analyzed using concept of fractals. The obtained information, combined with the results of differential scanning calorimetry, was used to construct a phase diagram of the studied material. Finally, hydration induced evolution of polymer structure revealed by the X-ray scattering was linked to the changes observed during swelling with optical microscopy. PMID:26453872

  7. Cross-Linked Fiber Network Embedded in Elastic Matrix

    PubMed Central

    Zhang, L.; Lake, S.P.; Barocas, V.H.; Shephard, M.S.; Picu, R.C.

    2013-01-01

    The mechanical behavior of a three-dimensional cross-linked fiber network embedded in matrix is studied in this work. The network is composed from linear elastic fibers which store energy only in the axial deformation mode, while the matrix is also isotropic and linear elastic. Such systems are encountered in a broad range of applications, from tissue to consumer products. As the matrix modulus increases, the network is constrained to deform more affinely. This leads to internal forces acting between the network and the matrix, which produce strong stress concentration at the network cross-links. This interaction increases the apparent modulus of the network and decreases the apparent modulus of the matrix. A model is developed to predict the effective modulus of the composite and its predictions are compared with numerical data for a variety of networks. PMID:24089623

  8. Probing the solution structure of Factor H using hydroxyl radical protein footprinting and cross-linking.

    PubMed

    Baud, Anna; Gonnet, Florence; Salard, Isabelle; Le Mignon, Maxime; Giuliani, Alexandre; Mercère, Pascal; Sclavi, Bianca; Daniel, Régis

    2016-06-15

    The control protein Factor H (FH) is a crucial regulator of the innate immune complement system, where it is active on host cell membranes and in the fluid phase. Mutations impairing the binding capacity of FH lead to severe autoimmune diseases. Here, we studied the solution structure of full-length FH, in its free state and bound to the C3b complement protein. To do so, we used two powerful techniques, hydroxyl radical protein footprinting (HRPF) and chemical cross-linking coupled with mass spectrometry (MS), to probe the structural rearrangements and to identify protein interfaces. The footprint of C3b on the FH surface matches existing crystal structures of C3b complexed with the N- and C-terminal fragments of FH. In addition, we revealed the position of the central portion of FH in the protein complex. Moreover, cross-linking studies confirmed the involvement of the C-terminus in the dimerization of FH. PMID:27099340

  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. Optimized Fragmentation Regime for Diazirine Photo-Cross-Linked Peptides.

    PubMed

    Giese, Sven H; Belsom, Adam; Rappsilber, Juri

    2016-08-16

    Cross-linking/mass spectrometry has evolved into a robust technology that reveals structural insights into proteins and protein complexes. We leverage a new tribrid instrument with improved fragmentation capacities in a systematic comparison to identify which fragmentation method would be best for the identification of cross-linked peptides. Specifically, we explored three fragmentation methods and two combinations: collision-induced dissociation (CID), beam-type CID (HCD), electron-transfer dissociation (ETD), ETciD, and EThcD. Trypsin-digested, SDA-cross-linked human serum albumin (HSA) served as a test sample, yielding over all methods and in triplicate analysis in total 2602 matched PSMs and 1390 linked residue pairs at 5% false discovery rate, as confirmed by the crystal structure. HCD wins in number of matched peptide-spectrum-matches (958 PSMs) and identified links (446). CID is most complementary, increasing the number of identified links by 13% (58 links). HCD wins together with EThcD in cross-link site calling precision, with approximately 62% of sites having adjacent backbone cleavages that unambiguously locate the link in both peptides, without assuming any cross-linker preference for amino acids. Overall quality of spectra, as judged by sequence coverage of both peptides, is best for EThcD for the majority of peptides. Sequence coverage might be of particular importance for complex samples, for which we propose a data dependent decision tree, else HCD is the method of choice. The mass spectrometric raw data has been deposited in PRIDE (PXD003737). PMID:27454319

  11. 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. PMID:24564765

  12. 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.

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

    PubMed Central

    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. PMID:24204144

  14. 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.

  15. 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. PMID:15930646

  16. 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.

  17. 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.

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

    PubMed Central

    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.

    2015-01-01

    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. PMID:25717100

  19. 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. PMID:25717100

  20. 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.

  1. Distribution and quantification of pyridinium cross-links of collagen within the different maturational zones of the chick growth plate.

    PubMed

    Farquharson, C; Duncan, A; Seawright, E; Whitehead, C C; Robins, S P

    1996-08-13

    In order to assess alterations in the collagen network during endochondral ossification the pyridinium cross-links of collagen were quantified in sequential transverse sections through the chick growth plate. This was accomplished using both morphological (alkaline phosphatase (ALP) histochemistry and collagen type X immunostaining) and analytical (HPLC) analyses. In articular cartilage, pyridinoline concentrations were maximal in the deep mature zones. In contrast, the proliferating chondrocyte zone of the growth plate had approximately a 10-fold greater pyridinoline cross-link concentration than the mature hypertrophic zone. Deoxypyridinoline was first found in the prehypertrophic zone of the growth plate cartilage that reacted positively for ALP activity but before collagen type X was detected. However, deoxypyridinoline concentrations were highest in the most differentiated regions of the growth plate where it was the principal pyridinium cross-link. In tibial dyschondroplasia, where chondrocyte differentiation is arrested in the prehypertrophic zone, higher concentrations of both cross-links were found with increasing distance down the lesion. We conclude that the decrease in pyridinoline cross-link concentration down the growth plate may be an essential adaptation (via increased collagenase activity and collagen turnover) of the matrix for vascular invasion and osteoclastic resorption to occur. PMID:8765127

  2. 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

  3. 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

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

    PubMed Central

    Brieher, William M.

    2013-01-01

    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. PMID:24322428

  5. 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

  6. MAPKAPK-2-mediated LIM-kinase activation is critical for VEGF-induced actin remodeling and cell migration

    PubMed Central

    Kobayashi, Miho; Nishita, Michiru; Mishima, Toshiaki; Ohashi, Kazumasa; Mizuno, Kensaku

    2006-01-01

    Vascular endothelial growth factor-A (VEGF-A) induces actin reorganization and migration of endothelial cells through a p38 mitogen-activated protein kinase (MAPK) pathway. LIM-kinase 1 (LIMK1) induces actin remodeling by phosphorylating and inactivating cofilin, an actin-depolymerizing factor. In this study, we demonstrate that activation of LIMK1 by MAPKAPK-2 (MK2; a downstream kinase of p38 MAPK) represents a novel signaling pathway in VEGF-A-induced cell migration. VEGF-A induced LIMK1 activation and cofilin phosphorylation, and this was inhibited by the p38 MAPK inhibitor SB203580. Although p38 phosphorylated LIMK1 at Ser-310, it failed to activate LIMK1 directly; however, MK2 activated LIMK1 by phosphorylation at Ser-323. Expression of a Ser-323-non-phosphorylatable mutant of LIMK1 suppressed VEGF-A-induced stress fiber formation and cell migration; however, expression of a Ser-323-phosphorylation-mimic mutant enhanced these processes. Knockdown of MK2 by siRNA suppressed VEGF-A-induced LIMK1 activation, stress fiber formation, and cell migration. Expression of kinase-dead LIMK1 suppressed VEGF-A-induced tubule formation. These findings suggest that MK2-mediated LIMK1 phosphorylation/activation plays an essential role in VEGF-A-induced actin reorganization, migration, and tubule formation of endothelial cells. PMID:16456544

  7. Probing actin incorporation into myofibrils using Asp11 and His73 actin mutants.

    PubMed

    Xia, D; Peng, B; Sesok, D A; Peng, I

    1993-01-01

    We used a cell free system Bouché et al.: J. Cell Biol. 107:587-596, 1988] to study the incorporation of actin into myofibrils. We used alpha-skeletal muscle actin and actins with substitutions of either His73 [Solomon and Rubenstein: J. Biol.Chem. 262:11382, 1987], or Asp11 [Solomon et al.: J. Biol. Chem. 263:19662, 1988]. Actins were translated in reticulocyte lysate and incubated with myofibrils. The incorporated wild type actin could be cross-linked into dimers using N,N'-1,4-phenylenebismaleimide (PBM), indicating that the incorporated actin is actually inserted into the thin filaments of the myofibril. The His73 mutants incorporated to the same extent as wild type actin and was also cross-linked with PBM. Although some of the Asp11 mutants co-assembled with carrier actin, only 1-3% of the Asp11 mutant actins incorporated after 2 min and did not increase after 2 hr. Roughly 17% of wild type actin incorporated after 2 min and 31% after 2 hr. ATP increased the release of wild type actin from myofibrils, but did not increase the release of Asp11 mutants. We suggest that (1) the incorporation of wild type and His73 mutant actins was due to a physiological process whereas association of Asp11 mutants with myofibrils was non-specific, (2) the incorporation of wild type actin involved a rapid initial phase, followed by a slower phase, and (3) since some of the Asp11 mutants can co-assemble with wild type actin, the ability to self-assemble was not sufficient for incorporation into myofibrils. Thus, incorporation probably includes interaction between actin and a thin filament associated protein. We also showed that incorporation occurred at actin concentrations which would cause disassembly of F-actin. Since the myofibrils did not show large scale disassembly but incorporated actin, filament stability and monomer incorporation are likely to be mediated by actin associated proteins of the myofibril. PMID:8287497

  8. 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

  9. Photoaffinity labeling and photoaffinity cross-linking of phosphofructokinase-1 from Saccharomyces cerevisiae by 8-azidoadeninenucleotides.

    PubMed

    Knoche, M; Mönnich, K; Schäfer, H J; Kopperschläger, G

    2001-01-15

    Phosphofructokinase-1 from Saccharomyces cerevisiae is composed of four alpha- and four beta-subunits, each of them carrying catalytic and regulatory bindings sites for MgATP. In this paper, various photoaffinity labels, such as 8-azidoadenosine 5'-triphosphate, 8-azido-1,N6-ethenoadenosine 5'-triphosphate, and 8-N3-3'(2')-O-biotinyl-8-azidoadenosine 5'-triphosphate have been used to study their interaction with the enzyme in the dark and during irradiation. All nucleotidetriphosphates function as phosphate donor forming fructose 1,6-bisphosphate from fructose 6-phosphate. However, the kinetic analysis revealed distinctly differences between them. Photolabeling causes a decrease in enzyme activity to a similar extent, and ATP acts as competitive effector to inactivation. Three bifunctional diazidodiadeninedinucleotides (8-diN3AP4A, monoepsilon-8-diN3AP4A, and diepsilon-8-diN3AP4A) were applied for studying the spatial arrangement of the nucleotide binding sites. No cross-linking of the subunits was obtained by irradiation of the enzyme with 8-diN3AP4A. Photolabeling with diepsilon-8-diN3AP4A resulted in the formation of two alpha-beta cross-links with different mobilities in the SDS-polyacrylamide gel electrophoresis, while monoepsilon-8-diN3AP4A yielded only one alpha-beta cross-link. Because an interfacial location of the catalytic sites between two subunits is less likely, we suggest that the formation of cross-linked subunits may be the result of specific interactions of the bifunctional photolabels with regulatory sites at the interface of both subunits. PMID:11368011

  10. Mechanically Stiff, Zinc Cross-Linked Nanocomposite Scaffolds with Improved Osteostimulation and Antibacterial Properties.

    PubMed

    Sehgal, Rekha R; Carvalho, Edmund; Banerjee, Rinti

    2016-06-01

    Nanocomposite scaffolds are studied widely due to their resemblance with the natural extracellular matrix of bone; but their use as a bone tissue engineered scaffold is clinically hampered due to low mechanical stiffness, inadequate osteoconduction, and graft associated infections. The purpose of the current study was the development of a mechanically stiff nanocomposite scaffold using biodegradable gellan and xanthan polymers reinforced with bioglass nanoparticles (nB) (Size: 20-120 nm). These nanocomposite scaffolds were cross-linked with zinc sulfate ions to improve their osteoconduction and antibacterial properties for the regeneration of a functional bone. The compressive strength and modulus of the optimized nanocomposite scaffold (1% w/v polymer reinforced with 4%w/v nB nanoparticles, cross-linked with 1.5 mM zinc sulfate) was 1.91 ± 0.31 MPa and 20.36 ± 1.08 MPa, respectively, which was comparable to the trabecular bone and very high compared to nanocomposite scaffolds reported in earlier studies. Further, in vitro simulated body fluid (SBF) study suggested deposition of biomimetic apatite on the surface of zinc cross-linked nanocomposite scaffolds confirming their bioactivity. MG 63 osteoblast-like cells cultured with the nanocomposite scaffolds responded to matrix stiffness with better adhesion, spreading and cellular interconnections compared to the polymeric gellan and xanthan scaffolds. Incorporation of bioglass nanoparticles and zinc cross-linker in nanocomposite scaffolds demonstrated 62% increment in expression of alkaline phosphatase activity (ALP) and 150% increment in calcium deposition of MG 63 osteoblast-like cells compared to just gellan and xanthan polymeric scaffolds. Furthermore, zinc cross-linked nanocomposite scaffolds significantly inhibited the growth of Gram-positive Bacillus subtilis (70% reduction) and Gram-negative Escherichia coli (81% reduction) bacteria. This study demonstrated a facile approach to tune the mechanical stiffness

  11. 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. PMID:26692166

  12. Well-defined cross-linked antioxidant nanozymes for treatment of ischemic brain injury

    PubMed Central

    Manickam, Devika S.; Brynskikh, Anna M.; Kopanic, Jennifer L.; Sorgen, Paul L.; Klyachko, Natalia L.; Batrakova, Elena V.; Bronich, Tatiana K.; Kabanov, Alexander V.

    2013-01-01

    Development of well-defined nanomedicines is critical for their successful clinical translation. A simple synthesis and purification procedure is established for chemically cross-linked polyion complexes of Cu/Zn superoxide dismutase (SOD1) or catalase with a cationic block copolymer, methoxy-poly(ethylene glycol)-block-poly(L-lysine hydrochloride) (PEG-pLL50). Such complexes, termed cross-linked nanozymes (cl-nanozymes) retain catalytic activity and have narrow size distribution. Moreover, their cytotoxicity is decreased compared to non-cross-linked complexes due to suppression of release of the free block copolymer. SOD1 cl-nanozymes exhibit prolonged ability to scavenge experimentally induced reactive oxygen species (ROS) in cultured brain microvessel endothelial cells and central neurons. In vivo they decrease ischemia/reperfusion-induced tissue injury and improve sensorimotor functions in a rat middle cerebral artery occlusion (MCAO)model after a single intravenous (i.v.) injection. Altogether, well-defined cl-nanozymes are promising modalities for attenuation of oxidative stress after brain injury. PMID:22902590

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

    PubMed

    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

  14. 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. PMID:25505146

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

    PubMed Central

    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. PMID:25505146

  16. Characteristic low density and shear sensitivity of cross-linked chromatin containing polycomb complexes.

    PubMed

    Schwartz, Yuri B; Kahn, Tatyana G; Pirrotta, Vincenzo

    2005-01-01

    Chromatin cross-linking is widely used for mapping the distribution of chromosomal proteins by immunoprecipitation, but our knowledge of the physical properties of chromatin complexes remains rudimentary. Density gradients have been long used to separate fragments of cross-linked chromatin with their bound proteins from free protein or free DNA. We find that the association of DNA fragments with very-high-molecular-weight protein complexes shifts their buoyant density to values much lower then that of bulk chromatin. We show that in a CsCl gradient, Polycomb response elements, promoters of active genes, and insulator or boundary elements are found at buoyant densities similar to those of free protein and are depleted from the bulk chromatin fractions. In these regions, the low density is associated with the presence of large protein complexes and with high sensitivity to sonication. Our results suggest that separation of different chromatin regions according to their buoyant density may bias chromatin immunoprecipitation results. Density centrifugation of cross-linked chromatin may provide a simple approach to investigate the properties of large chromatin complexes in vivo. PMID:15601863

  17. Well-defined cross-linked antioxidant nanozymes for treatment of ischemic brain injury.

    PubMed

    Manickam, Devika S; Brynskikh, Anna M; Kopanic, Jennifer L; Sorgen, Paul L; Klyachko, Natalia L; Batrakova, Elena V; Bronich, Tatiana K; Kabanov, Alexander V

    2012-09-28

    Development of well-defined nanomedicines is critical for their successful clinical translation. A simple synthesis and purification procedure is established for chemically cross-linked polyion complexes of Cu/Zn superoxide dismutase (SOD1) or catalase with a cationic block copolymer, methoxy-poly(ethylene glycol)-block-poly(L-lysine hydrochloride) (PEG-pLL₅₀). Such complexes, termed cross-linked nanozymes (cl-nanozymes) retain catalytic activity and have narrow size distribution. Moreover, their cytotoxicity is decreased compared to non-cross-linked complexes due to suppression of release of the free block copolymer. SOD1 cl-nanozymes exhibit prolonged ability to scavenge experimentally induced reactive oxygen species (ROS) in cultured brain microvessel endothelial cells and central neurons. In vivo they decrease ischemia/reperfusion-induced tissue injury and improve sensorimotor functions in a rat middle cerebral artery occlusion (MCAO) model after a single intravenous (i.v.) injection. Altogether, well-defined cl-nanozymes are promising modalities for attenuation of oxidative stress after brain injury. PMID:22902590

  18. Critical forces for actin filament buckling and force transmission influence transport in actomyosin networks

    NASA Astrophysics Data System (ADS)

    Stam, Samantha; Gardel, Margaret

    Viscoelastic networks of biopolymers coordinate the motion of intracellular objects during transport. These networks have nonlinear mechanical properties due to events such as filament buckling or breaking of cross-links. The influence of such nonlinear properties on the time and length scales of transport is not understood. Here, we use in vitro networks of actin and the motor protein myosin II to clarify how intracellular forces regulate active diffusion. We observe two transitions in the mean-squared displacement of cross-linked actin with increasing motor concentration. The first is a sharp transition from initially subdiffusive to diffusive-like motion that requires filament buckling but does not cause net contraction of the network. Further increase of the motor density produces a second transition to network rupture and ballistic actin transport. This corresponds with an increase in the correlation of motion and thus may be caused when forces propagate far enough for global motion. We conclude that filament buckling and overall network contraction require different amounts of force and produce distinct transport properties. These nonlinear transitions may act as mechanical switches that can be turned on to produce observed motion within cells.

  19. Proper Regulation of Cdc42 Activity is Required for Tight Actin Concentration at the Equator during Cytokinesis in Adherent Mammalian Cells

    PubMed Central

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

    2012-01-01

    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. PMID:21763307

  20. Actin Filament Tracking Based on Particle Filters and Stretching Open Active Contour Models

    PubMed Central

    Li, Hongsheng; Shen, Tian; Vavylonis, Dimitrios; Huang, Xiaolei

    2010-01-01

    We introduce a novel algorithm for actin filament tracking and elongation measurement. Particle Filters (PF) and Stretching Open Active Contours (SOAC) work cooperatively to simplify the modeling of PF in a one-dimensional state space while naturally integrating filament body constraints to tip estimation. Existing microtubule (MT) tracking methods track either MT tips or entire bodies in high-dimensional state spaces. In contrast, our algorithm reduces the PF state spaces to one-dimensional spaces by tracking filament bodies using SOAC and probabilistically estimating tip locations along the curve length of SOACs. Experimental evaluation on TIRFM image sequences with very low SNRs demonstrates the accuracy and robustness of the proposed approach. PMID:20426170

  1. Gas-phase transfer of polymer cross-linking agents and by-products to solid oral pharmaceuticals.

    PubMed

    Maus, Russell G; Li, Min; Clement, Christopher M; Kinzer, Jeffery A

    2007-11-01

    In the pharmaceutical industry, solid oral compressed tablets (OCT) are frequently transported in bulk containers prior to packaging. While in this state, the product is generally protected from interaction with liquid and solid contaminants by physical barriers (e.g., polyethylene bags, drums, etc.). Vapor phase contamination, although generally less frequently observed, is possible. A specific example of the detection and identification of volatile by-products (acetophenone and 2-phenyl-2-propanol) of a common polymer cross-linking agent (dicumyl peroxide) is presented. The product tablets were compressed, placed into double polyethylene bags, and subsequently placed into a polyethylene drum for shipment overseas. To cushion the product during transit, a cross-linked polyethylene foam disk (designed to fit into the bottom of the drum) was placed below the bag of tablets. Initially, these contaminants were detected by HPLC with UV detection at the receiving laboratory, and assumed to be degradates of the active components of the product. Further analysis showed that neither the collected UV absorbance data nor the observed levels of the contaminants were consistent with known degradates of the product. Liquid extraction followed by GC-MS analysis of the product as well as the cross-linked foam disk exhibited measurable quantities of the contaminants in question. Vapor phase transfer of these cross-linking agent by-products, originating in the cross-linked foam pads, was determined to be the root cause for the presence of these compounds in the product. PMID:17686599

  2. Alphaherpesvirus US3-mediated reorganization of the actin cytoskeleton is mediated by group A p21-activated kinases

    PubMed Central

    Van den Broeke, Céline; Radu, Maria; Deruelle, Matthias; Nauwynck, Hans; Hofmann, Clemens; Jaffer, Zahara M.; Chernoff, Jonathan; Favoreel, Herman W.

    2009-01-01

    The US3 protein is a viral serine/threonine kinase that is conserved among all members of the Alphaherpesvirinae. The US3 protein of different alphaherpesviruses causes dramatic alterations in the actin cytoskeleton, such as the disassembly of actin stress fibers and formation of cell projections, which have been associated with increased intercellular virus spread. Here, we find that inhibiting group A p21-activated kinases (PAKs), which are key regulators in Cdc42/Rac1 Rho GTPase signaling pathways, impairs US3-mediated actin alterations. By using PAK1−/− and PAK2−/− mouse embryo fibroblasts (MEFs), we show that US3-mediated stress fiber disassembly requires PAK2, whereas US3-mediated cell projection formation mainly is mediated by PAK1, also indicating that PAK1 and PAK2 can have different biological effects on the organization of the actin cytoskeleton. In addition, US3 was found to bind and phosphorylate group A PAKs. Lack of group A PAKs in MEFs was correlated with inefficient virus spread. Thus, US3 induces its effect on the actin cytoskeleton via group A PAKs. PMID:19435845

  3. Physicochemical, antimicrobial, and cytotoxic characteristics of a chitosan film cross-linked by a naturally occurring cross-linking agent, aglycone geniposidic acid.

    PubMed

    Mi, Fwu-Long; Huang, Chin-Tsung; Liang, Hsiang-Fa; Chen, Mei-Chin; Chiu, Ya-Ling; Chen, Chun-Hung; Sung, Hsing-Wen

    2006-05-01

    The purpose of this study was to evaluate the characteristics of a chitosan film cross-linked by a naturally occurring compound, aglycone geniposidic acid (aGSA). This newly developed aGSA-cross-linked chitosan film may be used as an edible film. The chitosan film without cross-linking (fresh) and the glutaraldehyde-cross-linked chitosan film were used as controls. The characteristics of test chitosan films evaluated were their degree of cross-linking, swelling ratio, mechanical properties, water vapor permeability, antimicrobial capability, cytotoxicity, and enzymatic degradability. It was found that cross-linking of chitosan films by aGSA (at a concentration up to 0.8 mM) significantly increased its ultimate tensile strength but reduced its strain at fracture and swelling ratio. There was no significant difference in the antimicrobial capability between the cross-linked chitosan films and their fresh counterpart. However, the aGSA-cross-linked chitosan film had a lower cytotoxicity, a slower degradation rate, and a relatively lower water vapor permeability as compared to the glutaraldehyde-cross-linked film. These results suggested that the aGSA-cross-linked chitosan film may be a promising material as an edible film. PMID:16637687

  4. Role of Transglutaminase 2 in Liver Injury via Cross-linking and Silencing of Transcription Factor Sp1

    PubMed Central

    TATSUKAWA, HIDEKI; FUKAYA, YAYOI; FRAMPTON, GORDON; MARTINEZ–FUENTES, ANTONIO; SUZUKI, KENJI; KUO, TING–FANG; NAGATSUMA, KEISUKE; SHIMOKADO, KENTARO; OKUNO, MASATAKA; WU, JIAN; IISMAA, SIIRI; MATSUURA, TOMOKAZU; TSUKAMOTO, HIDEKAZU; ZERN, MARK A.; GRAHAM, ROBERT M.; KOJIMA, SOICHI

    2016-01-01

    Background & Aims Despite high morbidity and mortality of alcoholic liver disease worldwide, the molecular mechanisms underlying alcohol-induced liver cell death are not fully understood. Transglutaminase 2 (TG2) is a cross-linking enzyme implicated in apoptosis. TG2 levels and activity are increased in association with various types of liver injury. However, how TG2 induces hepatic apoptosis is not known. Methods Human hepatic cells or primary hepatocytes from rats or TG2+/+ and TG2−/− mice were treated with ethanol. Mice were administered anti-Fas antibody or alcohol. Liver sections were prepared from patients with alcoholic steatohepatitis. Changes in TG2 levels, Sp1 cross-linking and its activities, expression of hepatocyte growth factor receptor, c-Met, and hepatic apoptosis were measured. Results Ethanol induced apoptosis in hepatic cells, enhanced activity and nuclear accumulation of TG2 as well as accumulation of cross-linked and inactivated Sp1, and reduced expression of the Sp1-responsive gene, c-Met. These effects were rescued by TG2 knockdown, restoration of functional Sp1, or addition of hepatocyte growth factor, whereas apoptosis was reproduced by Sp1 knockdown or TG2 overexpression. Compared with TG2+/+ mice, TG2−/− mice showed markedly reduced hepatocyte apoptosis and Sp1 cross-linking following ethanol or anti-Fas treatment. Treatment of TG2+/+ mice with the TG2 inhibitors putrescine or cystamine blocked anti-Fas–induced hepatic apoptosis and Sp1 silencing. Moreover, enhanced expression of cross-linked Sp1 and TG2 was evident in hepatocyte nuclei of patients with alcoholic steatohepatitis. Conclusions TG2 induces hepatocyte apoptosis via Sp1 cross-linking and inactivation, with resultant inhibition of the expression of c-Met required for hepatic cell viability. PMID:19208340

  5. 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

  6. 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.

  7. Actin filament barbed-end capping activity in neutrophil lysates: the role of capping protein-beta 2.

    PubMed

    DiNubile, M J; Cassimeris, L; Joyce, M; Zigmond, S H

    1995-12-01

    A barbed-end capping activity was found in high speed supernates of neutrophils lysed in submicromolar calcium. In dilute supernate (> or = 100-fold dilution of cytoplasm), this activity accounted for most of the inhibition of barbed-end elongation of pyrenyl-G-actin from spectrin-F-actin seeds. Pointed-end elongation from gelsolin-capped F-actin seeds was not inhibited at comparable concentrations of supernate, thus excluding actin monomer sequestration as a cause of the observed inhibition. Most of the capping activity was due to capping protein-beta 2 (a homologue of cap Z). Thus, while immunoadsorption of > or = 95% of the gelsolin in the supernate did not decrease capping activity, immunoadsorption of capping protein-beta 2 reduced capping activity proportionally to the amount of capping protein-beta 2 adsorbed. Depletion of > 90% of capping protein-beta 2 from the supernate removed 90% of its capping activity. The functional properties of the capping activity were defined. The dissociation constant for binding to barbed ends (determined by steady state and kinetic analyses) was approximately 1-2 nM; the on-rate of capping was between 7 x 10(5) and 5 x 10(6) M-1 s-1; and the off-rate was approximately 2 x 10(-3) s-1. The concentration of capper free in the intact cell (determined by adsorption of supernate with spectrin-actin seeds) was estimated to be approximately 1-2 microM. Thus, there appeared to be enough high affinity capper to cap all the barbed ends in vivo. Nevertheless, immediately after lysis with detergent, neutrophils contained sites that nucleate barbed-end elongation of pyrenyl-G-actin. These barbed ends subsequently become capped with a time course and concentration dependence similar to that of spectrin-F-actin seeds in high speed supernates. These observations suggest that, despite the excess of high affinity capper, some ends either are not capped in vivo or are transiently uncapped upon lysis and dilution. PMID:8590796

  8. Homologous Recombination Assay for Interstrand Cross-Link Repair

    PubMed Central

    Nakanishi, Koji; Cavallo, Francesca; Brunet, Erika; Jasin, Maria

    2012-01-01

    DNA interstrand cross-links (ICLs) covalently link both strands of the DNA duplex, impeding cellular processes like DNA replication. Homologous recombination (HR) is considered to be a major pathway for the repair of ICLs in mammalian cells as mutants for HR components are highly sensitive to DNA-damaging agents that cause ICLs. This chapter describes GFP assays to measure HR following site-specific ICL formation with psoralen through DNA triplex technology. This approach can be used to determine the genetic requirements for ICL-induced HR in relation to those involved in HR repair of other DNA lesions such as double-strand breaks. PMID:21660700

  9. LET dependence of DNA-protein cross-links

    SciTech Connect

    Blakely, E.A.; Chang, P.Y.; Bjornstad, K.A.

    1995-08-01

    We have preliminary data indicating a fluence-dependent yield of particle-induced protein cross-links (DPC`s) with a dependency on LET and particle residual energy. Our data indicate that the DPC yield for hamster fibroblasts in vitro irradiated at 32 keV/{mu}m is similar to that reported for hamster cells irradiated with cobalt-60 gamma rays. At 100-120 keV/{mu}m there is some evidence for an enhanced DPC yield with increasing particle fluence, but there are differences in the yields that are dependent on particle track structure.

  10. Physicochemical studies on polyurethane/siloxane cross-linked films for hydrophobic surfaces by the sol-gel process.

    PubMed

    Seeni Meera, Kamal Mohamed; Murali Sankar, Rajavelu; Jaisankar, Sellamuthu N; Mandal, Asit Baran

    2013-03-01

    A series of castor oil based polyurethane/siloxane cross-linked films were prepared using castor oil, isophorone diisocyanate, and 3-aminopropyl trimethoxysilane by the sol-gel process. Fourier transform infrared (FT-IR) spectra reveal the cross-linking interaction between polyurethane and siloxane moieties, thereby shifting the peak position of characteristic N-H and C═O groups to higher wavenumber. (29)Si (silica) solid state nuclear magnetic resonance spectra were used to prove the formation of siloxane network linkage in the polyurethane system, thereby analyzing the Si environment present in the polyurethane/siloxane cross-linked films. The activation energy values at two stages (Tmax1 and Tmax2) for the degradation of polyurethane films were increased with increasing silane ratio. The calculated activation energy values for the higher silane ratio (1.5) are 136 and 170 kJ/mol at Tmax1 and Tmax2, respectively. From contact angle measurements, we observed that increasing siloxane cross-linking increased the hydrophobicity of the films. The optical transmittance obtained from ultraviolet-visible spectra indicated that the film samples are transparent in the region 300-800 nm. The moisture sorption/desorption isotherm curve shows a characteristic behavior of type III isotherm corresponds to hydrophobic materials. Dynamic mechanical studies show that the increase in storage modulus reveals siloxane cross-linking gives rigidity to the films. Atomic force microscopic images show that the introduction of siloxane changes the surface roughness of the polyurethane films. It is found that the siloxane cross-linking can be used to obtain hydrophobic surface films having good thermal stability and optical transmittance. PMID:23394610

  11. Fyn Mediates High Glucose-Induced Actin Cytoskeleton Reorganization of Podocytes via Promoting ROCK Activation In Vitro.

    PubMed

    Lv, Zhimei; Hu, Mengsi; Ren, Xiaoxu; Fan, Minghua; Zhen, Junhui; Chen, Liqun; Lin, Jiangong; Ding, Nannan; Wang, Qun; Wang, Rong

    2016-01-01

    Fyn, a member of the Src family of tyrosine kinases, is a key regulator in cytoskeletal remodeling in a variety of cell types. Recent studies have demonstrated that Fyn is responsible for nephrin tyrosine phosphorylation, which will result in polymerization of actin filaments and podocyte damage. Thus detailed involvement of Fyn in podocytes is to be elucidated. In this study, we investigated the potential role of Fyn/ROCK signaling and its interactions with paxillin. Our results presented that high glucose led to filamentous actin (F-actin) rearrangement in podocytes, accompanied by paxillin phosphorylation and increased cell motility, during which Fyn and ROCK were markedly activated. Gene knockdown of Fyn by siRNA showed a reversal effect on high glucose-induced podocyte damage and ROCK activation; however, inhibition of ROCK had no significant effects on Fyn phosphorylation. These observations demonstrate that in vitro Fyn mediates high glucose-induced actin cytoskeleton remodeling of podocytes via promoting ROCK activation and paxillin phosphorylation. PMID:26881253

  12. Fyn Mediates High Glucose-Induced Actin Cytoskeleton Reorganization of Podocytes via Promoting ROCK Activation In Vitro

    PubMed Central

    Lv, Zhimei; Hu, Mengsi; Ren, Xiaoxu; Fan, Minghua; Zhen, Junhui; Chen, Liqun; Lin, Jiangong; Ding, Nannan; Wang, Qun; Wang, Rong

    2016-01-01

    Fyn, a member of the Src family of tyrosine kinases, is a key regulator in cytoskeletal remodeling in a variety of cell types. Recent studies have demonstrated that Fyn is responsible for nephrin tyrosine phosphorylation, which will result in polymerization of actin filaments and podocyte damage. Thus detailed involvement of Fyn in podocytes is to be elucidated. In this study, we investigated the potential role of Fyn/ROCK signaling and its interactions with paxillin. Our results presented that high glucose led to filamentous actin (F-actin) rearrangement in podocytes, accompanied by paxillin phosphorylation and increased cell motility, during which Fyn and ROCK were markedly activated. Gene knockdown of Fyn by siRNA showed a reversal effect on high glucose-induced podocyte damage and ROCK activation; however, inhibition of ROCK had no significant effects on Fyn phosphorylation. These observations demonstrate that in vitro Fyn mediates high glucose-induced actin cytoskeleton remodeling of podocytes via promoting ROCK activation and paxillin phosphorylation. PMID:26881253

  13. Transcriptional activation of jun and actin genes by estrogen during mitogenic stimulation of rat uterine cells.

    PubMed

    Cicatiello, L; Ambrosino, C; Coletta, B; Scalona, M; Sica, V; Bresciani, F; Weisz, A

    1992-03-01

    Estrogens induce transcriptional activation of c-fos and c-myc proto-oncogenes during mitogenic stimulation of human, chicken, mouse and rat cells in vivo and in vitro. In this paper we show that 17 beta-estradiol injected into adult ovariectomized rats increases c-jun, jun-B and jun-D gene transcription in the uterus. Kinetics and amplitude of response are different for each gene, since c-jun is activated first, within 30 min after injection, followed by jun-D and jun-B, 60 and 90 min after injection, respectively. Maximal activation of jun-B marks a drop in transcription of all the jun genes. Furthermore, transcriptional activation by 17 beta-estradiol of the growth-regulated beta- and gamma-cytoskeletal actin genes is prevented by an inhibitor of protein synthesis, indicating that it is a secondary response to the hormone. These data support the hypothesis that during growth stimulation of target cells the estrogen receptor induces transcription of regulatory genes, triggering in this way a cascade of gene regulation events that results in progression through the cell cycle. PMID:1373300

  14. The Effect of Multiple Sequential Light Sources to Activate Aminolevulinic Acid in the Treatment of Actinic Keratoses: A Retrospective Study

    PubMed Central

    Goldman, Mitchel P.; Fabi, Sabrina G.; Guiha, Isabella

    2014-01-01

    There is a lack of research regarding the sequential use of multiple light sources for topical 5-aminolevulinic acid activation in photodynamic therapy for actinic keratosis. This study evaluated 5-aminolevulinic acid-photodynamic therapy for actinic keratosis using blue light combined with red light, pulsed dye laser, and/or intense pulsed light in a retrospective fashion. Field-directed 5-aminolevulinic acid-photodynamic therapy was performed with blue light only, blue light + pulsed dye laser, blue light + intense pulsed light, blue light + pulsed dye laser + intense pulsed light, or blue light + red light + pulsed dye laser + intense pulsed light for nonhyperkeratotic actinic keratoses of face, scalp, or upper trunk. Blue light + intense pulsed light + pulsed dye laser produced greater patient-reported improvement in actinic keratoses than blue light or blue light + intense pulsed light and greater subject-reported improvement in overall skin quality than blue light + intense pulsed light. The addition of red light led to no further benefit in either outcome measure. Photodynamic therapy with multiple, sequential laser and light sources led to greater patient-graded improvement in actinic keratoses than that with a single light source (blue light), without significant differences in post-treatment adverse events. However, the small, widely disparate number of patients between groups and follow-up times between patients, as well as retrospective assessments based on subjective patient recall, severely limit the significance of these findings. Nevertheless, the results raise interesting questions regarding the use of multiple light and laser sources for photodynamic therapy of actinic keratoses and warrant further research with a prospective, randomized, controlled study. PMID:25276272

  15. Troponin T cross-linking in human apoptotic cardiomyocytes.

    PubMed Central

    Gorza, L.; Menabó, R.; Di Lisa, F.; Vitadello, M.

    1997-01-01

    Intracellular calcium overload of guinea pig cardiomyocytes is accompanied by troponin T cross-linking, which is revealed by changes in immunoreactivity of anti-troponin T antibodies. We presently investigated whether the same process is detectable in the human heart. Immunohistochemistry shows myofibrillar staining with BN-59 anti-troponin T antibody with rare cardiomyocytes in samples obtained at surgery, whereas approximately 50% of myocytes are labeled in heart samples taken at autopsy within 3 hours of death, and every cardiomyocyte is stained after exposure of biopsy sections to 10 mmol/L calcium. Western blot analysis shows reactive polypeptides of approximately 70 and 85 to 90 kd in addition to troponin T in both treated and autopsy heart sections. Neither reactivity in immunohistochemistry nor additional reactive polypeptides in Western blot are detectable when calpain or transglutaminase is inhibited during exposure of sections to high calcium. Troponin T crosslinking occurs also in isolated myofibrils, which show staining with BN-59 at either sarcomeric A or I bands. Labeling with TdT-mediated dUTP nick and labeling (TUNEL) to demonstrate apoptosis reveals DNA fragmentation in BN-59-positive myocytes. Thus, troponin T cross-linking occurs in human cardiac myocytes concomitantly with apoptosis and autopsy autolysis, suggesting that similar cytosolic alterations can be produced by different types of myocyte death. Images Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 PMID:9176400

  16. Collagen Cross-Linking: Current Status and Future Directions

    PubMed Central

    Hovakimyan, Marine; Guthoff, Rudolf F.; Stachs, Oliver

    2012-01-01

    Collagen cross-linking (CXL) using UVA light and riboflavin (vitamin B2) was introduced as a clinical application to stabilize the cornea by inducing cross-links within and between collagen fibers. CXL has been investigated extensively and has been shown clinically to arrest the progression of keratoconic or post-LASIK ectasia. With its minimal cost, simplicity, and proven positive clinical outcome, CXL can be regarded as a useful approach to reduce the number of penetrating keratoplasties performed. Small case series have also indicated that CXL is beneficial in corneal edema by reducing stromal swelling behavior and in keratitis by inhibiting pathogen growth. Despite these encouraging results, CXL remains a relatively new method that is potentially associated with complications. Aspects such as side effects and recurrence rates have still to be elucidated. In light of the growing interest in CXL, our paper summarizes present knowledge about this promising approach. We have intentionally endeavored to include the more relevant studies from the recent literature to provide an overview of the current status of CXL. PMID:22288005

  17. Polyimide aerogels cross-linked through amine functionalized polyoligomeric silsesquioxane.

    PubMed

    Guo, Haiquan; Meador, Mary Ann B; McCorkle, Linda; Quade, Derek J; Guo, Jiao; Hamilton, Bart; Cakmak, Miko; Sprowl, Guilherme

    2011-02-01

    We report the first synthesis of polyimide aerogels cross-linked through a polyhedral oligomeric silsesquioxane, octa(aminophenyl)silsesquioxane (OAPS). Gels formed from polyamic acid solutions of 3,3',4,4'-biphenyltetracarboxylic dianhydride (BPDA), bisaniline-p-xylidene (BAX) and OAPS were chemically imidized and dried using supercritical CO(2) extraction to give aerogels having density around 0.1 g/cm(3). The aerogels are greater than 90 % porous, have high surface areas (230 to 280 m(2)/g) and low thermal conductivity (14 mW/m-K at room temperature). Notably, the polyimide aerogels cross-linked with OAPS have higher modulus than polymer reinforced silica aerogels of similar density and can be fabricated as both monoliths and thin films. Thin films of the aerogel are flexible and foldable making them an ideal insulation for space suits, and inflatable structures for habitats or decelerators for planetary re-entry, as well as more down to earth applications. PMID:21294517

  18. Optimization model for UV-Riboflavin corneal cross-linking

    NASA Astrophysics Data System (ADS)

    Schumacher, S.; Wernli, J.; Scherrer, S.; Bueehler, M.; Seiler, T.; Mrochen, M.

    2011-03-01

    Nowadays UV-cross-linking is an established method for the treatment of keraectasia. Currently a standardized protocol is used for the cross-linking treatment. We will now present a theoretical model which predicts the number of induced crosslinks in the corneal tissue, in dependence of the Riboflavin concentration, the radiation intensity, the pre-treatment time and the treatment time. The model is developed by merging the difussion equation, the equation for the light distribution in dependence on the absorbers in the tissue and a rate equation for the polymerization process. A higher concentration of Riboflavin solution as well as a higher irradiation intensity will increase the number of induced crosslinks. However, performed stress-strain experiments which support the model showed that higher Riboflavin concentrations (> 0.125%) do not result in a further increase in stability of the corneal tissue. This is caused by the inhomogeneous distribution of induced crosslinks throughout the cornea due to the uneven absorption of the UV-light. The new model offers the possibility to optimize the treatment individually for every patient depending on their corneal thickness in terms of efficiency, saftey and treatment time.

  19. Thermoset-cross-linked lignocellulose: a moldable plant biomass.

    PubMed

    Karumuri, Sriharsha; Hiziroglu, Salim; Kalkan, A Kaan

    2015-04-01

    The present work demonstrates a high biomass content (i.e., up to 90% by weight) and moldable material by controlled covalent cross-linking of lignocellulosic particles by a thermoset through epoxide-hydroxyl reactions. As an example for lignocellulosic biomass, Eastern redcedar was employed. Using scanning fluorescence microscopy and vibrational spectroscopy, macroscopic to molecular scale interactions of the thermoset with the lignocellulose have been revealed. Impregnation of the polymer resin into the biomass cellular network by capillary action as well as applied pressure results in a self-organizing structure in the form of thermoset microrods in a matrix of lignocellulose. We also infer permeation of the thermoset into the cell walls from the reaction of epoxides with the hydroxyls of the lignin. Compression tests reveal, at 30% thermoset content, thermoset-cross-linked lignocellulose has superior mechanical properties over a commercial wood plastic composite while comparable stiffness and strength to bulk epoxy and wood, respectively. The failure mechanism is understood to be crack propagation along the particle-thermoset interface and/or interparticle thermoset network. PMID:25734539

  20. One-step electrospinning of cross-linked chitosan fibers.

    PubMed

    Schiffman, Jessica D; Schauer, Caroline L

    2007-09-01

    Chitin is a nitrogen-rich polysaccharide that is abundant in crustaceans, mollusks, insects, and fungi and is the second most abundant organic material found in nature next to cellulose. Chitosan, the N-deacetylated derivative of chitin, is environmentally friendly, nontoxic, biodegradable, and antibacterial. Fibrous mats are typically used in industries for filter media, catalysis, and sensors. Decreasing fiber diameters within these mats causes many beneficial effects such as increased specific surface area to volume ratios. When the intrinsically beneficial effects of chitosan are combined with the enhanced properties of nanofibrous mats, applications arise in a wide range of fields, including medical, packaging, agricultural, and automotive. This is particularly important as innovative technologies that focus around bio-based materials are currently of high urgency, as they can decrease dependencies on fossil fuels. We have demonstrated that Schiff base cross-linked chitosan fibrous mats can be produced utilizing a one-step electrospinning process that is 25 times faster and, therefore, more economical than a previously reported two-step vapor-cross-linking method. These fibrous mats are insoluble in acidic, basic, and aqueous solutions for 72 h. Additionally, this improved production method results in a decreased average fiber diameter, which measures 128 +/- 40 nm. Chemical and structural analyses were conducted utilizing Fourier transform infrared spectroscopy, solubility studies, and scanning electron microscopy. PMID:17696400

  1. 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. PMID:25922304

  2. Effects of processing conditions on the reliability of cross-linked polyethylene cable insulation. Progress report

    SciTech Connect

    Phillips, P.J.

    1981-03-01

    Crystallization and morphology were investigated in cross-linked PE. /sup 13/C NMR was used to quantify the cross-links. Production of cable is being studied. Dielectric constant and loss of cross-linked PE are being measured. (DLC)

  3. Chemical cross-linking/mass spectrometry targeting acidic residues in proteins and protein complexes.

    PubMed

    Leitner, Alexander; Joachimiak, Lukasz A; Unverdorben, Pia; Walzthoeni, Thomas; Frydman, Judith; Förster, Friedrich; Aebersold, Ruedi

    2014-07-01

    The study of proteins and protein complexes using chemical cross-linking followed by the MS identification of the cross-linked peptides has found increasingly widespread use in recent years. Thus far, such analyses have used almost exclusively homobifunctional, amine-reactive cross-linking reagents. Here we report the development and application of an orthogonal cross-linking chemistry specific for carboxyl groups. Chemical cross-linking of acidic residues is achieved using homobifunctional dihydrazides as cross-linking reagents and a coupling chemistry at neutral pH that is compatible with the structural integrity of most protein complexes. In addition to cross-links formed through insertion of the dihydrazides with different spacer lengths, zero-length cross-link products are also obtained, thereby providing additional structural information. We demonstrate the application of the reaction and the MS identification of the resulting cross-linked peptides for the chaperonin TRiC/CCT and the 26S proteasome. The results indicate that the targeting of acidic residues for cross-linking provides distance restraints that are complementary and orthogonal to those obtained from lysine cross-linking, thereby expanding the yield of structural information that can be obtained from cross-linking studies and used in hybrid modeling approaches. PMID:24938783

  4. Chemical cross-linking/mass spectrometry targeting acidic residues in proteins and protein complexes

    PubMed Central

    Leitner, Alexander; Joachimiak, Lukasz A.; Unverdorben, Pia; Walzthoeni, Thomas; Frydman, Judith; Förster, Friedrich; Aebersold, Ruedi

    2014-01-01

    The study of proteins and protein complexes using chemical cross-linking followed by the MS identification of the cross-linked peptides has found increasingly widespread use in recent years. Thus far, such analyses have used almost exclusively homobifunctional, amine-reactive cross-linking reagents. Here we report the development and application of an orthogonal cross-linking chemistry specific for carboxyl groups. Chemical cross-linking of acidic residues is achieved using homobifunctional dihydrazides as cross-linking reagents and a coupling chemistry at neutral pH that is compatible with the structural integrity of most protein complexes. In addition to cross-links formed through insertion of the dihydrazides with different spacer lengths, zero-length cross-link products are also obtained, thereby providing additional structural information. We demonstrate the application of the reaction and the MS identification of the resulting cross-linked peptides for the chaperonin TRiC/CCT and the 26S proteasome. The results indicate that the targeting of acidic residues for cross-linking provides distance restraints that are complementary and orthogonal to those obtained from lysine cross-linking, thereby expanding the yield of structural information that can be obtained from cross-linking studies and used in hybrid modeling approaches. PMID:24938783

  5. Cross-linking of the extracellular matrix by the maillard reaction in aging and diabetes: an update on "a puzzle nearing resolution".

    PubMed

    Monnier, Vincent M; Mustata, Georgian T; Biemel, Klaus L; Reihl, Oliver; Lederer, Marcus O; Zhenyu, Dai; Sell, David R

    2005-06-01

    The aging extracellular matrix is characterized by an age-related increase in insolubilization, yellowing, and stiffening, all of which can be mimicked by the Maillard reaction in vitro. These phenomena are accelerated in metabolic diseases such as diabetes and end-stage renal disease, which have in common with physiological aging the accumulation of various glycation products and cross-links. Eight years ago we concluded that the evidence favored oxidative cross-linking in experimental diabetes [Monnier, V.M. et al. 1996. The mechanism of collagen cross-linking in diabetes: a puzzle nearing completion. Diabetes 45(Suppl. 3): 67-72] and proposed a major role for a putative non-UV active cross-link derived from glucose. Below, we provide an update of the field that leads to the conclusion that, while oxidation might be important for Maillard reaction-mediated cross-linking via Strecker degradation and allysine formation, the single most important collagen cross-link known to date in diabetes and aging is glucosepane, a lysyl-arginine cross-link that forms under nonoxidative conditions. PMID:16037276

  6. 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.

  7. Furan oxidation based cross-linking: a new approach for the study and targeting of nucleic acid and protein interactions.

    PubMed

    Carrette, L L G; Gyssels, E; De Laet, N; Madder, A

    2016-01-28

    Furan mediated nucleic acid cross-linking, initially developed for DNA interstrand duplex cross-linking, has matured into a versatile tool for the study of protein and nucleic acid interactions, ready to face its applications. The methodology was initially developed for easy and clean chemical generation of DNA interstrand cross-linked duplexes, but has been further expanded for use with other probes, targets and triggers, now allowing mild biologically significant cross-linking with potential therapeutic benefit. It was shown that the methodology could be repurposed for RNA interstrand cross-linking, which is very relevant in today's antisense approaches or miRNA target identification endeavors. This further illustrates the furan oxidation method's generality and mildness, especially when using red light for oxidation. A complementary antigene approach has been explored through duplex targeting with furan modified triplex forming oligonucleotides (TFOs) and DNA binding proteins. Also targeting of peptides and proteins by furan-modified DNA and peptides has been explored. Thorough methodology examination exploring variable reaction conditions in combination with a series of different furan-modified building blocks and application of different activation signals resulted in a detailed understanding of the mechanisms involved and factors influencing the yield and selectivity of the reaction. In order to draw the bigger picture of the scope and limitations of furan-oxidation cross-linking, we here provide a unique side by side comparison and discussion of our published data, supplemented with unpublished results, providing a clear performance report of the currently established furan toolbox and its application potential in various biomacromolecular complexes. PMID:26679922

  8. p53-dependent global nucleotide excision repair of cisplatin-induced intrastrand cross links in human cells.

    PubMed

    Bhana, Sara; Hewer, Alan; Phillips, David H; Lloyd, Daniel R

    2008-03-01

    Cisplatin is an extremely effective chemotherapeutic agent used for the treatment of testicular and other solid tumours. It induces a variety of structural modifications in DNA, the most abundant being the GpG- and ApG-1,2-intrastrand cross links formed between adjacent purine bases. These cross links account for approximately 90% of cisplatin-induced DNA damage and are thought to be responsible for the cytotoxic activity of the drug. In human cells, the nucleotide excision repair (NER) process removes the intrastrand cross links from the genome, the efficiency of which is likely to be an important determinant of cisplatin cytotoxicity. We have investigated whether the p53 tumour suppressor status affects global NER of cisplatin-induced intrastrand cross links in human cells. We have used a (32)P-postlabelling method to monitor the removal of GpG- and ApG-intrastrand cross links from two human cell models (the 041TR system, in which p53 is regulated by a tetracycline-inducible promoter, together with WI38 fibroblasts and the SV40-transformed derivative VA13) that each differ in p53 status. We demonstrate that the absence of functional p53 leads to persistence of both cisplatin-induced intrastrand cross links in the genome, suggesting that p53 regulates NER of these DNA lesions. This observation extends the role of p53 in NER beyond enhancing the removal of environmentally induced DNA lesions to include those of clinical origin. Given the frequency of p53 mutations in human tumours, these results may have implications for the use of cisplatin in cancer chemotherapy. PMID:18267949

  9. Disassembly activity of actin-depolymerizing factor (ADF) is associated with distinct cellular processes in apicomplexan parasites

    PubMed Central

    Haase, Silvia; Zimmermann, Dennis; Olshina, Maya A.; Wilkinson, Mark; Fisher, Fabio; Tan, Yan Hong; Stewart, Rebecca J.; Tonkin, Christopher J.; Wong, Wilson; Kovar, David R.; Baum, Jake

    2015-01-01

    Proteins of the actin-depolymerizing factor (ADF)/cofilin family have been shown to be crucial for the motility and survival of apicomplexan parasites. However, the mechanisms by which ADF proteins fulfill their function remain poorly understood. In this study, we investigate the comparative activities of ADF proteins from Toxoplasma gondii and Plasmodium falciparum, the human malaria parasite, using a conditional T. gondii ADF-knockout line complemented with ADF variants from either species. We show that P. falciparum ADF1 can fully restore native TgADF activity, demonstrating functional conservation between parasites. Strikingly, mutation of a key basic residue (Lys-72), previously implicated in disassembly in PfADF1, had no detectable phenotypic effect on parasite growth, motility, or development. In contrast, organelle segregation was severely impaired when complementing with a TgADF mutant lacking the corresponding residue (Lys-68). Biochemical analyses of each ADF protein confirmed the reduced ability of lysine mutants to mediate actin depolymerization via filament disassembly although not severing, in contrast to previous reports. These data suggest that actin filament disassembly is essential for apicomplexan parasite development but not for motility, as well as pointing to genus-specific coevolution between ADF proteins and their native actin. PMID:26157165

  10. Disassembly activity of actin-depolymerizing factor (ADF) is associated with distinct cellular processes in apicomplexan parasites.

    PubMed

    Haase, Silvia; Zimmermann, Dennis; Olshina, Maya A; Wilkinson, Mark; Fisher, Fabio; Tan, Yan Hong; Stewart, Rebecca J; Tonkin, Christopher J; Wong, Wilson; Kovar, David R; Baum, Jake

    2015-09-01

    Proteins of the actin-depolymerizing factor (ADF)/cofilin family have been shown to be crucial for the motility and survival of apicomplexan parasites. However, the mechanisms by which ADF proteins fulfill their function remain poorly understood. In this study, we investigate the comparative activities of ADF proteins from Toxoplasma gondii and Plasmodium falciparum, the human malaria parasite, using a conditional T. gondii ADF-knockout line complemented with ADF variants from either species. We show that P. falciparum ADF1 can fully restore native TgADF activity, demonstrating functional conservation between parasites. Strikingly, mutation of a key basic residue (Lys-72), previously implicated in disassembly in PfADF1, had no detectable phenotypic effect on parasite growth, motility, or development. In contrast, organelle segregation was severely impaired when complementing with a TgADF mutant lacking the corresponding residue (Lys-68). Biochemical analyses of each ADF protein confirmed the reduced ability of lysine mutants to mediate actin depolymerization via filament disassembly although not severing, in contrast to previous reports. These data suggest that actin filament disassembly is essential for apicomplexan parasite development but not for motility, as well as pointing to genus-specific coevolution between ADF proteins and their native actin. PMID:26157165

  11. Induction of chondrogenic differentiation in mesenchymal stem cells by TGF-beta cross-linked to collagen-PLLA [poly(L-lactic acid)] scaffold by transglutaminase 2

    PubMed Central

    Niger, Corinne; Beazley, Kelly E.; Nurminskaya, Maria

    2013-01-01

    Transglutaminase-mediated cross-linking has been employed to optimize the mechanical properties and stability of tissue scaffolds. We have characterized tissue transglutaminase (TG2)-mediated cross-linking as a useful tool to deliver biologically-active TGF to mesenchymal stem cells (MSCs) and direct their differentiation towards a chondrogenic lineage. TGF- 3 is irreversibly cross-linked by TG2 to collagen type II-coated PLLA [poly(L-lactic acid)] nanofibrous scaffolds and activates Smad phosphorylation and Smad-dependent expression of a luciferase reporter. Human bone marrow-derived MSCs cultured on these scaffolds deposit cartilaginous matrix after 14 days of culture at 50% efficiency compared to chondrogenesis in the presence of soluble TGF- 3. These findings are significant because they suggest a novel approach for the programming of MSCs in a spatially controlled manner by immobilizing biologically active TGF- 3 via cross-linking to a collagen-coated polymeric scaffold. PMID:23892982

  12. Stable Bioactive Enzyme-Containing Multilayer Films Based on Covalent Cross-Linking from Mussel-Inspired Adhesives.

    PubMed

    Longo, Johan; Garnier, Tony; Mateescu, Mihaela; Ponzio, Florian; Schaaf, Pierre; Jierry, Loïc; Ball, Vincent

    2015-11-17

    The use of immobilized enzymes is mandatory for the easy separation of the enzyme, the unreacted substrates, and the obtained products to allow repeated enzymatic assays without cumbersome purification steps. The immobilization procedure is however critical to obtain a high fraction of active enzyme. In this article, we present an enzyme immobilization strategy based on a catechol functionalized alginate. We demonstrate that alkaline phosphatase (ALP) remains active in multilayered films made with alginate modified with catechol moieties (AlgCat) for long duration, that is, up to 7 weeks, provided the multilayered architecture is cross-linked with sodium periodate. This cross-linking reaction allows to create covalent bonds between the amino groups of ALP and the quinone group carried by the modified alginate. In the absence of cross-linking, the enzymatic activity is rapidly lost and this reduction is mainly due to enzyme desorption. We also show that NaIO4 cross-linked (AlgCat-Alp)n films can be freeze-dried and reused at least 3 weeks later without lost in enzymatic activity. PMID:26509712

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

    PubMed

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

    2007-02-01

    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

  15. 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

  16. Jak3 Enables Chemokine-Dependent Actin Cytoskeleton Reorganization by Regulating Cofilin and Rac/Rhoa GTPases Activation

    PubMed Central

    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. PMID:24498424

  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. Enzymatically cross-linked injectable alginate-g-pyrrole hydrogels for neovascularization.

    PubMed

    Devolder, Ross; Antoniadou, Eleni; Kong, Hyunjoon

    2013-11-28

    Microparticles capable of releasing protein drugs are often incorporated into injectable hydrogels to minimize their displacement at an implantation site, reduce initial drug burst, and further control drug release rates over a broader range. However, there is still a need to develop methods for releasing drug molecules over extended periods of time, in order to sustain the bioactivity of drug molecules at an implantation site. In this study, we hypothesized that a hydrogel formed through the cross-linking of pyrrole units linked to a hydrophilic polymer would release protein drugs in a more sustained manner, because of an enhanced association between cross-linked pyrrole groups and the drug molecules. To examine this hypothesis, we prepared hydrogels of alginate substituted with pyrrole groups, alginate-g-pyrrole, through a horse-radish peroxidase (HRP)-activated cross-linking of the pyrrole groups. The hydrogels were encapsulated with poly(lactic-co-glycolic acid) (PLGA) microparticles loaded with vascular endothelial growth factor (VEGF). The resulting hydrogel system released VEGF in a more sustained manner than Ca(2+) alginate or Ca(2+) alginate-g-pyrrole gel systems. Finally, implantations of the VEGF-releasing HRP-activated alginate-g-pyrrole hydrogel system on chicken chorioallantoic membranes resulted in the formation of blood vessels in higher densities and with larger diameters, compared to other control conditions. Overall, the drug releasing system developed in this study will be broadly useful for regulating release rates of a wide array of protein drugs, and further enhance the quality of protein drug-based therapies. PMID:23886705

  19. Characterization of the activities of actin-affecting drugs on tumor cell migration

    SciTech Connect

    Hayot, Caroline; Debeir, Olivier; Ham, Philippe van; Damme, Marc van; Kiss, Robert; Decaestecker, Christine . E-mail: cdecaes@ulb.ac.be

    2006-02-15

    Metastases kill 90% of cancer patients. It is thus a major challenge in cancer therapy to inhibit the spreading of tumor cells from primary tumor sites to those particular organs where metastases are likely to occur. Whereas the actin cytoskeleton is a key component involved in cell migration, agents targeting actin dynamics have been relatively poorly investigated. Consequently, valuable in vitro pharmacological tools are needed to selectively identify this type of agent. In response to the absence of any standardized process, the present work aims to develop a multi-assay strategy for screening actin-affecting drugs with anti-migratory potentials. To validate our approach, we used two cancer cell lines (MCF7 and A549) and three actin-affecting drugs (cytochalasin D, latrunculin A, and jasplakinolide). We quantified the effects of these drugs on the kinetics of actin polymerization in tubes (by means of spectrofluorimetry) and on the dynamics of actin cytoskeletons within whole cells (by means of fluorescence microscopy). Using quantitative videomicroscopy, we investigated the actual effects of the drugs on cell motility. Finally, the combined drug effects on cell motility and cell growth were evaluated by means of a scratch-wound assay. While our results showed concordant drug-induced effects on actin polymerization occurring in vitro in test tubes and within whole cells, the whole cell assay appeared more sensitive than the tube assay. The inhibition of actin polymerization induced by cytochalasin D was paralleled by a decrease in cell motility for both cell types. In the case of jasplakinolide, which induces actin polymerization, while it significantly enhanced the locomotion of the A549 cells, it significantly inhibited that of the MCF-7 ones. All these effects were confirmed by means of the scratch-wound assay except of the jasplakinolide-induced effects on MCF-7 cell motility. These later seemed compensated by an additional effect occurring during wound

  20. Studies on N-vinylformamide cross-linked copolymers

    NASA Astrophysics Data System (ADS)

    Świder, Joanna; Tąta, Agnieszka; Sokołowska, Katarzyna; Witek, Ewa; Proniewicz, Edyta

    2015-12-01

    Copolymers of N-vinylformamide (NVF) cross-linked with three multifunctional monomers, including divinylbenzene (DVB), ethylene glycol dimethacrylate (EGDMA), and N,N‧-methylenebisacrylamide (MBA) were synthetized by a three-dimensional free radical polymerization in inverse suspension using 2,2‧-azobis(2-methylpropionamide) dihydrochloride (AIBA) as an initiator. Methyl silicon oil was used as the continuous phase during the polymerization processes. Fourier-transform adsorption infrared (FT-IR) spectra revealed the presence of silicone oil traces and suggested that silicone oil strongly interacted with the copolymers surface. Purification procedure allowed to completely remove the silicon oil traces from P(NVF-co-DVB) only. The morphology and the structure of the investigated copolymers were examined by optical microscopy, FT-IR, and FT-Raman (Fourier-transform Raman spectroscopy) methods.

  1. 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. PMID:26375527

  2. [Cross-links of collagen and bone quality].

    PubMed

    Banse, X

    2010-01-01

    Bone tissue is a marvellous material. Basic bone function is to be structurally stiff and strong. Stiffness allows vertebrates to maintain their shape, to protect the organs and to move. Being strong, bone only breaks in exceptional circumstances. Osteoporosis is a disease where fractures happen too often, because of abnormal bone fragility. In this situation, bone--especially cancellous bone--does not take up its first duty. Trabeculae are scarce and thin, leading to very low tissue density. Biomechanical tests and clinical evidence have shown that some subjects have, with equal bone density, stronger or weaker bone tissue. This led to the concept of bone quality. Even if other hypotheses have been systematically explored, it seems that bone collagen chemical nature, especially its cross-link profile, significantly influences human bone quality. PMID:21513097

  3. Protein cross-linking tools for the construction of nanomaterials.

    PubMed

    Domeradzka, Natalia E; Werten, Marc Wt; Wolf, Frits A de; de Vries, Renko

    2016-06-01

    Across bioengineering there is a need to couple proteins to other proteins, or to peptides. Although traditional chemical conjugations have dominated in the past, more and more highly specific coupling strategies are becoming available that are based on protein engineering. Here we review the use of protein modification approaches such as enzymatic and autocatalytic protein-protein coupling, as well as the use of hetero-dimerizing (or hetero-oligomerizing) modules, applied to the specific case of linking together de novo designed recombinant polypeptides into precisely structured nanomaterials. Such polypeptides are increasingly being investigated for biomedical and other applications. In this review, we describe the protein-engineering based cross-linking strategies that dramatically expand the repertoire of possible molecular structures and, hence, the range of materials that can be produced from them. PMID:26871735

  4. Cross-linked polyethylenimine-tripolyphosphate nanoparticles for gene delivery.

    PubMed

    Huang, Xianzhang; Shen, Sujing; Zhang, Zhanfeng; Zhuang, Junhua

    2014-01-01

    The high transfection efficiency of polyethylenimine (PEI) makes it an attractive potential nonviral genetic vector for gene delivery and therapy. However, the highly positive charge of PEI leads to cytotoxicity and limits its application. To reduce the cytotoxicity of PEI, we prepared anion-enriched nanoparticles that combined PEI with tripolyphosphate (TPP). We then characterized the PEI-TPP nanoparticles in terms of size, zeta potential, and Fourier-transform infrared (FTIR) spectra, and assessed their transfection efficiency, cytotoxicity, and ability to resist deoxyribonuclease (DNase) I digestion. The cellular uptake of PEI-TPP with phosphorylated internal ribosome entry site-enhanced green fluorescent protein C1 or FAM (fluorouracil, Adriamycin [doxorubicin] and mitomycin)-labeled small interfering ribonucleic acids (siRNAs) was monitored by fluorescence microscopy and confocal laser microscopy. The efficiency of transfected delivery of plasmid deoxyribonucleic acid (DNA) and siRNA in vitro was 1.11- to 4.20-fold higher with the PEI-TPP particles (7.6% cross-linked) than with the PEI, at all N:P ratios (nitrogen in PEI to phosphorus in DNA) tested. The cell viability of different cell lines was more than 90% at the chosen N:P ratios of PEI-TPP/DNA complexes. Moreover, PEI-TPP nanoparticles resisted digestion by DNase I for more than 2 hours. The time-dependent absorption experiment showed that 7.6% of cross-linked PEI-TPP particles were internalized by 293T cells within 1 hour. In summary, PEI-TPP nanoparticles effectively transfected cells while conferring little or no toxicity, and thus have potential application in gene delivery. PMID:25342902

  5. Emergence of large chiroptical responses by ligand exchange cross-linking of monolayer-protected gold clusters with chiral dithiol.

    PubMed

    Yao, Hiroshi; Yaomura, Shota

    2013-05-28

    We here present a study of cross-linking chemistry of optically inactive monothiol-protected gold clusters by chiral bidentate dithiol with two stereogenic centers, (2R,3R)-1,4-dimercapto-2,3-butanediol (L-dithiothreitol; L-DTT), and explore the impacts of the cross-linking on their chiroptical responses. The pristine protective ligand is racemic penicillamine (rac-Pen), and the products of the ligand exchange reactions include clusters containing both rac-Pen and L-DTT (partial exchange). Electrophoresis using polyacrylamide gel with a very low gel concentration (3%) can make the products separable into two components, each of which has the similar mean core diameter of 0.78 and 0.83 nm, so the difference in the relative mobility is mainly ascribed to the size of the cluster assembly. In addition, very large optical activity with the maximum anisotropy factors of about 1.0 × 10(-3) is found for the assemblies. In comparison with chiral 1,3-dithiol protection incapable of cross-linking between gold clusters, we propose that the observed optical activity is due to surface intrinsic handedness caused by a cyclic cross-linking with at least two L-DTT molecules. PMID:23635318

  6. The use of bifunctional polyethyleneglycol derivatives for coupling of proteins to and cross-linking of collagen matrices.

    PubMed

    Chen, J-S; Noah, E M; Pallua, N; Steffens, G C M

    2002-11-01

    The realization of three-dimensional (3D) degradable matrices which slowly release bio-active components represents a major challenge in the field of tissue engineering. In this paper we report on the usage of commercially available bifunctional agents for both the covalent coupling of proteins to and the cross-linking of collagen matrices. Proteins - horse radish peroxidase (HRP) was used as a model protein - were cross-linked with either a homobifunctional (disuccinimidyldisuccinatepolyethylene-glycol) or a heterobifunctional (N-hydroxysuccinimidylvinylsulfonepolyethyleneglycol) agent. In the case of the heterobifunctional cross-linking agent the collagen matrices were previously modified with succinimidylacetylthioacetate in order to introduce sulfhydryl groups. As compared with control experiments a 10-fold and 50-fold increase of immobilized proteins were achieved with the homobifunctional and heterobifunctional cross-linker resp. The HRP-PEG conjugates demonstrated a better long-term stability as compared to the non-treated HRP. The effects of the cross-linking agents and the thiolation reagent succinimidylacetylthio acetate on the in vitro degradation of the collagen matrices by collagenase were also investigated. In particular the reaction with succinimidylacetylthio acetate appears to offer interesting opportunities both for coupling active proteins and modulating the degradation times of collagen matrices. PMID:15348172

  7. Bio-mimetic surface engineering of plasmid-loaded nanoparticles for active intracellular trafficking by actin comet-tail motility.

    PubMed

    Ng, Chee Ping; Goodman, Thomas T; Park, In-Kyu; Pun, Suzie H

    2009-02-01

    Intracellular transport after endosomal escape presents one of the major barriers for efficient non-viral gene delivery because plasmid DNA and synthetic nanoparticulate carriers suffer from significantly restricted diffusion in the cytoplasm. We postulate that forces generated by actin polymerization, a mechanism used by several bacterial pathogens such as Listeria monocytogenes, can be harnessed to propel nanoparticles within the cytoplasm and thereby overcome diffusional limitations associated with gene transport in the cell cytoplasm. In this work, we synthesized and characterized plasmid DNA-containing nanoparticles modified with ActA protein, the single protein in L. monocytogenes responsible for activating actin polymerization and initiating actin comet-tail propulsion. The motility of the ActA-modified nanoparticles was assessed in Xenopus laevis cytoplasmic extract supplemented with fluorescently labeled actin. Nanoparticle motility was monitored using multi-color, time-lapse fluorescence microscopy for the formation of actin comet tails attached to the fluorescently labeled vehicle. We observed particle motility with velocities approximately 0.06 microm/s with anionic-charged plasmid carriers formed from either poly(lactic-co-glycolic acid) (PLGA) or 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) liposomes, but interestingly not with cationic particles assembled by encapsulation of plasmid with either polyethylenimine (PEI) or 1,2-dioleoyl-3-trimethylammonium-propane/1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOTAP/DOPE) lipids. Control particles coated with albumin instead of ActA also showed no motility. Taken together, we have demonstrated the feasibility of translating the comet-tail propulsion mechanism to synthetic drug carriers as a potential approach to overcome intracellular transport barriers, and also have identified appropriate gene delivery systems that can be employed for this mechanism. PMID:19046764

  8. 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

  9. Evaluation of epigallocatechin-3-gallate (EGCG) cross-linked collagen membranes and concerns on osteoblasts.

    PubMed

    Chu, Chenyu; Deng, Jia; Xiang, Lin; Wu, Yingying; Wei, Xiawei; Qu, Yili; Man, Yi

    2016-10-01

    Collagen membranes have ideal biological and mechanical properties for supporting infiltration and proliferation of osteoblasts and play a vital role in guided bone regeneration (GBR). However, pure collagen can lead to inflammation, resulting in progressive bone resorption. Therefore, a method for regulating the level of inflammatory cytokines at surgical sites is paramount for the healing process. Epigallocatechin-3-gallate (EGCG) is a component extracted from green tea with numerous biological activities including an anti-inflammatory effect. Herein, we present a novel cross-linked collagen membrane containing different concentrations of EGCG (0.0064%, 0.064%, and 0.64%) to regulate the level of inflammatory factors secreted by pre-osteoblast cells; improve cell proliferation; and increase the tensile strength, wettability, and thermal stability of collagen membranes. Scanning electron microscope images show that the surfaces of collagen membranes became smoother and the collagen fiber diameters became larger with EGCG treatment. Measurement of the water contact angle demonstrated that introducing EGCG improved membrane wettability. Fourier transform infrared spectroscopy analyses indicated that the backbone of collagen was intact, and the thermal stability was significant improved in differential scanning calorimetry. The mechanical properties of 0.064% and 0.64% EGCG-treated collagen membranes were 1.5-fold greater than those of the control. The extent of cross-linking was significantly increased, as determined by a 2,4,6-trinitrobenzenesulfonic acid solution assay. The Cell Counting Kit-8 (CCK-8) and live/dead assays revealed that collagen membrane cross-linked by 0.0064% EGCG induced greater cell proliferation than pure collagen membranes. Additionally, real-time polymerase chain reaction and enzyme-linked immunosorbent assay results showed that EGCG significantly affected the production of inflammatory factors secreted by MC3T3-E1 cells. Taken together, our

  10. 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

  11. Actinic keratosis

    MedlinePlus

    ... example, if you work outdoors) Had many severe sunburns early in life Are older Symptoms Actinic keratosis ... and tanning salons. Other things to know about sun exposure: Sun exposure is stronger in or near surfaces ...

  12. Actinic Cheilitis

    MedlinePlus

    ... is a precancerous condition related to cumulative lifetime sun exposure. The lower lip is most often affected. Individuals ... Wearing barrier clothing (eg, wide-brimmed hats) and sunscreen-containing lip balms can aid in preventing actinic ...

  13. Antioxidant films based on cross-linked methyl cellulose and native Chilean berry for food packaging applications.

    PubMed

    López de Dicastillo, Carol; Rodríguez, Francisco; Guarda, Abel; Galotto, Maria José

    2016-01-20

    Development of antioxidant and antimicrobial active food packaging materials based on biodegradable polymer and natural plant extracts has numerous advantages as reduction of synthetic additives into the food, reduction of plastic waste, and food protection against microorganisms and oxidation reactions. In this way, active films based on methylcellulose (MC) and maqui (Aristotelia chilensis) berry fruit extract, as a source of antioxidants agents, were studied. On the other hand, due to the high water affinity of MC, this polymer was firstly cross-linked with glutaraldehyde (GA) at different concentrations. The results showed that the addition of GA decreased water solubility, swelling, water vapor permeability of MC films, and the release of antioxidant substances from the active materials increased with the concentration of GA. Natural extract and active cross-linked films were characterized in order to obtain the optimal formulation with the highest antioxidant activity and the best physical properties for latter active food packaging application. PMID:26572446

  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. Cross-linked enzyme aggregates of phenylalanine ammonia lyase: novel biocatalysts for synthesis of L-phenylalanine.

    PubMed

    Cui, Jian-Dong; Zhang, Si; Sun, Li-Mei

    2012-06-01

    Cross-linked enzyme aggregates of phenylalanine ammonia lyase (PAL-CLEAs) from Rhodotorula glutinis were prepared. The effects of the type of aggregating agent, its concentration, and that of cross-linking agent were studied. PAL-CLEAs production was most effective using ammonium sulfate (40 % saturation), followed by cross-linking for 1 h with 0.2 % (v/v) glutaraldehyde. Moreover, the storage and operational stability of the resulting PAL-CLEAs were also investigated. Compared to the free enzyme, the PAL-CLEAs exhibited the expected increased stability of the enzyme against various deactivating conditions such as pH, temperature, denaturants, and organic solvents and showed higher storage stability than its soluble counterpart. Additionally, the reusability of PAL-CLEAs with respect to the biotransformation of L-phenylalanine was evaluated. PAL-CLEAs could be recycled at least for 12 consecutive batch reactions without dramatic activity loss, which should dramatically increase the commercial potential of PAL for synthesis of L: -phenylalanine. To the best of our knowledge, this is the first report of immobilization of PAL as cross-linked enzyme aggregates. PMID:22622644

  16. Standard versus trans-epithelial collagen cross-linking in keratoconus patients suitable for standard collagen cross-linking

    PubMed Central

    Rossi, S; Orrico, A; Santamaria, C; Romano, V; De Rosa, L; Simonelli, F; De Rosa, G

    2015-01-01

    Purpose Evaluating the clinical results of trans-epithelial collagen cross-linking (CXL) and standard CXL in patients with progressive keratoconus. Methods This prospective study comprised 20 eyes of 20 patients with progressive keratoconus. Ten eyes were treated by standard CXL and ten by trans-epithelial cross-linking (TE-CXL, epithelium on) with 1 year of follow-up. All patients underwent complete ophthalmologic testing that included pre- and postoperative uncorrected visual acuity, corrected visual acuity, spherical error, spherical equivalent, corneal astigmatism, simulated maximum, minimum, and average keratometry, coma and spherical aberration, optical pachymetry, and endothelial cell density. Intra-and postoperative complications were recorded. The solution used for standard CXL comprised riboflavin 0.1% and dextran 20.0% (Ricrolin), while the solution for TE-CXL (Ricrolin, TE) comprised riboflavin 0.1%, dextran 15.0%, trometamol (Tris), and ethylenediaminetetraacetic acid. Ultraviolet-A treatment was performed with UV-X System at 3 mW/cm2. Results In both the standard CXL group (ten patients, ten eyes; mean age, 30.4±7.3 years) and the TE-CXL group (ten patients, ten eyes; mean age, 28±3.8 years), uncorrected visual acuity and corrected visual acuity improved significantly after treatment. Furthermore, a significant improvement in topographic outcomes, spherical error, and spherical equivalent was observed in both groups at month 12 posttreatment. No significant variations were recorded in other parameters. No complications were noted. Conclusion A 1-year follow-up showed stability of clinical and refractive outcomes after standard CXL and TE-CXL. PMID:25834386

  17. A Rickettsia WASP-like protein activates the Arp2/3 complex and mediates actin-based motility.

    PubMed

    Jeng, Robert L; Goley, Erin D; D'Alessio, Joseph A; Chaga, Oleg Y; Svitkina, Tatyana M; Borisy, Gary G; Heinzen, Robert A; Welch, Matthew D

    2004-08-01

    Spotted fever group Rickettsia are obligate intracellular pathogens that exploit the host cell actin cytoskeleton to promote motility and cell-to-cell spread. Although other pathogens such as Listeria monocytogenes use an Arp2/3 complex-dependent nucleation mechanism to generate comet tails consisting of Y-branched filament arrays, Rickettsia polymerize tails consisting of unbranched filaments by a previously unknown mechanism. We identified genes in several Rickettsia species encoding proteins (termed RickA) with similarity to the WASP family of Arp2/3-complex activators. Rickettsia rickettsii RickA activated both the nucleation and Y-branching activities of the Arp2/3 complex like other WASP-family proteins, and was sufficient to direct the motility of microscopic beads in cell extracts. Actin tails generated by RickA-coated beads consisted of Y-branched filament networks. These data suggest that Rickettsia use an Arp2/3 complex-dependent actin-nucleation mechanism similar to that of other pathogens. We propose that additional Rickettsia or host factors reorganize the Y-branched networks into parallel arrays in a manner similar to a recently proposed model of filopodia formation. PMID:15236643

  18. Reversible cross-linking, microdomain structure, and heterogeneous dynamics in thermally reversible cross-linked polyurethane as revealed by solid-state NMR.

    PubMed

    Zhang, Rongchun; Yu, Shen; Chen, Shengli; Wu, Qiang; Chen, Tiehong; Sun, Pingchuan; Li, Baohui; Ding, Datong

    2014-01-30

    Polyurethane material is widely utilized in industry and daily life due to its versatile chemistry and relatively easy handling. Here, we focused on a novel thermally reversible cross-linked polyurethane with comprehensive remarkable mechanical properties as reported in our recent work (Adv. Mater. 2013, 25, 4912). The microphase-separated structure and heterogeneous segmental dynamics were well revealed by T2 relaxometry experiments, which was also first utilized to in situ monitor the reversible cross-linking associated with Diels-Alder (DA) and retro-Diels-Alder (RDA) reactions. On the basis of T2 relaxometry results, we determined the actual temperature of the (R)DA reaction as well as the corresponding activation energies of the motion of soft segments. Besides, the roles of the temperature and cross-linker contents on the microdomain structure and dynamics are discussed in detail. It is found that the microphase separation is enhanced by the increase of temperature as well as the incorporation of cross-linkers. Also, the polyurethane samples are still thermal-stable even at a high temperature beyond the disassociation of the cross-linkages. Furthermore, Baum-Pines and three-pulse multiple-quantum NMR experiments are utilized to investigate the heterogeneous structures and dynamics of the mobile and rigid segments, respectively. Both the results obtained from the T2 relaxometry and multiple-quantum NMR experiments are in good agreement with the macroscopic mechanical properties of the polyurethane. Finally, it is also well demonstrated that proton T2 relaxometry combined with multiple-quantum NMR is a powerful method to study the heterogeneous structures and dynamics of a multiphase polymer system. PMID:24400980

  19. Plasmodium falciparum liver stage antigen-1 is cross-linked by tissue transglutaminase

    PubMed Central

    2011-01-01

    Background Plasmodium falciparum sporozoites injected by mosquitoes into the blood rapidly enter liver hepatocytes and undergo pre-erythrocytic developmental schizogony forming tens of thousands of merozoites per hepatocyte. Shortly after hepatocyte invasion, the parasite starts to produce Liver Stage Antigen-1 (LSA-1), which accumulates within the parasitophorous vacuole surrounding the mass of developing merozoites. The LSA-1 protein has been described as a flocculent mass, but its role in parasite development has not been determined. Methods Recombinant N-terminal, C-terminal or a construct containing both the N- and C- terminal regions flanking two 17 amino acid residue central repeat sequences (LSA-NRC) were subjected to in vitro modification by tissue transglutaminase-2 (TG2) to determine if cross-linking occurred. In addition, tissue sections of P. falciparum-infected human hepatocytes were probed with monoclonal antibodies to the isopeptide ε-(γ-glutamyl)lysine cross-bridge formed by TG2 enzymatic activity to determine if these antibodies co-localized with antibodies to LSA-1 in the growing liver schizonts. Results This study identified a substrate motif for (TG2) and a putative casein kinase 2 phosphorylation site within the central repeat region of LSA-1. The function of TG2 is the post-translational modification of proteins by the formation of a unique isopeptide ε-(γ-glutamyl)lysine cross-bridge between glutamine and lysine residues. When recombinant LSA-1 protein was crosslinked in vitro by purified TG2 in a calcium dependent reaction, a flocculent mass of protein was formed that was highly resistant to degradation. The cross-linking was not detectably affected by phosphorylation with plasmodial CK2 in vitro. Monoclonal antibodies specific to the very unique TG2 catalyzed ε- lysine cross-bridge co-localized with antibodies to LSA-1 in infected human hepatocytes providing visual evidence that LSA-1 was cross-linked in vivo. Conclusions While the

  20. 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.

  1. Ocular biocompatibility of carbodiimide cross-linked hyaluronic acid hydrogels for cell sheet delivery carriers.

    PubMed

    Lai, Jui-Yang; Ma, David Hui-Kang; Cheng, Hsiao-Yun; Sun, Chi-Chin; Huang, Shu-Jung; Li, Ya-Ting; Hsiue, Ging-Ho

    2010-01-01

    Due to its innocuous nature, hyaluronic acid (HA) is one of the most commonly used biopolymers for ophthalmic applications. We recently developed a cell sheet delivery system using carbodiimide cross-linked HA carriers. Chemical cross-linking provides an improvement in stability of polymer gels, but probably causes toxic side-effects. The aim of this study was to investigate the ocular biocompatibility of HA hydrogels cross-linked by 1-ethyl-3-(3-dimethyl aminopropyl) carbodiimide (EDC). HA discs without cross-linking and glutaraldehyde (GTA) cross-linked HA samples were used for comparison. The disc implants were inserted in the anterior chamber of rabbit eyes for 24 weeks and characterized by slit-lamp biomicroscopy, histology and scanning electron microscopy. The ophthalmic parameters obtained from biomicroscopic examinations were also scored to provide a quantitative grading system. Results of this study showed that the HA discs cross-linked with EDC had better ocular biocompatibility than those with GTA. The continued residence of GTA cross-linked HA implants in the intraocular cavity elicited severe tissue responses and significant foreign body reactions, whereas no adverse inflammatory reaction was observed after contact with non-cross-linked HA or EDC cross-linked HA samples. It is concluded that the cross-linking agent type gives influence on ocular biocompatibility of cell carriers and the EDC-HA hydrogel is an ideal candidate for use as an implantable material in cell sheet delivery applications. PMID:20178691

  2. Tuning nanoscale viscoelasticity of polyelectrolyte complexes with multiple types of cross-links

    NASA Astrophysics Data System (ADS)

    Ma, Tianzhu; Han, Biao; Lee, Daeyeon; Han, Lin

    Mechanical properties of hydrogels are manifestation of cross-link type and density, fixed charges and water-polymer interactions. In this study, we revealed how different types of cross-links regulate the nanoscale viscoelasticity of polyelectrolyte networks. Ionically cross-linked PAH/PAA layer-by-layer complexes were modified to include covalent cross-links using EDC. AFM-nanoindentation and force relaxation were performed at various ionic strength (0.01-1M) and pH (1.5-5.5). As-assembled networks, held only by ionic cross-links, underwent >95% relaxation, dominated by cross-link breaking and re-formation. Addition of covalent cross-links increased the instantaneous modulus by 1.6-fold and attenuated relaxation to ~80% of net neutral states (pH >=3.5), as covalent cross-links provide additional elastic components. The network remained stabilized when all ionic cross-links were dissociated at pH <=1.5, whereby further attenuation to 31% in relaxation could be due to viscoelastic polymer conformational changes and fluid flow-induced poroelasticity. Taken together, this study demonstrates the potential of using multiple cross-linking types to tune the viscoelastic mechanisms in polyelectrolyte complexes.

  3. 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. PMID:25140307

  4. 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. PMID:26189105

  5. 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

  6. 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

  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. PMID:27318817

  8. Development of a Novel Cross-linking Strategy for Fast and Accurate Identification of Cross-linked Peptides of Protein Complexes*

    PubMed Central

    Kao, Athit; Chiu, Chi-li; Vellucci, Danielle; Yang, Yingying; Patel, Vishal R.; Guan, Shenheng; Randall, Arlo; Baldi, Pierre; Rychnovsky, Scott D.; Huang, Lan

    2011-01-01

    Knowledge of elaborate structures of protein complexes is fundamental for understanding their functions and regulations. Although cross-linking coupled with mass spectrometry (MS) has been presented as a feasible strategy for structural elucidation of large multisubunit protein complexes, this method has proven challenging because of technical difficulties in unambiguous identification of cross-linked peptides and determination of cross-linked sites by MS analysis. In this work, we developed a novel cross-linking strategy using a newly designed MS-cleavable cross-linker, disuccinimidyl sulfoxide (DSSO). DSSO contains two symmetric collision-induced dissociation (CID)-cleavable sites that allow effective identification of DSSO-cross-linked peptides based on their distinct fragmentation patterns unique to cross-linking types (i.e. interlink, intralink, and dead end). The CID-induced separation of interlinked peptides in MS/MS permits MS3 analysis of single peptide chain fragment ions with defined modifications (due to DSSO remnants) for easy interpretation and unambiguous identification using existing database searching tools. Integration of data analyses from three generated data sets (MS, MS/MS, and MS3) allows high confidence identification of DSSO cross-linked peptides. The efficacy of the newly developed DSSO-based cross-linking strategy was demonstrated using model peptides and proteins. In addition, this method was successfully used for structural characterization of the yeast 20 S proteasome complex. In total, 13 non-redundant interlinked peptides of the 20 S proteasome were identified, representing the first application of an MS-cleavable cross-linker for the characterization of a multisubunit protein complex. Given its effectiveness and simplicity, this cross-linking strategy can find a broad range of applications in elucidating the structural topology of proteins and protein complexes. PMID:20736410

  9. mDia1 and formins: screw cap of the actin filament

    PubMed Central

    Mizuno, Hiroaki; Watanabe, Naoki

    2012-01-01

    Formin homology proteins (formins) are actin nucleation factors which remain bound to the growing barbed end and processively elongate actin filament (F-actin). Recently, we have demonstrated that a mammalian formin mDia1 rotates along the long-pitch helix of F-actin during processive elongation (helical rotation) by single-molecule fluorescence polarization. We have also shown processive depolymerization of mDia1-bound F-actin during which helical rotation was visualized. In the cell where F-actins are highly cross-linked, formins should rotate during filament elongation. Therefore, when formins are tightly anchored to cellular structures, formins may not elongate F-actin. Adversely, helical rotation of formins might affect the twist of F-actin. Formins could thus control actin elongation and regulate stability of cellular actin filaments through helical rotation. On the other hand, ADP-actin elongation at the mDia1-bound barbed end turned out to become decelerated by profilin, in marked contrast to its remarkably positive effect on mDia1-mediated ATP-actin elongation. This deceleration is caused by enhancement of the off-rate of ADP-actin. While mDia1 and profilin enhance the ADP-actin off-rate, they do not apparently increase the ADP-actin on-rate at the barbed end. These results imply that G-actin-bound ATP and its hydrolysis may be part of the acceleration mechanism of formin-mediated actin elongation.

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

    PubMed Central

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

    2015-01-01

    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 15N and 14N-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. PMID:26807012

  11. Rho-kinase-dependent actin turnover and actomyosin disassembly are necessary for mouse spinal neural tube closure

    PubMed Central

    Escuin, Sarah; Vernay, Bertrand; Savery, Dawn; Gurniak, Christine B.; Witke, Walter; Greene, Nicholas D. E.; Copp, Andrew J.

    2015-01-01

    ABSTRACT The cytoskeleton is widely considered essential for neurulation, yet the mouse spinal neural tube can close despite genetic and non-genetic disruption of the cytoskeleton. To investigate this apparent contradiction, we applied cytoskeletal inhibitors to mouse embryos in culture. Preventing actomyosin cross-linking, F-actin assembly or myosin II contractile activity did not disrupt spinal closure. In contrast, inhibiting Rho kinase (ROCK, for which there are two isoforms ROCK1 and ROCK2) or blocking F-actin disassembly prevented closure, with apical F-actin accumulation and adherens junction disturbance in the neuroepithelium. Cofilin-1-null embryos yielded a similar phenotype, supporting the hypothesis that there is a key role for actin turnover. Co-exposure to Blebbistatin rescued the neurulation defects caused by RhoA inhibition, whereas an inhibitor of myosin light chain kinase, ML-7, had no such effect. We conclude that regulation of RhoA, Rho kinase, LIM kinase and cofilin signalling is necessary for spinal neural tube closure through precise control of neuroepithelial actin turnover and actomyosin disassembly. In contrast, actomyosin assembly and myosin ATPase activity are not limiting for closure. PMID:26040287

  12. Intensity increases of actin layer-lines on activation of the Limulus muscle.

    PubMed Central

    Maéda, Y; Boulin, C; Gabriel, A; Sumner, I; Koch, M H

    1986-01-01

    Small angle x-ray diffraction patterns were recorded from isometrically contracting Limulus (horseshoe crab) telson levator muscle using a multiwire proportional-area detector on the storage ring DORIS. In the pattern a substantial increase in intensity is observed on the thin-filament-associated layer-line at 1/38 nm-1 (the first actin layer-line) with a maximum increase at a radial spacing of R = 0.07 nm-1 but there is a much smaller change in the intensity of the 5.9-nm layer-line, which also arises from the thin filament structure. The results suggest that during contraction the myosin heads, presumably being attached to the thin filaments, are arranged along the long-stranded helical tracks of the thin filaments but that the spatial relationship between the heads and the actin monomers varies. Intensity increases have also been observed (Maéda et al., manuscript in preparation) in the part of the patterns from frog muscle and barnacle muscle, which are attributable to the first actin layer-line. It is thus likely that the intensity increase of the first actin layer-line on the Limulus pattern is associated not with structural features which are special to Limulus muscle, but with the tension generating processes that are shared by muscles in general. Images FIGURE 1 FIGURE 2 PMID:3801566

  13. 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. PMID:25063151

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

    NASA Astrophysics Data System (ADS)

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

    2015-07-01

    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 d33 coefficients up to 650 pC/N at 200 Hz are achieved. The figure of merit (FOM, d33 ṡ g33) for a more typical d33 value of 400 pC/N is about 11.2 GPa-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 cm2 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.

  15. Mechanical characterization of cross-linked serum albumin microcapsules.

    PubMed

    de Loubens, Clément; Deschamps, Julien; Georgelin, Marc; Charrier, Anne; Edwards-Levy, Florence; Leonetti, Marc

    2014-07-01

    Controlling the deformation of microcapsules and capsules is essential in numerous biomedical applications. The mechanical properties of the membrane of microcapsules made of cross-linked human serum albumin (HSA) are revealed by two complementary experiments in the linear elastic regime. The first provides the surfacic shear elastic modulus Gs by the study of small deformations of a single capsule trapped in an elongational flow: Gs varies from 0.002 to 5 N m(-1). The second gives the volumic Young's modulus E of the membrane by shallow and local indentations of the membrane with an AFM probe: E varies from 20 kPa to 1 MPa. The surfacic and volumic elastic moduli increase with the size of the capsule up to three orders of magnitude and with the protein concentration of the membrane. The membrane thickness is evaluated from these two membrane mechanical characteristics and increases with the size and the initial HSA concentration from 2 to 20 μm. PMID:24817568

  16. 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.

  17. 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

  18. Cross-linking of polytetrafluoroethylene during room-temperature irradiation

    SciTech Connect

    Pugmire, David L; Wetteland, Chris J; Duncan, Wanda S; Lakis, Rollin E; Schwartz, Daniel S

    2008-01-01

    Exposure of polytetrafluoroethylene (PTFE) to {alpha}-radiation was investigated to detennine the physical and chemical effects, as well as to compare and contrast the damage mechanisms with other radiation types ({beta}, {gamma}, or thermal neutron). A number of techniques were used to investigate the chemical and physical changes in PTFE after exposure to {alpha}-radiation. These techniques include: Fourier transform infrared spectroscopy (FTIR), differential scanning calorimetry (DSC), and fluorescence spectroscopy. Similar to other radiation types at low doses, the primary damage mechanism for the exposure of PTFE to {alpha}-radiation appears to be chain scission. Increased doses result in a change-over of the damage mechanism to cross-linking. This result is not observed for any radiation type other than {alpha} when irradiation is performed at room temperature. Finally, at high doses, PTFE undergoes mass-loss (via smallfluorocarbon species evolution) and defluorination. The amount and type of damage versus sample depth was also investigated. Other types of radiation yield damage at depths on the order of mm to cm into PTFE due to low linear energy transfer (LET) and the correspondingly large penetration depths. By contrast, the {alpha}-radiation employed in this study was shown to only induce damage to a depth of approximately 26 {mu}m, except at very high doses.

  19. 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.

  20. An overview of corneal collagen cross-linking (CXL).

    PubMed

    Kymionis, George D; Mikropoulos, Dimitrios G; Portaliou, Dimitra M; Voudouragkaki, Irini C; Kozobolis, Vassilios P; Konstas, Anastasios G P

    2013-10-01

    Corneal collagen cross-linking (CXL) was first described over a decade ago and is now considered to be one of the most important surgical innovations of modern ophthalmology. Prior to its introduction, no interventions were available to arrest, or slow down ectatic disease progression, with corneal transplantation required in the majority of cases. Unlike earlier treatments of corneal ectasias that attempted to only improve the consequences of the disease, CXL aims to address the corneal biomechanical weakening itself. The long-term safety and efficacy of CXL have been established in several studies that have documented significant improvements in all outcome measures (visual acuity, spherical equivalent, astigmatism, and keratometric findings). The emerging combination of CXL with other interventions (termed 'CXL plus') optimizes the visual and topographic outcomes. This, along with the expansion of the techniques' indications for other clinical conditions, such as microbial keratitis, highlights the continuous improvement of the initial technique and confirms its wide acceptance. Overall, CXL has already demonstrated much promise and has several clinical indications, representing a clear example of recent advances in ocular therapy. PMID:24170589

  1. X-ray diffraction indicates that active cross-bridges bind to actin target zones in insect flight muscle.

    PubMed

    Tregear, R T; Edwards, R J; Irving, T C; Poole, K J; Reedy, M C; Schmitz, H; Towns-Andrews, E; Reedy, M K

    1998-03-01

    We report the first time-resolved study of the two-dimensional x-ray diffraction pattern during active contraction in insect flight muscle (IFM). Activation of demembranated Lethocerus IFM was triggered by 1.5-2.5% step stretches (risetime 10 ms; held for 1.5 s) giving delayed active tension that peaked at 100-200 ms. Bundles of 8-12 fibers were stretch-activated on SRS synchrotron x-ray beamline 16.1, and time-resolved changes in diffraction were monitored with a SRS 2-D multiwire detector. As active tension rose, the 14.5- and 7.2-nm meridionals fell, the first row line dropped at the 38.7 nm layer line while gaining a new peak at 19.3 nm, and three outer peaks on the 38.7-nm layer line rose. The first row line changes suggest restricted binding of active myosin heads to the helically preferred region in each actin target zone, where, in rigor, two-headed lead bridges bind, midway between troponin bulges that repeat every 38.7 nm. Halving this troponin repeat by binding of single active heads explains the intensity rise at 19.3 nm being coupled to a loss at 38.7 nm. The meridional changes signal movement of at least 30% of all myosin heads away from their axially ordered positions on the myosin helix. The 38.7- and 19.3-nm layer line changes signal stereoselective attachment of 7-23% of the myosin heads to the actin helix, although with too little ordering at 6-nm resolution to affect the 5.9-nm actin layer line. We conclude that stretch-activated tension of IFM is produced by cross-bridges that bind to rigor's lead-bridge target zones, comprising < or = 1/3 of the 75-80% that attach in rigor. PMID:9512040

  2. Modification of sodium and gating currents by amino group specific cross-linking and monofunctional reagents.

    PubMed Central

    Drews, G; Rack, M

    1988-01-01

    To test the possible role of lysine residues in Na channel function the effects of several imidoesters on Na and gating currents were studied in voltage-clamped single frog nerve fibers. Mono- and bisimidoesters were used. These reagents modify amino groups exclusively and do not change the net charge. The three bisimidoesters used easily introduce cross-links between neighboring amino groups. Their structure is almost identical; only the length of the spacers between the two amino-reactive groups is different. An irreversible reduction of Na currents and gating currents was observed with the longest (dimethyl suberimidate [DMS]) and the shortest (dimethyl adipimidate [DMA]) of the cross-linkers used. Of the three cross-linking reagents only the shortest made Na current inactivation slow and incomplete. The steady-state inactivation curve, h infinity (E), was shifted by greater than 25 mV in the hyperpolarizing direction by each of the reagents. The voltage dependence of activation, however, remained unchanged. Furthermore, the effects of two different monoimidoesters (ethyl acetimidate [EAI] and isethionyl acetimidate [IAI]) on gating currents were tested. EAI can penetrate a membrane, whereas IAI is membrane impermeant. IAI was almost without effect, whereas EAI caused a considerable reduction of the gating currents. EAI and DMS reduced the Qoff/Qon ratio without affecting the decay of the Na currents. The results show that lysine residues are critically involved in Na channel gating. PMID:2850028

  3. Enzymatically cross-linked alginic-hyaluronic acid composite hydrogels as cell delivery vehicles.

    PubMed

    Ganesh, Nitya; Hanna, Craig; Nair, Shantikumar V; Nair, Lakshmi S

    2013-04-01

    An injectable composite gel was developed from alginic and hyaluronic acid. The enzymatically cross-linked injectable gels were prepared via the oxidative coupling of tyramine modified sodium algiante and sodium hyaluronate in the presence of horse radish peroxidase (HRP) and hydrogen peroxide (H2O2). The composite gels were prepared by mixing equal parts of the two tyraminated polymer solutions in 10U HRP and treating with 1.0% H2O2. The properties of the alginate gels were significantly affected by the addition of hyaluronic acid. The percentage water absorption and storage modulus of the composite gels were found to be lower than the alginate gels. The alginate and composite gels showed lower protein release compared to hyaluronate gels in the absence of hyaluronidase. Even hyaluronate gels showed only approximately 10% protein release after 14 days incubation in phosphate buffer solution. ATDC-5 cells encapsulated in the injectable gels showed high cell viability. The composite gels showed the presence of enlarged spherical cells with significantly higher metabolic activity compared to cells in hyaluronic and alginic acid gels. The results suggest the potential of the composite approach to develop covalently cross-linked hydrogels with tuneable physical, mechanical, and biological properties. PMID:23357799

  4. 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. PMID:24571427

  5. Photo-Cross-Linked Laminarin-Based Hydrogels for Biomedical Applications.

    PubMed

    Custódio, Catarina A; Reis, Rui L; Mano, João F

    2016-05-01

    Laminarin is a low-molecular-weight (<10 kDa) glucan found in brown algae made up of β(1→3)-glucan with β(1→6)-branches. This is one of the most abundant carbon sources in the marine ecosystem. Laminarin has been found to possess various biological interesting properties, such as antioxidant and antimicrobial activities. An attractive feature of laminarin is its inherently low viscosity and high solubility in organic and aqueous solvents that facilitate processing. This makes laminarin an appealing material for the development of new hydrogels that can be easily injected through minimally invasive procedures or used for microfabrication of hydrogels. An approach for synthesizing photo-cross-linkable laminarin hydrogels is presented in this work for the first time. Photo-cross-linkable laminarin was prepared by chemical modification with acrylate groups. The synthesized photo-cross-linkable laminarin material provides the basis for the development of a new injectable system for biomedical purposes that could be used alone or with encapsulated cells or biological molecules. The cross-linking of the methacrylated laminarin is straightforward via photoinitiated polymerization. The possibility to control the methacrylation degree of laminarin and to prepare solutions up to at least 15% w/v permits us to obtain hydrogels with tuned and wide range of stiffness and swelling. Furthermore, the encapsulation of human-adipose-derived stem cells encapsulated in the photo-cross-linked hydrogels demonstrated in vitro biocompatibility. PMID:27017983

  6. BRCA1 functions independently of homologous recombination in DNA interstrand cross-link repair

    PubMed Central

    Bunting, Samuel F; Callen, Elsa; Kozak, Marina L; Kim, Jung-Min; Wong, Nancy; Lopez-Contreras, Andres J; Ludwig, Thomas; Baer, Richard; Faryabi, Robert B; Malhowski, Amy; Chen, Hua-Tang; Fernandez-Capetillo, Oscar; D’Andrea, Alan; Nussenzweig, Andre

    2012-01-01

    Brca1 is required for DNA repair by homologous recombination (HR) and normal embryonic development. Here we report that deletion of the DNA damage response factor 53BP1 overcomes embryonic lethality in Brca1-nullizygous mice, and rescues HR deficiency, as measured by hypersensitivity to PARP (polyADP-ribose polymerase) inhibition. However, Brca1,53BP1 double-deficient cells are hypersensitive to DNA interstrand cross-links (ICLs), indicating that BRCA1 has an additional role in DNA cross-link repair that is distinct from HR. Disruption of the non-homologous end-joining (NHEJ) factor, Ku, promotes DNA repair in Brca1-deficient cells; however deletion of either Ku or 53BP1 exacerbates genomic instability in cells lacking FANCD2, a mediator of the Fanconi Anemia pathway for ICL repair. BRCA1 therefore has two separate roles in ICL repair, whereas FANCD2 provides a key activity that can not be bypassed by ablation of 53BP1 or Ku. PMID:22445484

  7. Spermidine cross-linked hydrogels as a controlled release biomimetic approach for cloxacillin.

    PubMed

    López-Cebral, Rita; Romero-Caamaño, Vanessa; Seijo, Begoña; Alvarez-Lorenzo, Carmen; Martín-Pastor, Manuel; Concheiro, Ángel; Landin, Mariana; Sanchez, Alejandro

    2014-07-01

    The intrinsic ability of albumin to bind active substances in the physiological fluids has been explored to endow hydrogels with improved capability to regulate drug release. To develop such biomimetic-functional hydrogels, it is critical that albumin conformation is not altered and that the protein remains retained inside the hydrogel keeping its conformational freedom, i.e., it should be not chemically cross-linked. Thus, the hydrogels were prepared with various proportions of albumin by physical cross-linking of anionic polysaccharides (gellan gum and chondroitin sulfate) with the cationic endogen polyamine spermidine under mild conditions in order to prevent albumin denaturation. Texture and swelling properties of hydrogels with various compositions were recorded, and the effect of the preparation variables was evaluated applying neurofuzzy logic tools for hydrogels prepared with and without albumin and associating the antibiotic cloxacillin. Developed hydrogel systems were extensively analyzed by means of nuclear magnetic resonance (NMR) to determine weak-to-medium and strong binding modes and the equilibrium constants of the albumin-cloxacillin association. NMR techniques were also employed to demonstrate the successful modulation of the cloxacillin release from the albumin-containing hydrogels. In vitro microbiological tests carried out with Staphylococcus aureus and Staphylococcus epidermidis confirmed the interest of the albumin-containing hydrogels as efficient platforms for cloxacillin release in its bioactive form. PMID:24921673

  8. Peroxidase induced oligo-tyrosine cross-links during polymerization of α-lactalbumin.

    PubMed

    Dhayal, Surender Kumar; Sforza, Stefano; Wierenga, Peter A; Gruppen, Harry

    2015-12-01

    Horseradish peroxidase (HRP) induced cross-linking of proteins has been reported to proceed through formation of di-tyrosine cross-links. In the case of low molar mass phenolic substrates, the enzymatic oxidation is reported to lead to polymerization of the phenols. The aim of this work was to investigate if during oxidative cross-linking of proteins oligo-tyrosine cross-links are formed in addition to dityrosine. To this end, α-lactalbumin (α-LA) was cross-linked using horseradish peroxidase (HRP) and hydrogen peroxide (H₂O₂). The reaction products were acid hydrolysed, after which the cross-linked amino acids were investigated by LC-MS and MALDI-MS. To test the effect of the size of the substrate, the cross-linking reaction was also performed with L-tyrosine, N-acetyl L-tyrosinamide and angiotensin. These products were analyzed by LC-MS directly, as well as after acid hydrolysis. In the acid hydrolysates of all samples oligo-tyrosine (Yn, n=3-8) was found in addition to di-tyrosine (Y2). Two stages of cross-linking of α-LA were identified: a) 1-2 cross-links were formed per monomer until the monomers were converted into oligomers, and b) subsequent cross-linking of oligomers formed in the first stage to form nanoparticles containing 3-4 cross-links per monomer. The transition from first stage to the second stage coincided with the point where di-tyrosine started to decrease and more oligo-tyrosines were formed. In conclusion, extensive polymerization of α-LA using HRP via oligo-tyrosine cross-links is possible, as is the case for low molar mass tyrosine containing substrates. PMID:26282909

  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. Synthesis of Cross-Linked DNA Containing Oxidized Abasic Site Analogues

    PubMed Central

    2015-01-01

    DNA interstrand cross-links are an important family of DNA damage that block replication and transcription. Recently, it was discovered that oxidized abasic sites react with the opposing strand of DNA to produce interstrand cross-links. Some of the cross-links between 2′-deoxyadenosine and the oxidized abasic sites, 5′-(2-phosphoryl-1,4-dioxobutane) (DOB) and the C4-hydroxylated abasic site (C4-AP), are formed reversibly. Chemical instability hinders biochemical, structural, and physicochemical characterization of these cross-linked duplexes. To overcome these limitations, we developed methods for preparing stabilized analogues of DOB and C4-AP cross-links via solid-phase oligonucleotide synthesis. Oligonucleotides of any sequence are attainable by synthesizing phosphoramidites in which the hydroxyl groups of the cross-linked product were orthogonally protected using photochemically labile and hydrazine labile groups. Selective unmasking of a single hydroxyl group precedes solid-phase synthesis of one arm of the cross-linked DNA. The method is compatible with commercially available phosphoramidites and other oligonucleotide synthesis reagents. Cross-linked duplexes containing as many as 54 nt were synthesized on solid-phase supports. Subsequent enzyme ligation of one cross-link product provided a 60 bp duplex, which is suitable for nucleotide excision repair studies. PMID:24949656

  11. Controlled sparse and percolating cross-linking in waterborne soft adhesives.

    PubMed

    Deplace, F; Carelli, C; Langenfeld, A; Rabjohns, M A; Foster, A B; Lovell, P A; Creton, C

    2009-09-01

    The effect of low levels of cross-linking on the adhesive and mechanical properties of waterborne pressure-sensitive adhesives was investigated. We have taken advantage of a core-shell latex particle morphology obtained by emulsion polymerization to create a heterogeneous structure of cross-links without major modification of the monomer composition. The latex particles comprise a shell containing cross-linkable diacetone acrylamide (DAAM) repeat units localized on the periphery of a slightly softer core copolymer of very similar composition. Adipic acid dihydrazide was added to the latex prior to film formation to react with DAAM repeat units and affect interfacial cross-linking between particles in the adhesive films. The honeycomb-like structure obtained after drying of the latex results in a good balance between the dissipative properties required for adhesion and the resistance to creep. The characterization of the mechanical properties of the films shows that the chosen cross-linking method creates a percolating lightly cross-linked network, swollen with a nearly un-cross-linked component. With this cross-linking method, the linear viscoelastic properties of the soft films are nearly unaffected by the cross-linking while the nonlinear tensile properties are greatly modified. As a result, the long-term shear resistance of the adhesive film improves very significantly while the peel force remains nearly the same. A simple rheological model is used to interpret qualitatively the changes in the material parameters induced by cross-linking. PMID:20355828

  12. 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. PMID:22216745

  13. Organic solvent-tolerant elastase efficiently hydrolyzes insoluble, cross-linked, protein fiber of eggshell membranes.

    PubMed

    Takenaka, Shinji; Hano, Shinpei; Cheng, Minyi; Yoshida, Ken-ichi; Aoki, Kenji

    2012-05-01

    Eggshell membrane is a mechanically stable and insoluble cross-linked fibrous protein. Pseudomonas aeruginosa strain ME-4 synthesizes a metalloprotease that degrades the eggshell membrane. We cloned the encoding gene in Escherichia coli. The recombinant protease, over-expressed in E. coli, was inactive but addition of acetone to crude cell extracts restored the activity and removed many E. coli proteins. We purified the active, acetone-treated protease to homogeneity in a single chromatography step with 57% recovery. The recombinant protease partially hydrolyzed eggshell membrane and produced more soluble peptides and proteins than commercial elastase, α-chymotrypsin, and collagenase. The soluble peptides produced from hydrolyzed eggshell membrane inhibited angiotensin-I-converting enzyme activity. The degradation of eggshell membrane by the recombinant elastase could be applied to the production of soluble bioactive peptides. PMID:22286207

  14. 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.

  15. Simulation of the effect of confinement in actin ring formation

    NASA Astrophysics Data System (ADS)

    Adeli Koudehi, Maral; Vavylonis, Dimitrios; Haosu Tang Team; Dimitrios Vavylonis Team

    Actin filaments are vital for different network structures in living cells. During cytokinesis, they form a contractile ring containing myosin motor proteins and actin filament cross-linkers to separate one cell into two cells. Recent experimental studies have quantified the bundle, ring, and network structures that form when actin filaments polymerize in confined environments in vitro, in the presence of varying concentrations of cross-linkers. In this study, we performed numerical simulations to investigate the effect of actin spherical confinement and cross-linking in ring formation. We used a spring-bead model and Brownian dynamics to simulate semiflexible actin filaments that polymerize in a confining sphere with a rate proportional to the monomer concentration. Applying the model for different size of the confining spheres shows that the probability of ring formation decreases by increasing the radius (at fixed initial monomer concentration), in agreement with prior experimental data. We describe the effect of persistence length, orientation-dependent cross-linking, and initial actin monomer concentration. Simulations show that equilibrium configurations can be reached through zipping and unzipping of actin filaments in bundles and transient ring formation.

  16. Actinic reticuloid

    SciTech Connect

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

    1982-09-01

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

  17. Synthesis of borate cross-linked rhamnogalacturonan II

    PubMed Central

    Funakawa, Hiroya; Miwa, Kyoko

    2015-01-01

    In the present review, we describe current knowledge about synthesis of borate crosslinked rhamnogalacturonan II (RG-II) and it physiological roles. RG-II is a portion of pectic polysaccharide with high complexity, present in primary cell wall. It is composed of homogalacturonan backbone and four distinct side chains (A–D). Borate forms ester bonds with the apiosyl residues of side chain A of two RG-II monomers to generate borate dimerized RG-II, contributing for the formation of networks of pectic polysaccharides. In plant cell walls, more than 90% of RG-II are dimerized by borate under boron (B) sufficient conditions. Borate crosslinking of RG-II in primary cell walls, to our knowledge, is the only experimentally proven molecular function of B, an essential trace-element. Although abundance of RG-II and B is quite small in cell wall polysaccharides, increasing evidence supports that RG-II and its borate crosslinking are critical for plant growth and development. Significant advancement was made recently on the location and the mechanisms of RG-II synthesis and borate cross-linking. Molecular genetic studies have successfully identified key enzymes for RG-II synthesis and regulators including B transporters required for efficient formation of RG-II crosslinking and consequent normal plant growth. The present article focuses recent advances on (i) RG-II polysaccharide synthesis, (ii) occurrence of borate crosslinking and (iii) B transport for borate supply to RG-II. Molecular mechanisms underlying formation of borate RG-II crosslinking and the physiological impacts are discussed. PMID:25954281

  18. 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.

  19. Gelation of Covalently Cross-Linked PEG–Heparin Hydrogels

    PubMed Central

    Schultz, Kelly M.; Baldwin, Aaron D.; Kiick, Kristi L.; Furst, Eric M.

    2010-01-01

    We study PEG–heparin hydrogels to identify compositions that lead to gel formation and measure the corresponding gelation kinetics. The material consists of a maleimide-functionalized high molecular weight heparin (HMWH) backbone covalently cross-linked with bis-thiol poly(ethylene glycol) (PEG). Using multiple particle tracking microrheology, we investigate a broad composition space, defined by the number of maleimide functional sites per HMWH (f = 3.9–11.8), the molecular weight of the PEG cross-linker (Mn = 2000, 5000, and 10 000), and the concentrations of the heparin and PEG polymers. Gelation kinetics are characterized by time–cure superposition, yielding the gel time, tc, and the critical relaxation exponent, n. Gelation times range from 5 < tc ≤ 45 min, with the fastest kinetics occurring for the highest HMWH maleimide functionalities. tc depends nonmonotonically on the PEG cross-linker molecular weight, suggesting that gelation is affected by the length of the cross-linker relative to intermolecular interactions between heparin molecules. The critical relaxation exponent decreases from n = 0.52 for PEG 2000 to n = 0.39 for PEG 10 000. Finally, 219 equilibrated samples taken over the entire composition space are identified as liquid or solid, defining the “gelation envelope”. The boundaries of this empirical gelation envelope are in good agreement with Flory–Stockmayer theory. In all, microrheological measurements enable characterization over a large parameter space and provide crucial insight into the gelation of complex, multifunctional hydrogelators used in therapeutic applications. PMID:21494422

  20. Transepithelial corneal collagen cross-linking in ultrathin keratoconic corneas

    PubMed Central

    Spadea, Leopoldo; Mencucci, Rita

    2012-01-01

    Background The purpose of this paper was to report the results of transepithelial corneal collagen cross-linking (CXL) with modified riboflavin and ultraviolet A irradiation in patients affected by keratoconus, each with thinnest pachymetry values of less than 400 μm (with epithelium) and not treatable using standard de-epithelialization techniques. Methods Sixteen patients affected by progressive keratoconus with thinnest pachymetry values ranging from 331 μm to 389 μm underwent transepithelial CXL in one eye using a riboflavin 0.1% solution in 15% Dextran T500 containing ethylenediamine tetra-acetic acid 0.01% and trometamol to enhance epithelial penetration. The patients underwent complete ophthalmological examination, including endothelial cell density measurements and computerized videokeratography, before CXL and at one day, one week, and one, 6, and 12 months thereafter. Results Epithelial healing was complete in all patients after one day of use of a soft bandage contact lens. No side effects or damage to the limbal region was observed during the follow-up period. All patients showed slightly improved uncorrected and spectacle-corrected visual acuity; keratometric astigmatism showed reductions (up to 5.3 D) and apical ectasia power decreased (Kmax values reduced up to 4.3 D). Endothelial cell density was unchanged. Conclusion Application of transepithelial CXL using riboflavin with substances added to enhance epithelial permeability was safe, seemed to be moderately effective in keratoconic eyes with ultrathin corneas, and applications of the procedure could be extended to patients with advanced keratoconus. PMID:23152657

  1. SkfB Abstracts a Hydrogen Atom from Cα on SkfA To Initiate Thioether Cross-Link Formation.

    PubMed

    Bruender, Nathan A; Bandarian, Vahe

    2016-08-01

    Sulfur to α-carbon thioether-containing peptides (sactipeptides) are ribosomally synthesized post-translationally modified peptides with bacteriocidal activities. The thioether cross-link, which is required for biological activity, is installed by a member of the radical S-adenosyl-l-methionine (SAM) superfamily in the peptide substrate. Herein, we show that the radical SAM enzyme, SkfB, utilizes the 5'-deoxyadenosyl radical generated from the reductive cleavage of SAM to abstract a hydrogen atom from the α-carbon of the amino acid at position 12 in the substrate, SkfA, to initiate the installation of a thioether cross-link. The insights from this work can be applied to all radical SAM sactipeptide maturases. PMID:27410522

  2. Order-Disorder Transitions in Cross-Linked Block Copolymer Solids

    SciTech Connect

    Das, J.

    2005-01-12

    With a view toward creating solid block copolymers wherein the order-disorder transition can be accessed many times they investigated the nature of order-disorder transitions in cross-linked diblock copolymer melts using synergistic theory and experiment. A mean-field theory based on a coarse grained free-energy and the Random Phase Approximation (RPA) is developed for the system of interest. The quenched distribution of cross-links is averaged using the replica method. The phase behavior of a particular A-B block copolymer melt with a randomly cross-linked B-Block is determined as a function of the Florry-Huggins interaction parameter ({chi}) and the average number of cross-links per chain N{sub c}. They find for a cross-link density greater than N*{sub c} the B monomers are localized within a region of size {zeta} {approx} (N{sub c} - N*{sub c}){sup -1/2}. The cross-links strongly oppose ordering in the system as {zeta} becomes comparable to the radius of gyration of the block copolymer chain. As such the order-disorder transition temperature T{sub ODT} decreases precipitously when N{sub c} > N*{sub c}. When N{sub c} < N*{sub c}, T{sub ODT} increases weakly with N{sub c}. Experiments were conducted on cross-linked polystyrene-block-polyisoprene copolymer samples wherein the polyisoprene block was selectively cross-linked at a temperature well above the order-disorder transition temperature of the pure block copolymer. Small angle X-ray scattering (SAXS) and birefringence measurements on the cross-linked samples are consistent with the theoretical prediction. T{sub ODT} decreases rapidly when the cross-linking density exceeds the critical cross-linking density.

  3. Matching Cross-linked Peptide Spectra: Only as Good as the Worse Identification*

    PubMed Central

    Trnka, Michael J.; Baker, Peter R.; Robinson, Philip J. J.; Burlingame, A. L.; Chalkley, Robert J.

    2014-01-01

    Chemical cross-linking mass spectrometry identifies interacting surfaces within a protein assembly through labeling with bifunctional reagents and identifying the covalently modified peptides. These yield distance constraints that provide a powerful means to model the three-dimensional structure of the assembly. Bioinformatic analysis of cross-linked data resulting from large protein assemblies is challenging because each cross-linked product contains two covalently linked peptides, each of which must be correctly identified from a complex matrix of potential confounders. Protein Prospector addresses these issues through a complementary mass modification strategy in which each peptide is searched and identified separately. We demonstrate this strategy with an analysis of RNA polymerase II. False discovery rates (FDRs) are assessed via comparison of cross-linking data to crystal structure, as well as by using a decoy database strategy. Parameters that are most useful for positive identification of cross-linked spectra are explored. We find that fragmentation spectra generally contain more product ions from one of the two peptides constituting the cross-link. Hence, metrics reflecting the quality of the spectral match to the less confident peptide provide the most discriminatory power between correct and incorrect matches. A support vector machine model was built to further improve classification of cross-linked peptide hits. Furthermore, the frequency with which peptides cross-linked via common acylating reagents fragment to produce diagnostic, cross-linker-specific ions is assessed. The threshold for successful identification of the cross-linked peptide product depends upon the complexity of the sample under investigation. Protein Prospector, by focusing the reliability assessment on the least confident peptide, is better able to control the FDR for results as larger complexes and databases are analyzed. In addition, when FDR thresholds are calculated separately

  4. Regulation of DNA cross-link repair by the Fanconi anemia/BRCA pathway

    PubMed Central

    Kim, Hyungjin; D'Andrea, Alan D.

    2012-01-01

    The maintenance of genome stability is critical for survival, and its failure is often associated with tumorigenesis. The Fanconi anemia (FA) pathway is essential for the repair of DNA interstrand cross-links (ICLs), and a germline defect in the pathway results in FA, a cancer predisposition syndrome driven by genome instability. Central to this pathway is the monoubiquitination of FANCD2, which coordinates multiple DNA repair activities required for the resolution of ICLs. Recent studies have demonstrated how the FA pathway coordinates three critical DNA repair processes, including nucleolytic incision, translesion DNA synthesis (TLS), and homologous recombination (HR). Here, we review recent advances in our understanding of the downstream ICL repair steps initiated by ubiquitin-mediated FA pathway activation. PMID:22751496

  5. Lamin A/C and emerin regulate MKL1-SRF activity by modulating actin dynamics.

    PubMed

    Ho, Chin Yee; Jaalouk, Diana E; Vartiainen, Maria K; Lammerding, Jan

    2013-05-23

    Laminopathies, caused by mutations in the LMNA gene encoding the nuclear envelope proteins lamins A and C, represent a diverse group of diseases that include Emery-Dreifuss muscular dystrophy (EDMD), dilated cardiomyopathy (DCM), limb-girdle muscular dystrophy, and Hutchison-Gilford progeria syndrome. Most LMNA mutations affect skeletal and cardiac muscle by mechanisms that remain incompletely understood. Loss of structural function and altered interaction of mutant lamins with (tissue-specific) transcription factors have been proposed to explain the tissue-specific phenotypes. Here we report in mice that lamin-A/C-deficient (Lmna(-/-)) and Lmna(N195K/N195K) mutant cells have impaired nuclear translocation and downstream signalling of the mechanosensitive transcription factor megakaryoblastic leukaemia 1 (MKL1), a myocardin family member that is pivotal in cardiac development and function. Altered nucleo-cytoplasmic shuttling of MKL1 was caused by altered actin dynamics in Lmna(-/-) and Lmna(N195K/N195K) mutant cells. Ectopic expression of the nuclear envelope protein emerin, which is mislocalized in Lmna mutant cells and also linked to EDMD and DCM, restored MKL1 nuclear translocation and rescued actin dynamics in mutant cells. These findings present a novel mechanism that could provide insight into the disease aetiology for the cardiac phenotype in many laminopathies, whereby lamin A/C and emerin regulate gene expression through modulation of nuclear and cytoskeletal actin polymerization. PMID:23644458

  6. Lamin A/C and emerin regulate MKL1/SRF activity by modulating actin dynamics

    PubMed Central

    Ho, Chin Yee; Jaalouk, Diana E.; Vartiainen, Maria K.; Lammerding, Jan

    2013-01-01

    Laminopathies, caused by mutations in the LMNA gene encoding the nuclear envelope proteins lamins A and C, represent a diverse group of diseases that include Emery-Dreifuss Muscular Dystrophy (EDMD), dilated cardiomyopathy (DCM), limb-girdle muscular dystrophy, and Hutchison-Gilford progeria syndrome (HGPS).1 The majority of LMNA mutations affect skeletal and cardiac muscle by mechanisms that remain incompletely understood. Loss of structural function and disturbed interaction of mutant lamins with (tissue-specific) transcription factors have been proposed to explain the tissue-specific phenotypes.1 We report here that lamin A/C-deficient (Lmna−/−) and Lmna N195K mutant cells have impaired nuclear translocation and downstream signaling of the mechanosensitive transcription factor megakaryoblastic leukaemia 1 (MKL1), a myocardin family member that is pivotal in cardiac development and function.2 Disturbed nucleo-cytoplasmic shuttling of MKL1 was caused by altered actin dynamics in Lmna−/− and N195K mutant cells. Ectopic expression of the nuclear envelope protein emerin, which is mislocalized in Lmna mutant cells and also linked to EDMD and DCM, restored MKL1 nuclear translocation and rescued actin dynamics in mutant cells. These findings present a novel mechanism that could provide insight into the disease etiology for the cardiac phenotype in many laminopathies, whereby lamins A/C and emerin regulate gene expression through modulation of nuclear and cytoskeletal actin polymerization. PMID:23644458

  7. Isoforms of α-Actinin from Cardiac, Smooth, and Skeletal Muscle Form Polar Arrays of Actin Filaments

    PubMed Central

    Taylor, Kenneth A.; Taylor, Dianne W.; Schachat, Fred

    2000-01-01

    We have used a positively charged lipid monolayer to form two-dimensional bundles of F-actin cross-linked by α-actinin to investigate the relative orientation of the actin filaments within them. This method prevents growth of the bundles perpendicular to the monolayer plane, thereby facilitating interpretation of the electron micrographs. Using α-actinin isoforms isolated from the three types of vertebrate muscle, i.e., cardiac, skeletal, and smooth, we have observed almost exclusively cross-linking between polar arrays of filaments, i.e., actin filaments with their plus ends oriented in the same direction. One type of bundle can be classified as an Archimedian spiral consisting of a single actin filament that spirals inward as the filament grows and the bundle is formed. These spirals have a consistent hand and grow to a limiting internal diameter of 0.4–0.7 μm, where the filaments appear to break and spiral formation ceases. These results, using isoforms usually characterized as cross-linkers of bipolar actin filament bundles, suggest that α-actinin is capable of cross-linking actin filaments in any orientation. Formation of specifically bipolar or polar filament arrays cross-linked by α-actinin may require additional factors that either determine the filament orientation or restrict the cross-linking capabilities of α-actinin. PMID:10791977

  8. Glycosylation-Dependent IFN-γR Partitioning in Lipid and Actin Nanodomains Is Critical for JAK Activation.

    PubMed

    Blouin, Cédric M; Hamon, Yannick; Gonnord, Pauline; Boularan, Cédric; Kagan, Jérémy; Viaris de Lesegno, Christine; Ruez, Richard; Mailfert, Sébastien; Bertaux, Nicolas; Loew, Damarys; Wunder, Christian; Johannes, Ludger; Vogt, Guillaume; Contreras, Francesc-Xabier; Marguet, Didier; Casanova, Jean-Laurent; Galès, Céline; He, Hai-Tao; Lamaze, Christophe

    2016-08-11

    Understanding how membrane nanoscale organization controls transmembrane receptors signaling activity remains a challenge. We studied interferon-γ receptor (IFN-γR) signaling in fibroblasts from homozygous patients with a T168N mutation in IFNGR2. By adding a neo-N-glycan on IFN-γR2 subunit, this mutation blocks IFN-γ activity by unknown mechanisms. We show that the lateral diffusion of IFN-γR2 is confined by sphingolipid/cholesterol nanodomains. In contrast, the IFN-γR2 T168N mutant diffusion is confined by distinct actin nanodomains where conformational changes required for Janus-activated tyrosine kinase/signal transducer and activator of transcription (JAK/STAT) activation by IFN-γ could not occur. Removing IFN-γR2 T168N-bound galectins restored lateral diffusion in lipid nanodomains and JAK/STAT signaling in patient cells, whereas adding galectins impaired these processes in control cells. These experiments prove the critical role of dynamic receptor interactions with actin and lipid nanodomains and reveal a new function for receptor glycosylation and galectins. Our study establishes the physiological relevance of membrane nanodomains in the control of transmembrane receptor signaling in vivo. VIDEO ABSTRACT. PMID:27499022

  9. Demonstration of prominent actin filaments in the root columella

    NASA Technical Reports Server (NTRS)

    Collings, D. A.; Zsuppan, G.; Allen, N. S.; Blancaflor, E. B.; Brown, C. S. (Principal Investigator)

    2001-01-01

    The distribution of actin filaments within the gravity-sensing columella cells of plant roots remains poorly understood, with studies over numerous years providing inconsistent descriptions of actin organization in these cells. This uncertainty in actin organization, and thus in actin's role in graviperception and gravisignaling, has led us to investigate actin arrangements in the columella cells of Zea mays L., Medicago truncatula Gaertn., Linum usitatissiilium L. and Nicotianla benthamiana Domin. Actin organization was examined using a combination of optimized immunofluorescence techniques, and an improved fluorochrome-conjugated phalloidin labeling method reliant on 3-maleimidobenzoyl-N-hydroxy-succinimide ester (MBS) cross-linking combined with glycerol permeabilization. Confocal microscopy of root sections labeled with anti-actin antibodies revealed patterns suggestive of actin throughout the columella region. These patterns included short and fragmented actin bundles, fluorescent rings around amyloplasts and intense fluorescence originating from the nucleus. Additionally, confocal microscopy of MBS-stabilized and Alexa Fluor-phalloidin-labeled root sections revealed a previously undetected state of actin organization in the columella. Discrete actin structures surrounded the amyloplasts and prominent actin cables radiated from the nuclear surface toward the cell periphery. Furthermore, the cortex of the columella cells contained fine actin bundles (or single filaments) that had a predominant transverse orientation. We also used confocal microscopy of plant roots expressing endoplasmic reticulum (ER)-targeted green fluorescent protein to demonstrate rapid ER movements within the columella cells, suggesting that the imaged actin network is functional. The successful identification of discrete actin structures in the root columella cells forms the perception and signaling.

  10. Luteinizing Hormone Receptor-Stimulated Progesterone Production by Preovulatory Granulosa Cells Requires Protein Kinase A-Dependent Activation/Dephosphorylation of the Actin Dynamizing Protein Cofilin

    PubMed Central

    Karlsson, Amelia B.; Maizels, Evelyn T.; Flynn, Maxfield P.; Jones, Jonathan C.; Shelden, Eric A.; Bamburg, James R.; Hunzicker-Dunn, Mary

    2010-01-01

    Activation of the LH receptor (LHR) on preovulatory granulosa cells stimulates the cAMP/protein kinase A (PKA) pathway to regulate expression of genes required for ovulation and luteinization. LHR signaling also initiates rearrangement of the actin cytoskeleton. Because disruption of the actin cytoskeleton has been causally linked to steroidogenesis in various cell models, we sought to identify the cellular mechanisms that may modulate reorganization of the actin cytoskeleton and to determine whether cytoskeletal reorganization is required for steroidogenesis. Herein we report that LHR signaling in preovulatory granulosa cells promotes rapid dephosphorylation of the actin-depolymerizing factor cofilin at Ser3 that is dependent on PKA. The LHR-stimulated dephosphorylation of cofilin(Ser3) switches on cofilin activity to bind actin filaments and enhance their dynamics. Basal phosphorylation of cofilin(Ser3) is mediated by active/GTP-bound Rho and downstream protein kinases; LHR signaling promotes a decrease in active/GTP-bound Rho by a PKA-dependent mechanism. LHR-dependent Rho inactivation and subsequent activation of cofilin does not involve ERK, epidermal growth factor receptor, or phosphatidylinositol 3-kinase pathways downstream of PKA. To understand the biological significance of cofilin activation, preovulatory granulosa cells were transduced with a mutant cofilin adenoviral vector in which Ser3 was mutated to Glu (S-E cofilin). Inactive S-E cofilin abolished LHR-mediated reorganization of the actin cytoskeleton and caused a 70% decrease in LHR-stimulated progesterone that is obligatory for ovulation. Taken together, these results show that LHR signaling via PKA activates a cofilin-regulated rearrangement of the actin cytoskeleton and that active cofilin is required to initiate progesterone secretion by preovulatory granulosa cells. PMID:20610540

  11. Characterization of Aziridinylbenzoquinone DNA Cross-links by LC-IRMPD-MS

    PubMed Central

    Pierce, Sarah E.; Guziec, Lynn J.; Guziec, Frank S.; Brodbelt, Jennifer S.

    2010-01-01

    DNA cross-linking was evaluated by LC-MS/MS to determine the relative cross-linking abilities of two aziridinylbenzoquinones. Reactivities of RH1 (2,5-diaziridinyl-3-[hydroxymethyl]-6-methyl-1,4-benzoquinone), a clinically studied anti-tumor cross-linking agent, and an analog containing a phenyl group (PhRH1) rather than a methyl group was compared. The bulky phenyl substituent was added to determine the impact of steric hindrance on the formation of cross-links within a double helical structure. Cross-links formed by RH1 and PhRH1 (2,5-diaziridinyl-3-[hydroxymethyl]-6-phenyl-1,4-benzoquinone) were observed at 5’-dGNC sites as well as 5’-dGAAC/dGTTC sites. RH1 was more effective at forming cross-links than PhRH1 for a variety of duplexes. Infrared multiphoton dissociation (IRMPD) and collision induced dissociation (CID) results confirmed the presence and the location of the cross-links within the duplexes, and IRMPD was used to identify the dissociation pathways of the cross-linked duplexes. PMID:20369834

  12. 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...

  13. Cross-Linking Electrochemical Mass Spectrometry for Probing Protein Three-Dimensional Structures

    PubMed Central

    2015-01-01

    Chemical cross-linking combined with mass spectrometry (MS) is powerful to provide protein three-dimensional structure information but difficulties in identifying cross-linked peptides and elucidating their structures limit its usefulness. To tackle these challenges, this study presents a novel cross-linking MS in conjunction with electrochemistry using disulfide-bond-containing dithiobis[succinimidyl propionate] (DSP) as the cross-linker. In our approach, electrolysis of DSP-bridged protein/peptide products, as online monitored by desorption electrospray ionization mass spectrometry is highly informative. First, as disulfide bonds are electrochemically reducible, the cross-links are subject to pronounced intensity decrease upon electrolytic reduction, suggesting a new way to identify cross-links. Also, mass shift before and after electrolysis suggests the linkage pattern of cross-links. Electrochemical reduction removes disulfide bond constraints, possibly increasing sequence coverage for tandem MS analysis and yielding linear peptides whose structures are more easily determined than their cross-linked precursor peptides. Furthermore, this cross-linking electrochemical MS method is rapid, due to the fast nature of electrochemical conversion (much faster than traditional chemical reduction) and no need for chromatographic separation, which would be of high value for structural proteomics research. PMID:25141260

  14. Lamb and Cow Performance when Fed Corn Silage that has Reduced Ferulate Cross Linking

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Ferulate-mediated lignin/hemicellulose cross linking in grasses reduces in vitro NDF digestibility (IVNDFD). Impact of ferulate cross linking on animal performance was examined in lamb digestibility and dairy cow performance trials using the seedling ferulate ester (sfe) corn mutant that reduces cro...

  15. Electrospun zein fibers using glutaraldehyde as the cross-linking reagent

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Glutaraldehyde was used as a cross-linking reagent for zein (corn protein) to provide fibers with improved physical properties and solvent resistance. Glutaraldehyde was used at levels between 2 - 8%. The cross-linking reaction was carried out in acetic acid for twenty hours at room temperature. ...

  16. Electrospun zein fibers using glyoxal or formaldehyde as the cross-linking reagent

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Glyoxal or formaldehyde was used as a cross-linking reagent for zein (corn protein) to provide electrospun fibers with improved physical properties and solvent resistance. These reagents were used between 2 and 6%. The cross-linking reaction was carried out in acetic acid for various lengths of ti...

  17. 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...

  18. B cell receptor cross-linking triggers a caspase-8-dependent apoptotic pathway that is independent of the death effector domain of Fas-associated death domain protein.

    PubMed

    Besnault, L; Schrantz, N; Auffredou, M T; Leca, G; Bourgeade, M F; Vazquez, A

    2001-07-15

    We have previously reported that B cell receptors, depending on the degree to which they are cross-linked, can promote apoptosis in various human B cell types. In this study, we show that B cell receptors can trigger two apoptotic pathways according to cross-linking and that these pathways control mitochondrial activation in human Burkitt's lymphoma cells. Whereas soluble anti-mu Ab triggers caspase-independent mitochondrial activation, cross-linked anti-mu Ab induces an apoptotic response associated with a caspase-dependent loss of mitochondrial transmembrane potential. This B cell receptor-mediated caspase-dependent mitochondrial activation is associated with caspase-8 activation. We show here that caspase-8 inhibitors strongly decrease cross-linking-dependent B cell receptor-mediated apoptosis in Burkitt's lymphoma BL41 cells. These inhibitors act upstream from the mitochondria as they prevented the loss of mitochondrial membrane potential observed in B cell receptor-treated BL41 cells. Caspase-8 activation in these cells was also evident from the detection of cleaved fragments of caspase-8 and the cleavage of specific substrates, including Bid. Our data show that cross-linked B cell receptors induced an apoptotic pathway involving sequential caspase-8 activation, loss of mitochondrial membrane potential, and the activation of caspase-9 and caspase-3. Cells expressing a dominant negative mutant of Fas-associated death domain protein were sensitive to cross-linked B cell receptor-induced caspase-8 activation and apoptosis; therefore, this caspase-8 activation was independent of the death effector domain of Fas-associated death domain protein. PMID:11441077

  19. 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

    2016-05-01

    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. PMID:27033731

  20. 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

  1. 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

  2. Cross-Linking the Surface of Cured Polydimethylsiloxane via Hyperthemal Hydrogen Projectile Bombardment.

    PubMed

    Bao, Chao; Xu, Ke-Qin; Tang, Chang-Yu; Lau, Woon-ming; Yin, Cong-Bin; Zhu, Yan; Mei, Jun; Lee, Jonathan; Hui, David; Nie, Heng-Yong; Liu, Yu

    2015-04-29

    Cross-linking of polydimethylsiloxane (PDMS) is increasingly important with recent focus on its top surface stiffness. In this paper, we demonstrate that hyperthermal hydrogen projectile bombardment, a surface sensitive cross-linking technology, is superior in enhancing the mechanical properties of a cured PDMS surface without significantly degrading its hydrophobicity. Both water contact angle measurements and time-of-flight secondary ion mass spectrometry are used to investigate the variations in surface chemistry and structure upon cross-linking. Using nanoindentation and atomic force microscopy, we confirm that the thickness of the cross-linked PDMS is controllable by the bombardment time, which opens opportunities for tuning cross-linking degree in compliance with arising requirements from the practice. PMID:25849306

  3. 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.

  4. Microtubule-dependent transport of vimentin filament precursors is regulated by actin and by the concerted action of Rho- and p21-activated kinases

    PubMed Central

    Robert, Amélie; Herrmann, Harald; Davidson, Michael W.; Gelfand, Vladimir I.

    2014-01-01

    Intermediate filaments (IFs) form a dense and dynamic network that is functionally associated with microtubules and actin filaments. We used the GFP-tagged vimentin mutant Y117L to study vimentin-cytoskeletal interactions and transport of vimentin filament precursors. This mutant preserves vimentin interaction with other components of the cytoskeleton, but its assembly is blocked at the unit-length filament (ULF) stage. ULFs are easy to track, and they allow a reliable and quantifiable analysis of movement. Our results show that in cultured human vimentin-negative SW13 cells, 2% of vimentin-ULFs move along microtubules bidirectionally, while the majority are stationary and tightly associated with actin filaments. Rapid motor-dependent transport of ULFs along microtubules is enhanced ≥5-fold by depolymerization of actin cytoskeleton with latrunculin B. The microtubule-dependent transport of vimentin ULFs is further regulated by Rho-kinase (ROCK) and p21-activated kinase (PAK): ROCK inhibits ULF transport, while PAK stimulates it. Both kinases act on microtubule transport independently of their effects on actin cytoskeleton. Our study demonstrates the importance of the actin cytoskeleton to restrict IF transport and reveals a new role for PAK and ROCK in the regulation of IF precursor transport.—Robert, A., Herrmann, H., Davidson, M. W., and Gelfand, V. I. Microtubule-dependent transport of vimentin filament precursors is regulated by actin and by the concerted action of Rho- and p21-activated kinases. PMID:24652946

  5. A New in Vivo Cross-linking Mass Spectrometry Platform to Define Protein–Protein Interactions in Living Cells*

    PubMed Central

    Kaake, Robyn M.; Wang, Xiaorong; Burke, Anthony; Yu, Clinton; Kandur, Wynne; Yang, Yingying; Novtisky, Eric J.; Second, Tonya; Duan, Jicheng; Kao, Athit; Guan, Shenheng; Vellucci, Danielle; Rychnovsky, Scott D.; Huang, Lan

    2014-01-01

    Protein–protein interactions (PPIs) are fundamental to the structure and function of protein complexes. Resolving the physical contacts between proteins as they occur in cells is critical to uncovering the molecular details underlying various cellular activities. To advance the study of PPIs in living cells, we have developed a new in vivo cross-linking mass spectrometry platform that couples a novel membrane-permeable, enrichable, and MS-cleavable cross-linker with multistage tandem mass spectrometry. This strategy permits the effective capture, enrichment, and identification of in vivo cross-linked products from mammalian cells and thus enables the determination of protein interaction interfaces. The utility of the developed method has been demonstrated by profiling PPIs in mammalian cells at the proteome scale and the targeted protein complex level. Our work represents a general approach for studying in vivo PPIs and provides a solid foundation for future studies toward the complete mapping of PPI networks in living systems. PMID:25253489

  6. 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. PMID:26716873

  7. Constrained peptides with target-adapted cross-links as inhibitors of a pathogenic protein-protein interaction.

    PubMed

    Glas, Adrian; Bier, David; Hahne, Gernot; Rademacher, Christoph; Ottmann, Christian; Grossmann, Tom N

    2014-02-24

    Bioactive conformations of peptides can be stabilized by macrocyclization, resulting in increased target affinity and activity. Such macrocyclic peptides proved useful as modulators of biological functions, in particular as inhibitors of protein-protein interactions (PPI). However, most peptide-derived PPI inhibitors involve stabilized α-helices, leaving a large number of secondary structures unaddressed. Herein, we present a rational approach towards stabilization of an irregular peptide structure, using hydrophobic cross-links that replace residues crucially involved in target binding. The molecular basis of this interaction was elucidated by X-ray crystallography and isothermal titration calorimetry. The resulting cross-linked peptides inhibit the interaction between human adaptor protein 14-3-3 and virulence factor exoenzyme S. Taking into consideration that irregular peptide structures participate widely in PPIs, this approach provides access to novel peptide-derived inhibitors. PMID:24504455

  8. UV cross-linking identifies four polypeptides that require the TATA box to bind to the Drosophila hsp70 promoter

    SciTech Connect

    Gilmour, D.S.; Dietz, T.J.; Elgin, S.C. )

    1990-08-01

    A protein fraction that requires the TATA sequence to bind to the hsp70 promoter has been partially purified from nuclear extracts of Drosophila embryos. This TATA factor produces a large DNase I footprint that extends from -44 to +35 on the promoter. A mutation that changes TATA to TATG interferes both with the binding of this complex and with the transcription of the hsp70 promoter in vitro, indicating that this interaction is important for transcriptional activity. Using a highly specific protein-DNA cross-linking assay, we have identified four polypeptides that require the TATA sequence to bind to the hsp70 promoter. Polypeptides of 26 and 42 kilodaltons are in intimate contact with the TATA sequence. Polypeptides of 150 and 60 kilodaltons interact within the region from +24 to +47 in a TATA-dependent manner. Both the extended footprint and the polypeptides identified by UV cross-linking indicate that the Drosophila TATA factor is a multicomponent complex.

  9. Identification of glucose-derived cross-linking sites in ribonuclease A.

    PubMed

    Dai, Zhenyu; Wang, Benlian; Sun, Gang; Fan, Xingjun; Anderson, Vernon E; Monnier, Vincent M

    2008-07-01

    The accumulation of glycation derived cross-links has been widely implicated in extracellular matrix damage in aging and diabetes, yet little information is available on the cross-linking sites in proteins and the intra- versus intermolecular character of cross-linking. Recently, glucosepane, a 7-membered heterocycle formed between lysine and arginine residues, has been found to be the single major cross-link known so far to accumulate during aging. As an approach toward identification of glucose derived cross-linking sites, we have preglycated ribonuclease A first for for 14 days with 500 mM glucose, followed by a 4-week incubation in absence of glucose. MALDI-TOF analysis of tryptic digests revealed the presence of Amadori products (Delta m/ z = 162) at K1, K7, K37 and K41, in accordance with previous studies. In addition, K66, K98 and K104 were also modified by Amadori products. Intramolecular glucosepane cross-links were observed at K41-R39 and K98-R85. Surprisingly, the only intermolecular cross-link observed was the 3-deoxyglucosone-derived DODIC at K1-R39. The identity of cross-linked peptides was confirmed by sequencing with tandem mass spectrometry. Recombinant ribonuclease A mutants R39A, R85A, and K91A were produced, purified, and glycated to further confirm the importance of these sites on protein cross-linking. These data provide the first documentation that both intramolecular and intermolecular cross-links form in glucose-incubated proteins. PMID:18500835

  10. Identification of Glucose-Derived Cross-Linking Sites in Ribonuclease A

    PubMed Central

    Dai, Zhenyu; Wang, Benlian; Sun, Gang; Fan, Xingjun; Anderson, Vernon E.; Monnier, Vincent M.

    2008-01-01

    The accumulation of glycation derived cross-links has been widely implicated in extracellular matrix damage in aging and diabetes, yet little information is available on the cross-linking sites in proteins and the intra- versus intermolecular character of cross-linking. Recently, glucosepane, a 7-membered heterocycle formed between lysine and arginine residues, has been found to be the single major cross-link known so far to accumulate during aging. As an approach toward identification of glucose derived cross-linking sites, we have preglycated ribonuclease A first for for 14 days with 500 mM glucose, followed by a 4-week incubation in absence of glucose. MALDI-TOF analysis of tryptic digests revealed the presence of Amadori products (Δm/z = 162) at K1, K7, K37 and K41, in accordance with previous studies. In addition, K66, K98 and K104 were also modified by Amadori products. Intramolecular glucosepane cross-links were observed at K41-R39 and K98-R85. Surprisingly, the only intermolecular cross-link observed was the 3-deoxyglucosone-derived DODIC at K1-R39. The identity of cross-linked peptides was confirmed by sequencing with tandem mass spectrometry. Recombinant ribonuclease A mutants R39A, R85A, and K91A were produced, purified, and glycated to further confirm the importance of these sites on protein cross-linking. These data provide the first documentation that both intramolecular and intermolecular cross-links form in glucose-incubated proteins. PMID:18500835

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

    PubMed Central

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

    2016-01-01

    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. PMID:26719564

  12. Intranuclear Actin Regulates Osteogenesis

    PubMed Central

    Sen, Buer; Xie, Zhihui; Uzer, Gunes; Thompson, William R.; Styner, Maya; Wu, Xin; Rubin, Janet

    2016-01-01

    Depolymerization of the actin cytoskeleton induces nuclear trafficking of regulatory proteins and global effects on gene transcription. We here show that in mesenchymal stem cells (MSCs), cytochalasin D treatment causes rapid cofilin-/importin-9-dependent transfer of G-actin into the nucleus. The continued presence of intranuclear actin, which forms rod-like structures that stain with phalloidin, is associated with induction of robust expression of the osteogenic genes osterix and osteocalcin in a Runx2-dependent manner, and leads to acquisition of osteogenic phenotype. Adipogenic differentiation also occurs, but to a lesser degree. Intranuclear actin leads to nuclear export of Yes-associated protein (YAP); maintenance of nuclear YAP inhibits Runx2 initiation of osteogenesis. Injection of cytochalasin into the tibial marrow space of live mice results in abundant bone formation within the space of 1 week. In sum, increased intranuclear actin forces MSC into osteogenic lineage through controlling Runx2 activity; this process may be useful for clinical objectives of forming bone. PMID:26140478

  13. 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

  14. Broadening the Spectrum of Actin-Based Protrusive Activity Mediated by Arp2/3 Complex-Facilitated Polymerization: Motility of Cytoplasmic Ridges and Tubular Projections

    PubMed Central

    Henson, John H.; Gianakas, Anastasia D.; Henson, Lauren H.; Lakin, Christina L.; Voss, Meagen K.; Bewersdorf, Joerg; Oldenbourg, Rudolf; Morris, Robert L.

    2014-01-01

    Arp2/3 complex-facilitated actin polymerization plays an essential role in a variety of cellular functions including motility, adherence, endocytosis and trafficking. In the present study we employ the sea urchin coelomocyte experimental model system to test the hypotheses that Arp2/3 complex-nucleated actin assembly mediates the motility of two unusual cellular protrusions; the cytoplasmic ridges present during coelomocyte spreading, and inducible, tubular-shaped, and neurite-like projections. Our investigations couple pharmacological manipulation employing inhibitors of actin polymerization and the Arp2/3 complex with a wide array of imaging methods including digitally enhanced phase contrast, DIC and polarization light microscopy of live cells; conventional, confocal and super-resolution light microscopy of fluorescently labeled cells; and scanning and transmission electron microscopy. Taken together, the results of this study indicate that Arp2/3 complex-facilitated actin polymerization underlies the motility of coelomocyte cytoplasmic ridges and tubular projections, that these processes are related to each other, and that they have been preliminarily identified in other cell types. The results also highlight the broad spectrum of actin-based protrusive activities dependent on the Arp2/3 complex and provide additional insights into the pervasive nature of this ubiquitous actin nucleator. Furthermore we provide the first evidence of a possible mechanistic difference between the impacts of the small molecule drugs BDM and CK666 on the Arp2/3 complex. PMID:25111797

  15. Dynamic Regulation of Activated Leukocyte Cell Adhesion Molecule–mediated Homotypic Cell Adhesion through the Actin CytoskeletonV⃞

    PubMed Central

    Nelissen, Judith M. D. T.; Peters, Inge M.; de Grooth, Bart G.; van Kooyk, Yvette; Figdor, Carl G.

    2000-01-01

    Restricted expression of activated leukocyte cell adhesion molecule (ALCAM) by hematopoietic cells suggests an important role in the immune system and hematopoiesis. To get insight into the mechanisms that control ALCAM-mediated adhesion we have investigated homotypic ALCAM–ALCAM interactions. Here, we demonstrate that the cytoskeleton regulates ALCAM-mediated cell adhesion because inhibition of actin polymerization by cytochalasin D (CytD) strongly induces homotypic ALCAM–ALCAM interactions. This induction of cell adhesion is likely due to clustering of ALCAM at the cell surface, which is observed after CytD treatment. Single-particle tracking demonstrated that the lateral mobility of ALCAM in the cell membrane is increased 30-fold after CytD treatment. In contrast, both surface distribution and adhesion of a glycosylphosphatidylinositol (GPI)-anchored ALCAM mutant are insensitive to CytD, despite the increase in lateral mobility of GPI-ALCAM upon CytD treatment. This demonstrates that clustering of ALCAM is essential for cell adhesion, whereas enhanced diffusion of ALCAM alone is not sufficient for cluster formation. In addition, upon ligand binding, both free diffusion and the freely dragged distance of wild-type ALCAM, but not of GPI-ALCAM, are reduced over time, suggesting strengthening of the cytoskeleton linkage. From these findings we conclude that activation of ALCAM-mediated adhesion is dynamically regulated through actin cytoskeleton-dependent clustering. PMID:10848629

  16. Collagen cross-link metabolites in urine as markers of bone metastases in prostatic carcinoma.

    PubMed

    Miyamoto, K K; McSherry, S A; Robins, S P; Besterman, J M; Mohler, J L

    1994-04-01

    The efficacy of radionuclide bone scans in monitoring metastatic bone activity remains controversial. Objective measurement of bone tumor burden would be useful for the evaluation of new therapies for metastatic carcinoma of the prostate. The recent discovery of the urinary excretion of pyridinoline (cross-link of mature collagen found in cartilage and bone) and deoxypyridinoline (collagen cross-link specific to bone) measured by high pressure liquid chromatography has provided sensitive specific indexes of cartilage and bone breakdown in rheumatoid arthritis, osteoporosis and metabolic bone diseases. We compared the urinary excretion of deoxypyridinoline,pyridinoline and hydroxyproline relative to urinary creatinine (nmol./mmol.creatinine) in 27 patients with benign prostatic hyperplasia (patient age 70.0 +/- 8.5 years, standard deviation), 29 with clinically confined prostate cancer (age 70.2 +/- 9.7 years), and 26 with prostate cancer and bone metastases (age 71.1 +/- 7.7 years). No diurnal variation of deoxypyridinoline or pyridinoline urinary excretion was detected in 5 patients with metastases. Urinary excretion of pyridinoline and deoxypyridinoline was significantly greater in patients with metastatic carcinoma of the prostate compared with patients with either benign prostatic hyperplasia (Mann-Whitney-Wilcoxon rank sum analysis, p < 0.00004 and 0.002, respectively) or localized prostate cancer (Mann-Whitney-Wilcoxon, p < 0.00001 and 0.00005, respectively). Urinary hydroxyproline levels failed to separate the 3 groups. Pyridinoline and deoxypyridinoline excretion in prostate cancer patients with metastases directly correlated with bone scan Soloway scores (r = 0.55, p < 0.005 and r = 0.57, p < 0.004 respectively), whereas serum prostate specific antigen did not (r = 0.36, p = 0.08). Serial measurements of pyridinoline and deoxypyridinoline progressively increased in 3 patients with clinical progression documented by new metastatic lesions by bone scan

  17. Cross-linking and 1H n.m.r. spectroscopy of the pyruvate dehydrogenase complex of Escherichia coli

    PubMed Central

    Packman, Leonard C.; Perham, Richard N.; Roberts, Gordon C. K.

    1982-01-01

    The pyruvate dehydrogenase complex of Escherichia coli was treated with o-phenylene bismaleimide in the presence of the substrate pyruvate, producing almost complete cross-linking of the lipoate acetyltransferase polypeptide chains as judged by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. This took place without effect on the catalytic activities of the other two component enzymes and with little evidence of cross-links being formed with other types of protein subunit. Limited proteolysis with trypsin indicated that the cross-links were largely confined to the lipoyl domains of the lipoate acetyltransferase component of the same enzyme particle. This intramolecular cross-linking had no effect on the very sharp resonances observed in the 1H n.m.r. spectrum of the enzyme complex, which derive from regions of highly mobile polypeptide chain in the lipoyl domains. Comparison of the spin–spin relaxation times, T2, with the measured linewidths supported the idea that the highly mobile region is best characterized as a random coil. Intensity measurements in spin-echo spectra showed that it comprises a significant proportion (probably not less than one-third) of a lipoyl domain and is thus much more than a small hinge region, but there was insufficient intensity in the resonances to account for the whole lipoyl domain. On the other hand, no evidence was found in the 1H n.m.r. spectrum for a substantial structured region around the lipoyl-lysine residues that was free to move on the end of this highly flexible connection. If such a structured region were bound to other parts of the enzyme complex for a major part of its time, its resonances might be broadened sufficiently to evade detection by 1H n.m.r. spectroscopy. ImagesFig. 2.Fig. 3. PMID:6753833

  18. 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. PMID:27342729

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

    PubMed

    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

    2014-12-16

    Proanthocyanidins (PACs) are secondary plant metabolites that mediate nonenzymatic 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: PAC 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 combining 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 the 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 modulates the mechanism of cross-linking at the molecular, intermolecular, and inter-microfibrillar levels. PMID:25379878

  20. 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

  1. Proteome-wide profiling of protein assemblies by cross-linking mass spectrometry.

    PubMed

    Liu, Fan; Rijkers, Dirk T S; Post, Harm; Heck, Albert J R

    2015-12-01

    We describe an integrated workflow that robustly identifies cross-links from endogenous protein complexes in human cellular lysates. Our approach is based on the application of mass spectrometry (MS)-cleavable cross-linkers, sequential collision-induced dissociation (CID)-tandem MS (MS/MS) and electron-transfer dissociation (ETD)-MS/MS acquisitions, and a dedicated search engine, XlinkX, which allows rapid cross-link identification against a complete human proteome database. This approach allowed us to detect 2,179 unique cross-links (1,665 intraprotein cross-links at a 5% false discovery rate (FDR) and 514 interprotein cross-links at 1% FDR) in HeLa cell lysates. We validated the confidence of our cross-linking results by using a target-decoy strategy and mapping the observed cross-link distances onto existing high-resolution structures. Our data provided new structural information about many protein assemblies and captured dynamic interactions of the ribosome in contact with different elongation factors. PMID:26414014

  2. 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. PMID:25478965

  3. Controlled degradation of hydrogels using multi-functional cross-linking molecules.

    PubMed

    Lee, Kuen Yong; Bouhadir, Kamal H; Mooney, David J

    2004-06-01

    Hydrogels, chemically cross-linked or physically entangled, have found a number of applications as novel delivery vehicles of drugs and cells. However, the narrow ranges of degradation rates and mechanical strength currently available from many hydrogels limits their applications. We have hypothesized that utilization of multi-functional cross-linking molecules to form hydrogels could provide a wider range and tighter control over the degradation rates and mechanical stiffness of gels than bi-functional cross-linking molecules. To address the possibility, we isolated alpha-L-guluronate residues of sodium alginate, and oxidized them to prepare poly(aldehyde guluronate) (PAG). Hydrogels were formed with either poly(acrylamide-co-hydrazide) (PAH) as a multi-functional cross-linking molecule or adipic acid dihydrazide (AAD) as a bi-functional cross-linking molecule. The initial properties and degradation behavior of both PAG gel types were monitored. PAG/PAH hydrogels showed higher mechanical stiffness before degradation and degraded more slowly than PAG/AAD gels, at the same concentration of cross-linking functional groups. The enhanced mechanical stiffness and prolonged degradation behavior could be attributed to the multiple attachment points of PAH in the gel at the same concentration of functional groups. This approach to regulating gel properties with multifunctional cross-linking molecules could be broadly used in hydrogels. PMID:14751730

  4. 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

  5. XLSearch: a Probabilistic Database Search Algorithm for Identifying Cross-Linked Peptides.

    PubMed

    Ji, Chao; Li, Sujun; Reilly, James P; Radivojac, Predrag; Tang, Haixu

    2016-06-01

    Chemical cross-linking combined with mass spectrometric analysis has become an important technique for probing protein three-dimensional structure and protein-protein interactions. A key step in this process is the accurate identification and validation of cross-linked peptides from tandem mass spectra. The identification of cross-linked peptides, however, presents challenges related to the expanded nature of the search space (all pairs of peptides in a sequence database) and the fact that some peptide-spectrum matches (PSMs) contain one correct and one incorrect peptide but often receive scores that are comparable to those in which both peptides are correctly identified. To address these problems and improve detection of cross-linked peptides, we propose a new database search algorithm, XLSearch, for identifying cross-linked peptides. Our approach is based on a data-driven scoring scheme that independently estimates the probability of correctly identifying each individual peptide in the cross-link given knowledge of the correct or incorrect identification of the other peptide. These conditional probabilities are subsequently used to estimate the joint posterior probability that both peptides are correctly identified. Using the data from two previous cross-link studies, we show the effectiveness of this scoring scheme, particularly in distinguishing between true identifications and those containing one incorrect peptide. We also provide evidence that XLSearch achieves more identifications than two alternative methods at the same false discovery rate (availability: https://github.com/COL-IU/XLSearch ). PMID:27068484

  6. Arabinosylation Plays a Crucial Role in Extensin Cross-linking In Vitro

    PubMed Central

    Chen, Yuning; Dong, Wen; Tan, Li; Held, Michael A; Kieliszewski, Marcia J

    2015-01-01

    Extensins (EXTs) are hydroxyproline-rich glycoproteins (HRGPs) that are structural components of the plant primary cell wall. They are basic proteins and are highly glycosylated with carbohydrate accounting for >50% of their dry weight. Carbohydrate occurs as monogalactosyl serine and arabinosyl hydroxyproline, with arabinosides ranging in size from ~1 to 4 or 5 residues. Proposed functions of EXT arabinosylation include stabilizing the polyproline II helix structure and facilitating EXT cross-linking. Here, the involvement of arabinosylation in EXT cross-linking was investigated by assaying the initial cross-linking rate and degree of cross-linking of partially or fully de-arabinosylated EXTs using an in vitro cross-linking assay followed by gel permeation chromatography. Our results indicate that EXT arabinosylation is required for EXT cross-linking in vitro and the fourth arabinosyl residue in the tetraarabinoside chain, which is uniquely α-linked, may determine the initial cross-linking rate. Our results also confirm the conserved structure of the oligoarabinosides across species, indicating an evolutionary significance for EXT arabinosylation. PMID:26568683

  7. 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. PMID:18636736

  8. Sulfhydryl site-specific cross-linking and labeling of monoclonal antibodies by a fluorescent equilibrium transfer alkylation cross-link reagent.

    PubMed

    del Rosario, R B; Wahl, R L; Brocchini, S J; Lawton, R G; Smith, R H

    1990-01-01

    The site-specific intramolecular cross-linking of sulfhydryls of monoclonal antibodies via a new class of "equilibrium transfer alkylation cross-link (ETAC) reagents" is described. Following complete or partial reduction of interchain disulfides with dithiothreitol (DTT), two murine IgG2a monoclonal antibodies, 225.28S and 5G6.4, were reacted with alpha,alpha-bis[(p-tolylsulfonyl)methyl]-m-aminoacetophenone (ETAC 1a) and a fluorescent conjugated derivative, sulforhodamine B m-(alpha,alpha-bis(p-tolysulfonylmethyl)acetyl)anilide derivative (ETAC 1b). Reducing SDS-polyacrylamide gel electrophoresis analysis of the products from 1b indicated the formation of S-ETAC-S interchain heavy and light chain cross-links (approximately 23-34% overall yield by video-camera densitometry) which do not undergo disulfide-thiol exchange with DTT at 100 degrees C. In contrast, no interchain cross-links were observed upon reaction of unreduced or reduced antibody wherein the thiols have been previously alkylated with iodoacetamide. These results indicated site-specific cross-linking of interchain sulfhydryls and places their distance within 3-4 A. Flow cytometry of the ETAC 1b 5G6.4 cross-linked product using 77 IP3 human ovarian carcinoma target cells showed positive binding and retention of immunoreactivity. The in vivo biodistributions of 131I-labeled intact 5G6.4 and 125I-labeled reduced 5G6.4 + ETAC 1a product in rats were essentially identical over a period of 24 h. The present study illustrates the potential applications of labelable ETAC reagents as thiol-specific probes for a wide variety of immunological studies. PMID:2128870

  9. Fabrication of manganese dioxide nanoplates anchoring on biomass-derived cross-linked carbon nanosheets for high-performance asymmetric supercapacitors

    NASA Astrophysics Data System (ADS)

    Li, Yiju; Yu, Neng; Yan, Peng; Li, Yuguang; Zhou, Xuemei; Chen, Shuangling; Wang, Guiling; Wei, Tong; Fan, Zhuangjun

    2015-12-01

    In this paper, MnO2 nanoplates loading on biomass-derived cross-linked carbon nanosheets have been prepared by a two-step synthesis. At first, the cross-linked carbon nanosheets derived from willow catkin are synthesized by one-step pyrolysis and activation method, then the MnO2 anchored cross-linked carbon nanosheets is prepared via in-situ hydrothermal deposition. The asymmetric supercapacitor with terrific energy and power density is assembled by employing the MnO2 anchored cross-linked carbon nanosheets as the positive electrode and the cross-linked carbon nanosheets as the negative electrode in a 1 M Na2SO4 electrolyte. The asymmetric supercapacitor displays a high energy density of 23.6 Wh kg-1 at a power density of 188.8 W kg-1 within a wide voltage rage of 0-1.9 V. In addition, the asymmetric supercapacitor exhibits excellent cycling stability with only 1.4% capacitance loss after 10000 cycles at 1 A g-1. These discoveries open up the prospect of biomass/biowaste derived carbon-based composites for high-voltage asymmetric supercapacitors with superb energy and power density performance.

  10. Existence of G-quadruplex structures in promoter region of oncogenes confirmed by G-quadruplex DNA cross-linking strategy

    PubMed Central

    Yuan, Libo; Tian, Tian; Chen, Yuqi; Yan, Shengyong; Xing, Xiwen; Zhang, Zhengan; Zhai, Qianqian; Xu, Liang; Wang, Shaoru; Weng, Xiaocheng; Yuan, Bifeng; Feng, Yuqi; Zhou, Xiang

    2013-01-01

    Existence of G-quadruplex DNA in vivo always attract widespread interest in the field of biology and biological chemistry. We reported our findings for the existence of G-quadruplex structures in promoter region of oncogenes confirmed by G-quadruplex DNA cross-linking strategy. Probes for selective G-quadruplex cross-linking was designed and synthesized that show high selectivity for G-quadruplex cross-linking. Further biological studies demonstrated its good inhibition activity against murine melanoma cells. To further investigate if G-quadruplex DNA was formed in vivo and as the target, a derivative was synthesized and pull-down process toward chromosome DNAs combined with circular dichroism and high throughput deep sequencing were performed. Several simulated intracellular conditions, including X. laevis oocytes, Ficoll 70 and PEG, was used to investigate the compound's pure cross-linking ability upon preformed G-quadruplex. Thus, as a potent G-quadruplex cross-linking agent, our strategy provided both valuable evidence of G-quadruplex structures in vivo and intense potential in anti-cancer therapy. PMID:23657205

  11. Solution structure of the reduced form of human peroxiredoxin-6 elucidated using zero-length chemical cross-linking and homology modelling.

    PubMed

    Rivera-Santiago, Roland F; Harper, Sandra L; Zhou, Suiping; Sriswasdi, Sira; Feinstein, Sheldon I; Fisher, Aron B; Speicher, David W

    2015-05-15

    Peroxiredoxin-6 (PRDX6) is an unusual member of the peroxiredoxin family of antioxidant enzymes that has only one evolutionarily conserved cysteine. It reduces oxidized lipids and reactive oxygen species (ROS) by oxidation of the active-site cysteine (Cys(47)) to a sulfenic acid, but the mechanism for conversion back to a thiol is not completely understood. Moreover, it has phospholipase A2 (PLA2) activity in addition to its peroxidase activity. Interestingly, some biochemical data are inconsistent with a known high-resolution crystal structure of the catalytic intermediate of the protein, and biophysical data indicate that the protein undergoes conformational changes that affect enzyme activity. In order to further elucidate the solution structure of this important enzyme, we used chemical cross-linking coupled with high-resolution MS (CX-MS), with an emphasis on zero-length cross-links. Distance constraints from high confidence cross-links were used in homology modelling experiments to determine a solution structure of the reduced form of the protein. This structure was further evaluated using chemical cross-links produced by several homo-bifunctional amine-reactive cross-linking reagents, which helped to confirm the solution structure. The results show that several regions of the reduced version of human PRDX6 are in a substantially different conformation from that shown for the crystal structure of the peroxidase catalytic intermediate. The differences between these two structures are likely to reflect catalysis-related conformational changes. These studies also demonstrate that CX-MS using zero-length cross-linking is a powerful strategy for probing protein conformational changes that is complementary to alternative methods such as crystallographic, NMR and biophysical studies. PMID:25748205

  12. Helical buckling of actin inside filopodia generates traction

    PubMed Central

    Leijnse, Natascha; Oddershede, Lene B.; Bendix, Poul M.

    2015-01-01

    Cells can interact with their surroundings via filopodia, which are membrane protrusions that extend beyond the cell body. Filopodia are essential during dynamic cellular processes like motility, invasion, and cell–cell communication. Filopodia contain cross-linked actin filaments, attached to the surrounding cell membrane via protein linkers such as integrins. These actin filaments are thought to play a pivotal role in force transduction, bending, and rotation. We investigated whether, and how, actin within filopodia is responsible for filopodia dynamics by conducting simultaneous force spectroscopy and confocal imaging of F-actin in membrane protrusions. The actin shaft was observed to periodically undergo helical coiling and rotational motion, which occurred simultaneously with retrograde movement of actin inside the filopodium. The cells were found to retract beads attached to the filopodial tip, and retraction was found to correlate with rotation and coiling of the actin shaft. These results suggest a previously unidentified mechanism by which a cell can use rotation of the filopodial actin shaft to induce coiling and hence axial shortening of the filopodial actin bundle. PMID:25535347

  13. A single charge in the actin binding domain of fascin can independently tune the linear and non-linear response of an actin bundle network.

    PubMed

    Maier, M; Müller, K W; Heussinger, C; Köhler, S; Wall, W A; Bausch, A R; Lieleg, O

    2015-05-01

    Actin binding proteins (ABPs) not only set the structure of actin filament assemblies but also mediate the frequency-dependent viscoelastic moduli of cross-linked and bundled actin networks. Point mutations in the actin binding domain of those ABPs can tune the association and dissociation dynamics of the actin/ABP bond and thus modulate the network mechanics both in the linear and non-linear response regime. We here demonstrate how the exchange of a single charged amino acid in the actin binding domain of the ABP fascin triggers such a modulation of the network rheology. Whereas the overall structure of the bundle networks is conserved, the transition point from strain-hardening to strain-weakening sensitively depends on the cross-linker off-rate and the applied shear rate. Our experimental results are consistent both with numerical simulations of a cross-linked bundle network and a theoretical description of the bundle network mechanics which is based on non-affine bending deformations and force-dependent cross-link dynamics. PMID:26004635

  14. Radiation cross-linking in ultra-high molecular weight polyethylene for orthopaedic applications

    PubMed Central

    Oral, Ebru; Muratoglu, Orhun K.

    2007-01-01

    The motivation for radiation cross-linking of ultra-high molecular weight polyethylene (UHMWPE) is to increase its wear resistance to be used as bearing surfaces for total joint arthroplasty. However, radiation also leaves behind long-lived residual free radicals in this polymer, the reactions of which can detrimentally affect mechanical properties. In this review, we focus on the radiation cross-linking and oxidative stability of first and second generation highly cross-linked UHMWPEs developed in our laboratory. PMID:19050735

  15. Role of the C-terminal Extension of Formin 2 in Its Activation by Spire Protein and Processive Assembly of Actin Filaments.

    PubMed

    Montaville, Pierre; Kühn, Sonja; Compper, Christel; Carlier, Marie-France

    2016-02-12

    Formin 2 (Fmn2), a member of the FMN family of formins, plays an important role in early development. This formin cooperates with profilin and Spire, a WASP homology domain 2 (WH2) repeat protein, to stimulate assembly of a dynamic cytoplasmic actin meshwork that facilitates translocation of the meiotic spindle in asymmetric division of mouse oocytes. The kinase-like non-catalytic domain (KIND) of Spire directly interacts with the C-terminal extension of the formin homology domain 2 (FH2) domain of Fmn2, called FSI. This direct interaction is required for the synergy between the two proteins in actin assembly. We have recently demonstrated how Spire, which caps barbed ends via its WH2 domains, activates Fmn2. Fmn2 by itself associates very poorly to filament barbed ends but is rapidly recruited to Spire-capped barbed ends via the KIND domain, and it subsequently displaces Spire from the barbed end to elicit rapid processive assembly from profilin·actin. Here, we address the mechanism by which Spire and Fmn2 compete at barbed ends and the role of FSI in orchestrating this competition as well as in the processivity of Fmn2. We have combined microcalorimetric, fluorescence, and hydrodynamic binding assays, as well as bulk solution and single filament measurements of actin assembly, to show that removal of FSI converts Fmn2 into a Capping Protein. This activity is mimicked by association of KIND to Fmn2. In addition, FSI binds actin at filament barbed ends as a weak capper and plays a role in displacing the WH2 domains of Spire from actin, thus allowing the association of actin-binding regions of FH2 to the barbed end. PMID:26668326

  16. The mtDNA NARP mutation activates the actin-Nrf2 signaling of antioxidant defenses

    SciTech Connect

    Dassa, Emmanuel Philippe; Paupe, Vincent; Goncalves, Sergio; Rustin, Pierre

    2008-04-11

    An efficient handling of superoxides by antioxidant defenses is a crucial issue for cells with respiratory chain deficient mitochondria. We used human cultured skin fibroblasts to delineate the mechanism controlling the expression of antioxidant defenses in the case of a severe ATPase deficiency resulting from an 8993T>G mutation in the mitochondrial ATPase6 gene. We observed the nuclear translocation of the transcription factor Nrf2 associated with thinning of the actin stress fibers. The mobilization of the Nrf2 signaling pathway could be mimicked by a chemical blockade of the ATPase with a specific inhibitor, oligomycin. Interestingly enough, Nrf2 nuclear translocation was not observed in the case of a severe cytochrome oxidase deficiency, indicating that studying the status of this signaling pathway could throw some light on the importance of the oxidative insult associated with different respiratory chain defects.

  17. Preparation and characterization of stable cross-linked enzyme aggregates of novel laccase enzyme from Shewanella putrefaciens and using malachite green decolorization.

    PubMed

    Sinirlioglu, Zeynep Aydin; Sinirlioglu, Deniz; Akbas, Fahri

    2013-10-01

    A novel type laccase from Shewanella putrefaciens was identified, expressed in Escherichia coli, characterized, prepared in cross-linked enzyme aggregates (CLEA) for industrial applications and investigated of decolorization activity on malchite green dye. Enzyme characterization was investigated by enzyme assay, SDS-PAGE and other biochemical reactions. Moreover, cross-linked enzyme aggregates were prepared and characterized. Saturated ammonium sulphate solution was used as the precipitating agent and cross linked with glutaraldehyde. These CLEA-laccase aggregates showed more catalytic efficiency and more stabilities compared to free laccase against harsh conditions of thermal and chemical agents as well as high reusability. Also it showed more decolorization ability. These results suggest that this CLEA is potentially usable in industrial applications. PMID:23992799

  18. Rac1-Rab11-FIP3 regulatory hub coordinates vesicle traffic with actin remodeling and T-cell activation.

    PubMed

    Bouchet, Jérôme; Del Río-Iñiguez, Iratxe; Lasserre, Rémi; Agüera-Gonzalez, Sonia; Cuche, Céline; Danckaert, Anne; McCaffrey, Mary W; Di Bartolo, Vincenzo; Alcover, Andrés

    2016-06-01

    The immunological synapse generation and function is the result of a T-cell polarization process that depends on the orchestrated action of the actin and microtubule cytoskeleton and of intracellular vesicle traffic. However, how these events are coordinated is ill defined. Since Rab and Rho families of GTPases control intracellular vesicle traffic and cytoskeleton reorganization, respectively, we investigated their possible interplay. We show here that a significant fraction of Rac1 is associated with Rab11-positive recycling endosomes. Moreover, the Rab11 effector FIP3 controls Rac1 intracellular localization and Rac1 targeting to the immunological synapse. FIP3 regulates, in a Rac1-dependent manner, key morphological events, like T-cell spreading and synapse symmetry. Finally, Rab11-/FIP3-mediated regulation is necessary for T-cell activation leading to cytokine production. Therefore, Rac1 endosomal traffic is key to regulate T-cell activation. PMID:27154205

  19. Preparation of Au Nanoparticles Immobilized Cross-Linked Poly(4-vinylpyridine) Nanofibers and Their Catalytic Application for the Reduction of 4-Nitrophenol.

    PubMed

    Qin, Qi-Hu; Na, Hui; Zhang, Chunyu; Yu, Qizhou; Zhang, Xue-Quan; Zhang, He-Xin

    2015-05-01

    Catalytic nanofibers are prepared by the immobilization of Au nanoparticles (AuNPs) onto the surface of cross-linked electrospun poly(4-vinylpyridine) (P4VP) nanofibers. The crosslinking of the P4VP nanofibers by 1,4-diiodobutane via quaternization reaction greatly enhances the stability of the nanofibers against the solvent dissolution, which can then be used as promising platform for the immobilization of catalytic metal nanoparticles. The AuNPs immobilized cross-linked P4VP nanofibers have shown a good catalytic activity for the reduction of 4-nitrophenol (4-NP) to 4-aminophenol (4-AP). PMID:26505022

  20. Association of thrombospondin-1 with the actin cytoskeleton of human thrombin-activated platelets through an alphaIIbbeta3- or CD36-independent mechanism.

    PubMed Central

    Saumet, Anne; Jesus, Nando de; Legrand, Chantal; Dubernard, Véronique

    2002-01-01

    Thrombospondin-1 (TSP-1) is an adhesive glycoprotein which, when secreted from alpha-granules of activated platelets, can bind to the cell surface and participate in platelet aggregate formation. In this study, we show that thrombin activation leads to the rapid and specific association of a large amount of secreted alpha-granular TSP-1 with the actin cytoskeleton. This cytoskeletal association of TSP-1 was correlated with platelet secretion, but not aggregation, and was inhibited by cytochalasin D, an inhibitor of actin polymerization. Association of TSP-1 with the actin cytoskeleton was mediated by membrane receptors, as shown by using MAII, a TSP-1-specific monoclonal antibody that inhibited both TSP-1 surface binding to activated platelets and cytoskeletal association. TSP-1 and its potential membrane receptors, e.g. alphaIIbbeta3 integrin, CD36 and CD47, concomitantly associated with the actin cytoskeleton. However, studies on platelets from a patient with type I Glanzmann's thrombasthenia lacking alphaIIbbeta3 and another with barely detectable CD36 showed normal TSP-1 surface expression and association with the actin cytoskeleton. Likewise, no involvement of CD47 in TSP-1 association with the actin cytoskeleton could be inferred from experiments with control platelets using the function-blocking anti-CD47 antibody B6H12. Finally, assembly of signalling complexes, as observed through translocation of tyrosine-phosphorylated proteins and kinases to the actin cytoskeleton, was found to occur in concert with cytoskeletal association of TSP-1, in control platelets as well as in thrombasthenic and CD36-deficient platelets. Our results imply a role for the actin cytoskeleton in the membrane-surface expression process of TSP-1 molecules and suggest a possible coupling of TSP-1 receptors to signalling events occurring independently of alphaIIbbeta3 or CD36. These results provide new insights into the link between surface-bound TSP-1 and the contractile actin

  1. Cross-Linked Conjugated Polymer Fibrils: Robust Nanowires from Functional Polythiophene Diblock Copolymers

    SciTech Connect

    Hammer, Brenton A. G.; Bokel, Felicia A.; Hayward, Ryan C.; Emrick, Todd

    2011-09-27

    A series of poly(3-hexyl thiophene) (P3HT)-based diblock copolymers were prepared and examined in solution for their assembly into fibrils, and post-assembly cross-linking into robust nanowire structures. P3HT-b-poly(3-methanol thiophene) (P3MT), and P3HT-b-poly(3-aminopropyloxymethyl thiophene) (P3AmT) diblock copolymers were synthesized using Grignard metathesis (GRIM) polymerization. Fibrils formed from solution assembly of these copolymers are thus decorated with hydroxyl and amine functionality, and cross-linking is achieved by reaction of diisocyanates with the hydroxyl and amine groups. A variety of cross-linked structures, characterized by transmission electron microscopy (TEM), were produced by this method, including dense fibrillar sheets, fibril bundles, or predominately individual fibrils, depending on the chosen reaction conditions. In solution, the cross-linked fibrils maintained their characteristic vibronic structure in solvents that would normally disrupt (dissolve) the structures.

  2. Determination of the cross-linking effect of adipic acid dihydrazide on glycoconjugate preparation.

    PubMed

    Bystrický, S; Machová, E; Malovíková, A; Kogan, G

    1999-11-01

    The cross-linking effect of adipic acid dihydrazide (ADH) on polysaccharide derivatization can be evaluated by applying combination of elemental analysis and colorimetric assay. Elemental analysis is used for estimation of total ADH bound to polysaccharide and a colorimetric trinitrobenzene sulfonic acid assay is used to determine the part of ADH not involved in cross-linking. The difference of values expressed as molar ratios (per repeating unit) provides information on the amount of ADH involved in cross-linking the polysaccharides. Carboxymethylated polysaccharides were derivatized with different amounts of ADH to test the procedure. Analytical results showed that excess of ADH in the reaction only slightly decreased the cross-linking. The number of carboxyl groups remained unmodified even at high excess of ADH and high concentration of carbodiimide (EDC) coupling reagent. PMID:11003553

  3. Effect of cross-link density on carbon dioxide separation in polydimtheylsiloxane-norbornene membranes

    DOE PAGESBeta

    Hong, Tao; Niu, Zhenbin; Hu, Xunxiang; Gmernicki, Kevin R.; Cheng, Shiwang; Fan, Fei; Johnson, Joseph C.; Hong, Eunice K.; Mahurin, Shannon Mark; Jiang, De-en; et al

    2015-10-01

    Here, the development of high-performance materials for carbon dioxide separation and capture will significantly contribute to a solution for climate change. Herein, (bicycloheptenyl)ethylterminated polydimethylsiloxane (PDMSPNB) membranes with varied cross-link densities were synthesized via ring-opening metathesis polymerization. The developed polymer membranes show higher permeability and better selectivity than those of conventional cross-linked PDMS membrane. The achieved performance (CO2 permeability ≈ 6800 Barrer; CO2/N2 selectivity ≈ 14) is very promising for practical applications. The key to achieving this high performance is the use of an in situ cross-linking method for difunctional PDMS macromonomers, which provides lightly cross-linked membranes. By combining positron annihilationmore » lifetime spectroscopy, broadband dielectric spectroscopy, and gas solubility measurements, key parameters necessary for achieving excellent performance have been elucidated.« less

  4. Cross-linking proteins with bimetallic tetracarboxylate compounds of transition metals

    DOEpatents

    Kostic, N.M.; Chen, J.

    1991-03-05

    Stable cross-linked complexes of transition-metal tetracarboxylates and proteins are formed. The preferred transition-metal is rhodium. The protein may be collagen or an enzyme such as a proteolytic enzyme. No Drawings

  5. Cross-linking proteins with bimetallic tetracarboxylate compounds of transition metals

    DOEpatents

    Kostic, Nenad M.; Chen, Jian

    1991-03-05

    Stable cross-linked complexes of transition-metal tetracarboxylates and proteins are formed. The preferred transition-metal is rhodium. The protein may be collagen or an enzyme such as a proteolytic enzyme.

  6. Vapor deposition of cross-linked fluoropolymer barrier coatings onto pre-assembled microfluidic devices.

    PubMed

    Riche, Carson T; Marin, Brandon C; Malmstadt, Noah; Gupta, Malancha

    2011-09-21

    The interior surfaces of pre-assembled poly(dimethylsiloxane) (PDMS) microfluidic devices were modified with a cross-linked fluoropolymer barrier coating that significantly increased the chemical compatibility of the devices. PMID:21850298

  7. Microfabrication of Photo-Cross-Linked Hyaluronan Hydrogels by Single- and Two-Photon Tyramine Oxidation.

    PubMed

    Loebel, Claudia; Broguiere, Nicolas; Alini, Mauro; Zenobi-Wong, Marcy; Eglin, David

    2015-09-14

    Photo-cross-linking of tyramine-substituted hyaluronan (HA-Tyr) hydrogels is demonstrated for the first time. HA-Tyr hydrogels are fabricated via a rapid photosensitized process using visible light illumination. Nontoxic conditions offer photoencapsulation of human mesenchymal stromal cells (hMSCs) with high viability. Macroscopic gels can be formed in less than 10 s, and one- and two-photon photopatterning enable 2D and 3D microfabrication. Different degrees of cross-linking induce different swelling/shrinking, allowing for light-induced microactuation. These new tools are complementary to the previously reported horseradish peroxidase/hydrogen peroxide cross-linking and allow sequential cross-linking of HA-Tyr matrices. PMID:26222128

  8. Collagen type IX from human cartilage: a structural profile of intermolecular cross-linking sites.

    PubMed Central

    Diab, M; Wu, J J; Eyre, D R

    1996-01-01

    Type IX collagen, a quantitatively minor collagenous component of cartilage, is known to be associated with and covalently cross-linked to type II collagen fibrils in chick and bovine cartilage. Type IX collagen molecules have also been shown to form covalent cross-links with each other in bovine cartilage. In the present study we demonstrate by structural analysis and location of cross-linking sites that, in human cartilage, type IX collagen is covalently cross-linked to type II collagen and to other molecules of type IX collagen. We also present evidence that, if the proteoglycan form of type IX collagen is present in human cartilage, it can only be a minor component of the matrix, similar to findings with bovine cartilage. PMID:8660302

  9. Effect of cross-link density on carbon dioxide separation in polydimtheylsiloxane-norbornene membranes

    SciTech Connect

    Hong, Tao; Niu, Zhenbin; Hu, Xunxiang; Gmernicki, Kevin R.; Cheng, Shiwang; Fan, Fei; Johnson, Joseph C.; Hong, Eunice K.; Mahurin, Shannon Mark; Jiang, De-en; Long, Brian K.; Mays, Jimmy; Sokolov, Alexei P.; Saito, Tomonori

    2015-01-01

    Here, the development of high-performance materials for carbon dioxide separation and capture will significantly contribute to a solution for climate change. Herein, (bicycloheptenyl)ethylterminated polydimethylsiloxane (PDMSPNB) membranes with varied cross-link densities were synthesized via ring-opening metathesis polymerization. The developed polymer membranes show higher permeability and better selectivity than those of conventional cross-linked PDMS membrane. The achieved performance (CO2 permeability ≈ 6800 Barrer; CO2/N2 selectivity ≈ 14) is very promising for practical applications. The key to achieving this high performance is the use of an in situ cross-linking method for difunctional PDMS macromonomers, which provides lightly cross-linked membranes. By combining positron annihilation lifetime spectroscopy, broadband dielectric spectroscopy, and gas solubility measurements, key parameters necessary for achieving excellent performance have been elucidated.

  10. Characteristics, formation, and pathophysiology of glucosepane: a major protein cross-link.

    PubMed

    Sjöberg, Johan Svantesson; Bulterijs, Sven

    2009-04-01

    Advanced glycation end products are the results of a series of chemical reactions collectively known as the Maillard reaction, or nonenzymatic glycation, and sometimes cross-link proteins, thereby impairing their normal function. Glucosepane is the most abundant protein cross-link found in vivo so far and mainly has been shown to accumulate in the extracellular matrix, where it cross-links collagen. Levels of glucosepane increase with aging. By increasing collagen stiffness, glucosepane cross-links may have significant implications in several age-related diseases, such as cardiovascular disease, diabetes, and osteoporosis. Although the formation pathways for glucosepane are relatively well researched, much is still unknown about the accumulation and pathophysiology of glucosepane. PMID:19415980

  11. Effect of cross-link density on carbon dioxide separation in polydimethylsiloxane-norbornene membranes

    DOE PAGESBeta

    Hong, Tao; Niu, Zhenbin; Hu, Xunxiang; Gmernicki, Kevin; Cheng, Shiwang; Fan, Fei; Johnson, J. Casey; Hong, Eunice; Mahurin, Shannon; Jiang, De -en; et al

    2015-01-01

    The development of high performance materials for CO2 separation and capture will significantly contribute to a solution for climate change. In this work, (bicycloheptenyl) ethyl terminated polydimethylsiloxane (PDMSPNB) membranes with varied cross-link densities were synthesized via ring-opening metathesis polymerization. The developed polymer membranes show higher permeability and better selectivity than those of conventional cross-linked PDMS membrane. The achieved performance (CO2 permeability ~ 6800 Barrer and CO2/N2 selectivity ~ 14) is very promising for practical applications. The key to achieving this high performance is the use of an in-situ cross-linking method of the difunctional PDMS macromonomers, which provides lightly cross-linked membranes.more » By combining positron annihilation lifetime spectroscopy, broadband dielectric spectroscopy and gas solubility measurements, we have elucidated the key parameters necessary for achieving their excellent performance.« less

  12. Effect of cross-link density on carbon dioxide separation in polydimtheylsiloxane-norbornene membranes

    SciTech Connect

    Hong, Tao; Niu, Zhenbin; Hu, Xunxiang; Gmernicki, Kevin R.; Cheng, Shiwang; Fan, Fei; Johnson, Joseph C.; Hong, Eunice K.; Mahurin, Shannon Mark; Jiang, De-en; Long, Brian K.; Mays, Jimmy; Sokolov, Alexei P.; Saito, Tomonori

    2015-10-01

    Here, the development of high-performance materials for carbon dioxide separation and capture will significantly contribute to a solution for climate change. Herein, (bicycloheptenyl)ethylterminated polydimethylsiloxane (PDMSPNB) membranes with varied cross-link densities were synthesized via ring-opening metathesis polymerization. The developed polymer membranes show higher permeability and better selectivity than those of conventional cross-linked PDMS membrane. The achieved performance (CO2 permeability ≈ 6800 Barrer; CO2/N2 selectivity ≈ 14) is very promising for practical applications. The key to achieving this high performance is the use of an in situ cross-linking method for difunctional PDMS macromonomers, which provides lightly cross-linked membranes. By combining positron annihilation lifetime spectroscopy, broadband dielectric spectroscopy, and gas solubility measurements, key parameters necessary for achieving excellent performance have been elucidated.

  13. Effect of cross-link density on carbon dioxide separation in polydimethylsiloxane-norbornene membranes

    SciTech Connect

    Hong, Tao; Niu, Zhenbin; Hu, Xunxiang; Gmernicki, Kevin; Cheng, Shiwang; Fan, Fei; Johnson, J. Casey; Hong, Eunice; Mahurin, Shannon; Jiang, De -en; Long, Brian; Mays, Jimmy; Sokolov, Alexei; Saito, Tomonori

    2015-01-01

    The development of high performance materials for CO2 separation and capture will significantly contribute to a solution for climate change. In this work, (bicycloheptenyl) ethyl terminated polydimethylsiloxane (PDMSPNB) membranes with varied cross-link densities were synthesized via ring-opening metathesis polymerization. The developed polymer membranes show higher permeability and better selectivity than those of conventional cross-linked PDMS membrane. The achieved performance (CO2 permeability ~ 6800 Barrer and CO2/N2 selectivity ~ 14) is very promising for practical applications. The key to achieving this high performance is the use of an in-situ cross-linking method of the difunctional PDMS macromonomers, which provides lightly cross-linked membranes. By combining positron annihilation lifetime spectroscopy, broadband dielectric spectroscopy and gas solubility measurements, we have elucidated the key parameters necessary for achieving their excellent performance.

  14. Inverted bulk-heterojunction solar cell with cross-linked hole-blocking layer

    PubMed Central

    Udum, Yasemin; Denk, Patrick; Adam, Getachew; Apaydin, Dogukan H.; Nevosad, Andreas; Teichert, Christian; S. White, Matthew.; S. Sariciftci, Niyazi.; Scharber, Markus C.

    2014-01-01

    We have developed a hole-blocking layer for bulk-heterojunction solar cells based on cross-linked polyethylenimine (PEI). We tested five different ether-based cross-linkers and found that all of them give comparable solar cell efficiencies. The initial idea that a cross-linked layer is more solvent resistant compared to a pristine PEI layer could not be confirmed. With and without cross-linking, the PEI layer sticks very well to the surface of the indium–tin–oxide electrode and cannot be removed by solvents used to process PEI or common organic semiconductors. The cross-linked PEI hole-blocking layer functions for multiple donor–acceptor blends. We found that using cross-linkers improves the reproducibility of the device fabrication process. PMID:24817837

  15. Inverted bulk-heterojunction solar cell with cross-linked hole-blocking layer.

    PubMed

    Udum, Yasemin; Denk, Patrick; Adam, Getachew; Apaydin, Dogukan H; Nevosad, Andreas; Teichert, Christian; S White, Matthew; S Sariciftci, Niyazi; Scharber, Markus C

    2014-05-01

    We have developed a hole-blocking layer for bulk-heterojunction solar cells based on cross-linked polyethylenimine (PEI). We tested five different ether-based cross-linkers and found that all of them give comparable solar cell efficiencies. The initial idea that a cross-linked layer is more solvent resistant compared to a pristine PEI layer could not be confirmed. With and without cross-linking, the PEI layer sticks very well to the surface of the indium-tin-oxide electrode and cannot be removed by solvents used to process PEI or common organic semiconductors. The cross-linked PEI hole-blocking layer functions for multiple donor-acceptor blends. We found that using cross-linkers improves the reproducibility of the device fabrication process. PMID:24817837

  16. A Review of Collagen Cross-Linking in Cornea and Sclera

    PubMed Central

    Zhang, Xiao; Tao, Xiang-chen; Zhang, Jian; Li, Zhi-wei; Xu, Yan-yun; Wang, Yu-meng; Zhang, Chun-xiao; Mu, Guo-ying

    2015-01-01

    Riboflavin/UVA cross-linking is a technique introduced in the past decades for the treatment of keratoconus, keratectasia, and infectious keratitis. Its efficacy and safety have been investigated with clinical and laboratory studies since its first clinical application by Wollensak for the treatment of keratoconus. Although its complications are encountered during clinical practice, such as infection inducing risk, minimal invasion merits a further investigation on its future application in clinical practice. Recently, collagen cross-linking in sclera shows a promising prospect. In present study, we summarized the representative studies describing the clinical and laboratory application of collagen cross-linking published in past decades and provided our opinion on the positive and negative results of cross-linking in the treatment of ophthalmic disorders. PMID:25922758

  17. In Vivo Oxidative Stability Changes of Highly Cross-Linked Polyethylene Bearings: An Ex Vivo Investigation.

    PubMed

    Rowell, Shannon L; Reyes, Christopher R; Malchau, Henrik; Muratoglu, Orhun K

    2015-10-01

    The development of highly cross-linked UHMWPEs focused on stabilizing radiation-induced free radicals as the sole precursor to oxidative degradation. However, secondary in vivo oxidation mechanisms have been discovered. After a preliminary post-operative analysis, we subjected highly cross-linked retrievals with 1-4 years in vivo durations and never-implanted controls to accelerated aging to predict the extent to which their oxidative stability was compromised in vivo. Lipid absorption, oxidation, and hydroperoxides were measured using infrared spectroscopy. Gravimetric swelling was used to measure cross-link density. After aging, all retrievals, except vitamin E-stabilized components, regardless of initial lipid levels or oxidation, showed significant oxidative degradation, demonstrated by subsurface oxidative peaks, increased hydroperoxides and decreased cross-link density, compared to their post-operative material properties and never-implanted counterparts, confirming oxidative stability changes. PMID:26048729

  18. Anomalous cross-linking by mechlorethamine of DNA duplexes containing C-C mismatch pairs.

    PubMed

    Romero, R M; Mitas, M; Haworth, I S

    1999-03-23

    Nitrogen mustards such as mechlorethamine have previously been shown to covalently cross-link DNA through the N7 position of the two guanine bases of a d[GXC].d[GYC] duplex sequence, a so-called 1,3 G-G-cross-link, when X-Y = C-G or T-A. Here, we report the formation of a new mechlorethamine cross-link with the d[GXC].d[GYC] fragment when X-Y is a C-C mismatch pair. Mechlorethamine cross-links this fragment preferentially between the two mismatched cytosine bases, rather than between the guanine bases. The cross-link also forms when one or both of the guanine bases of the d[GCC].d[GCC] fragment are replaced by N7-deazaguanine, and, more generally, forms with any C-C mismatch, regardless of the flanking base pairs. Piperidine cleavage of the cross-link species containing the d[GCC].d[GCC] sequence gives DNA fragments consistent with alkylation at the mismatched cytosine bases. We also provide evidence that the cross-link reaction occurs between the N3 atoms of the two cytosine bases by showing that the formation of the C-C cross-link is pH dependent for both mechlorethamine and chlorambucil. Dimethyl sulfate (DMS) probing of the cross-linked d[GCC].d[GCC] fragment showed that the major groove of the guanine adjacent to the C-C mismatch is still accessible to DMS. In contrast, the known minor groove binder Hoechst 33258 inhibits the cross-link formation with a C-C mismatch pair flanked by A-T base pairs. These results suggest that the C-C mismatch is cross-linked by mechlorethamine in the minor groove. Since C-C pairs may be involved in unusual secondary structures formed by the trinucleotide repeat sequence d[CCG]n, and associated with triplet repeat expansion diseases, mechlorethamine may serve as a useful probe for these structures. PMID:10090751

  19. DNA-protein cross-links produced by various chemicals in cultured human lymphoma cells.

    PubMed

    Costa, M; Zhitkovich, A; Harris, M; Paustenbach, D; Gargas, M

    1997-04-11

    Chemicals such as cis-platinum, formaldehyde, chromate, copper, and certain arsenic compounds have been shown to produce DNA-protein cross-links in human in vitro cell systems at high doses, such as those in the cytotoxic range. Thus far there have only been a limited number of other chemicals evaluated for their ability to produce cross-links. The purpose of the work described here was to evaluate whether select industrial chemicals can form DNA-protein cross-links in human cells in vitro. We evaluated acetaldehyde, acrolein, diepoxybutane, paraformaldehyde, 2-furaldehyde, propionaldehyde, chloroacetaldehyde, sodium arsenite, and a deodorant tablet [Mega Blue; hazardous component listed as tris(hydroxymethyl)nitromethane]. Short- and long-term cytotoxicity was evaluated and used to select appropriate doses for in vitro testing. DNA-protein cross-linking was evaluated at no fewer than three doses and two cell lysate washing temperatures (45 and 65 degrees C) in Epstein-Barr virus (EBV) human Burkitt's lymphoma cells. The two washing temperatures were used to assess the heat stability of the DNA-protein cross-link, 2-Furaldehyde, acetaldehyde, and propionaldehyde produced statistically significant increases in DNA-protein cross-links at washing temperatures of 45 degrees C, but not 65 degrees C, and at or above concentrations of 5, 17.5, and 75 mM, respectively. Acrolein, diepoxybutane, paraformaldehyde, and Mega Blue produced statistically significant increases in DNA-protein cross-links washed at 45 and 65 degrees C at or above concentrations of 0.15 mM, 12.5 mM, 0.003%, and 0.1%, respectively. Sodium arsenite and chloroacetaldehyde did not produce significantly increased DNA-protein cross-links at either temperature nor at any dose tested. Excluding paraformaldehyde and 2-furaldehyde treatments, significant increases in DNA-protein cross-links were observed only at doses that resulted in complete cell death within 4 d following dosing. This work demonstrates that

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

    PubMed

    Lambert, Muriel W

    2016-09-01

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